Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

PubMed

De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

1992-07-15

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

The impact of viral RNA on the association free energies of capsid protein assembly: bacteriophage MS2 as a case study.

PubMed

ElSawy, Karim M

2017-02-01

A large number of single-stranded RNA viruses assemble their capsid and their genomic material simultaneously. The RNA viral genome plays multiple roles in this process that are currently only partly understood. In this work, we investigated the thermodynamic basis of the role of viral RNA on the assembly of capsid proteins. The viral capsid of bacteriophage MS2 was considered as a case study. The MS2 virus capsid is composed of 60 AB and 30 CC protein dimers. We investigated the effect of RNA stem loop (the translational repressor TR) binding to the capsid dimers on the dimer-dimer relative association free energies. We found that TR binding results in destabilization of AB self-association compared with AB and CC association. This indicates that the association of the AB and CC dimers is the most likely assembly pathway for the MS2 virus, which explains the experimental observation of alternating patterns of AB and CC dimers in dominant assembly intermediates of the MS2 virus. The presence of viral RNA, therefore, dramatically channels virus assembly to a limited number of pathways, thereby enhancing the efficiency of virus self-assembly process. Interestingly, Thr59Ser and Thr45Ala mutations of the dimers, in the absence of RNA stem loops, lead to stabilization of AB self-association compared with the AB and CC associations, thereby channelling virus assembly towards a fivefold (AB) 5 pentamer intermediate, providing a testable hypothesis of our thermodynamic arguments.

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

PubMed

Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

2017-10-01

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer.

PubMed

Bhattarai, Nisha; Gc, Jeevan B; Gerstman, Bernard S; Stahelin, Robert V; Chapagain, Prem P

2017-04-26

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

PubMed Central

Brindley, Melinda A.; Plattet, Philippe; Plemper, Richard Karl

2014-01-01

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. PMID:25157143

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain.

PubMed

Brindley, Melinda A; Plattet, Philippe; Plemper, Richard Karl

2014-09-09

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

PubMed Central

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

PubMed Central

Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

2004-01-01

Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

Crystal structure of the Rous sarcoma virus intasome

DOE PAGES

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit; ...

2016-02-17

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Dimeric Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Anti-Hepatitis B Virus Activities.

PubMed

Zhang, Yu-Bo; Zhan, Li-Qin; Li, Guo-Qiang; Wang, Feng; Wang, Ying; Li, Yao-Lan; Ye, Wen-Cai; Wang, Guo-Cai

2016-08-05

Six unusual matrine-type alkaloid dimers, flavesines A-F (1-6, respectively), together with three proposed biosynthetic intermediates (7-9) were isolated from the roots of Sophora flavescens. Compounds 1-5 were the first natural matrine-type alkaloid dimers, and compound 6 represented an unprecedented dimerization pattern constructed by matrine and (-)-cytisine. Their structures were elucidated by NMR, MS, single-crystal X-ray diffraction, and a chemical method. The hypothetical biogenetic pathways of 1-6 were also proposed. Compounds 1-9 exhibited inhibitory activities against hepatitis B virus.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein.

PubMed

Zhang, J Z; Ng, M H; Xia, N S; Lau, S H; Che, X Y; Chau, T N; Lai, S T; Im, S W

2001-06-01

A 23 kDa peptide locating to amino acid residues 394 to 604 of the major Hepatitis E Virus (HEV) structural protein was expressed in E. coli. This peptide was found to interact naturally with one another to form homodimers and it was recognized strongly and commonly in its dimeric form by HEV reactive human sera. The antigenic activity associated with the dimeric form was abrogated when the dimer was dissociated into monomer and the activity was reconstituted after the monomer was re-associated into dimer again. The dimeric form of the peptide elicited a vigorous antibody response in experimental animals and the resulting antisera were found to cross-react against HEV, effecting an efficient immune capture of the virus. These results attributed the antigenic activity associated with the dimeric form of the peptide to conformational antigenic determinants generated as a result of interaction between the peptide molecules. It is suggested that some of these antigenic determinants may be expressed by the HEV capsid and raised the possibility of this bacterially expressed peptide as an HEV vaccine candidate. Copyright 2001 Wiley-Liss, Inc.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

PubMed

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

PubMed Central

Farza, H; Hadchouel, M; Scotto, J; Tiollais, P; Babinet, C; Pourcel, C

1988-01-01

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication. Images PMID:2845128

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed Central

Torrent, C; Gabus, C; Darlix, J L

1994-01-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer. Images PMID:8289369

Crystal structure of the human astrovirus capsid spike.

PubMed

Dong, Jinhui; Dong, Liping; Méndez, Ernesto; Tao, Yizhi

2011-08-02

Astroviruses are single-stranded, plus-sense RNA viruses that infect both mammals and birds, causing gastroenteritis and other extraintestinal diseases. Clinical studies have established astroviruses as the second leading cause of viral diarrhea in young children. Here we report the crystal structure of the human astrovirus dimeric surface spike determined to 1.8-Å resolution. The overall structure of each spike/projection domain has a unique three-layered β-sandwiches fold, with a core, six-stranded β-barrel structure that is also found in the hepatitis E virus capsid protrusions, suggesting a closer phylogenetic relationship between these two viruses than previously acknowledged. Based on a hepatitis E virus capsid model, we performed homology modeling and produced a complete, T = 3 astrovirus capsid model with features remarkably similar to those observed in a cryoelectron microscopy reconstruction image of a human astrovirus. Mapping conserved residues onto the astrovirus projection domain revealed a putative receptor binding site with amino acid compositions characteristic for polysaccharide recognition. Our results will have an important impact on future characterization of astrovirus structure and function, and will likely have practical applications in the development of vaccines and antivirals.

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

PubMed Central

Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

2013-01-01

The 5′ untranslated region (5′ UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNALys3 annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5′-UTR RNA. In this study, we show that annealing of tRNALys3 or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5′ UTR enhances its dimerization in vitro. Structural analysis of the 5′-UTR RNA using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5′ UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNALys3 to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNALys3 to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions. PMID:23960173

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

PubMed

Scheuring, Simon; Busselez, Johan; Lévy, Daniel

2005-01-14

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed

Bieth, E; Gabus, C; Darlix, J L

1990-01-11

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed Central

Bieth, E; Gabus, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12. Images PMID:2155394

Engineering Human Immunodeficiency Virus 1 Protease Heterodimers as Macromolecular Inhibitors of Viral Maturation

NASA Astrophysics Data System (ADS)

McPhee, Fiona; Good, Andrew C.; Kuntz, Irwin D.; Craik, Charles S.

1996-10-01

Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Ping; Swanson, Kurt A.; Leser, George P.

2014-10-02

The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HNmore » interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.« less

Dimerization of BTas is required for the transactivational activity of bovine foamy virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan Juan; Qiao Wentao; Xu Fengwen

2008-06-20

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activitymore » on the LTR and IP. Furthermore, BTas ({delta}46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.« less

Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

PubMed Central

McPhee, F; Good, A C; Kuntz, I D; Craik, C S

1996-01-01

Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876160

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed Central

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-01-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA. PMID:7884897

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-04-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA.

Last 20 aa of the West Nile virus NS1' protein are responsible for its retention in cells and the formation of unique heat-stable dimers.

PubMed

Young, Lucy B; Melian, Ezequiel Balmori; Setoh, Yin Xiang; Young, Paul R; Khromykh, Alexander A

2015-05-01

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice. © 2015 The Authors.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

PubMed

Clifton, Matthew C; Bruhn, Jessica F; Atkins, Kateri; Webb, Terry L; Baydo, Ruth O; Raymond, Amy; Lorimer, Donald D; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2015-10-01

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

PubMed

Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

1990-12-05

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.

PubMed

Haglin, Elizabeth R; Yang, Wen; Briegel, Ariane; Thompson, Lynmarie K

2017-11-07

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

(19)F NMR reveals multiple conformations at the dimer interface of the nonstructural protein 1 effector domain from influenza A virus.

PubMed

Aramini, James M; Hamilton, Keith; Ma, Li-Chung; Swapna, G V T; Leonard, Paul G; Ladbury, John E; Krug, Robert M; Montelione, Gaetano T

2014-04-08

Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply (19)F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan, and we demonstrate that the (19)F signal of Trp187 is a sensitive, direct monitor of the ED helix:helix dimer interface. (19)F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein:protein interface, whose rate is more than three orders of magnitude faster than the kinetics of ED dimerization. (19)F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

PubMed

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-02-24

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-01-01

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. PMID:28233845

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com; Zhou, Hui; Ethen, Cheryl M.

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6more » fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain why 1918 pandemic influenza caused higher death rate among young population.« less

In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication.

PubMed

Liu, Q; Yong, C B; Astell, C R

1994-06-01

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.

C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain.

PubMed

Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2017-05-15

HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS. Copyright © 2017 American Society for Microbiology.

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.

2010-11-03

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have amore » degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.« less

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

PubMed

Bausewein, Thomas; Mills, Deryck J; Langer, Julian D; Nitschke, Beate; Nussberger, Stephan; Kühlbrandt, Werner

2017-08-10

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrastructural and Functional Analyses of Recombinant Influenza Virus Ribonucleoproteins Suggest Dimerization of Nucleoprotein during Virus Amplification

PubMed Central

Ortega, Joaquín; Martín-Benito, Jaime; Zürcher, Thomas; Valpuesta, José M.; Carrascosa, José L.; Ortín, Juan

2000-01-01

Influenza virus ribonucleoproteins (RNPs) were reconstituted in vivo from cloned cDNAs expressing the three polymerase subunits, the nucleoprotein (NP), and short template RNAs. The structure of purified RNPs was studied by electron microscopy and image processing. Circular and elliptic structures were obtained in which the NP and the polymerase complex could be defined. Comparison of the structure of RNPs of various lengths indicated that each NP monomer interacts with approximately 24 nucleotides. The analysis of the amplification of RNPs with different lengths showed that those with the highest replication efficiency contained an even number of NP monomers, suggesting that the NP is incorporated as dimers into newly synthesized RNPs. PMID:10590102

Two Populations Mean-Field Monomer-Dimer Model

NASA Astrophysics Data System (ADS)

Alberici, Diego; Mingione, Emanuele

2018-04-01

A two populations mean-field monomer-dimer model including both hard-core and attractive interactions between dimers is considered. The pressure density in the thermodynamic limit is proved to satisfy a variational principle. A detailed analysis is made in the limit of one population is much smaller than the other and a ferromagnetic mean-field phase transition is found.

Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection.

PubMed

Fernandez, Estefania; Dejnirattisai, Wanwisa; Cao, Bin; Scheaffer, Suzanne M; Supasa, Piyada; Wongwiwat, Wiyada; Esakky, Prabagaran; Drury, Andrea; Mongkolsapaya, Juthathip; Moley, Kelle H; Mysorekar, Indira U; Screaton, Gavin R; Diamond, Michael S

2017-11-01

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM.

PubMed

Hashiguchi, Takao; Ose, Toyoyuki; Kubota, Marie; Maita, Nobuo; Kamishikiryo, Jun; Maenaka, Katsumi; Yanagi, Yusuke

2011-02-01

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

The H2A-H2B dimeric kinetic intermediate is stabilized by widespread hydrophobic burial with few fully native interactions.

PubMed

Guyett, Paul J; Gloss, Lisa M

2012-01-20

The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ∼5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I₂ and I₂*, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I₂ ensemble folds to the native heterodimer, N₂, through an observable, first-order kinetic phase. To determine the regions of structure in the I₂ ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I₂, the transition state and N₂; however, only residues in the hydrophobic core of the dimer interface perturbed the I₂* population. Destabilization of I₂* correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I₂ and N₂, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I₂. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I₂, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N₂, more native-like interactions are developed and nonnative interactions are rearranged. Copyright © 2011 Elsevier Ltd. All rights reserved.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

PubMed Central

Gherghe, Cristina; Lombo, Tania; Leonard, Christopher W.; Datta, Siddhartha A. K.; Bess, Julian W.; Gorelick, Robert J.; Rein, Alan; Weeks, Kevin M.

2010-01-01

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs. PMID:20974908

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kweon, Kyoung E.; Aberg, Daniel; Lordi, Vincenzo

The atomic and electronic structures of 60° glide perfect and 30°/90° glide partial dislocations in CdTe are studied using combined semi-empirical and density functional theory calculations. The calculations predict that the dislocation cores tend to undergo significant reconstructions along the dislocation lines from the singly-periodic (SP) structures, yielding either doubly-periodic (DP) ordering by forming a dimer or quadruply-periodic (QP) ordering by alternating a dimer and a missing dimer. Charge modulation along the dislocation line, accompanied by the QP reconstruction for the Cd-/Te-core 60° perfect and 30° partials or the DP reconstruction for the Cd-core 90° partial, results in semiconducting character,more » as opposed to the metallic character of the SP dislocation cores. Dislocation-induced defect states for the 60° Cd-/Te-core are located relatively close to the band edges, whereas the defect states lie in the middle of the band gap for the 30° Cd-/Te-core partial dislocations. In addition to the intracore charge modulation within each QP core, the possibility of intercore charge transfer between two different dislocation cores when they are paired together in the same system is discussed. As a result, the analysis of the electronic structures reveals the potential role of the dislocations on charge transport in CdTe, particularly in terms of charge trapping and recombination.« less

Physiological temperatures reduce dimerization of dengue and Zika virus recombinant envelope proteins.

PubMed

Kudlacek, Stephan T; Premkumar, Lakshmanane; Metz, Stefan W; Tripathy, Ashutosh; Bobkov, Andrey A; Payne, Alexander Matthew; Graham, Stephen; Brackbill, James A; Miley, Michael J; de Silva, Aravinda M; Kuhlman, Brian

2018-06-08

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 μm at 25 °C to 50 μm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine. © 2018 Kudlacek et al.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

PubMed

Friberg, Anders; Thumann, Sybille; Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D; Sattler, Michael; Kempkes, Bettina

2015-05-01

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

Characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis.

PubMed

Gritsun, T S; Frolova, T V; Zhankov, A I; Armesto, M; Turner, S L; Frolova, M P; Pogodina, V V; Lashkevich, V A; Gould, E A

2003-01-01

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T(277)-->V and E(279)-->G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

Characterization of a Siberian Virus Isolated from a Patient with Progressive Chronic Tick-Borne Encephalitis

PubMed Central

Gritsun, T. S.; Frolova, T. V.; Zhankov, A. I.; Armesto, M.; Turner, S. L.; Frolova, M. P.; Pogodina, V. V.; Lashkevich, V. A.; Gould, E. A.

2003-01-01

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T277→V and E279→G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis. PMID:12477807

Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro, E-mail: fujii@sapmed.ac.j

2011-05-25

Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression ofmore » C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.« less

Theoretical analysis of bimetallic nanorod dimer biosensors for label-free molecule detection

NASA Astrophysics Data System (ADS)

Das, Avijit; Talukder, Muhammad Anisuzzaman

2018-02-01

In this work, we theoretically analyze a gold (Au) core within silver (Ag) shell (Au@Ag) nanorod dimer biosensor for label-free molecule detection. The incident light on an Au@Ag nanorod strongly couples to localized surface plasmon modes, especially around the tip region. The field enhancement around the tip of a nanorod or between the tips of two longitudinally aligned nanorods as in a dimer can be exploited for sensitive detection of biomolecules. We derive analytical expressions for the interactions of an Au@Ag nanorod dimer with the incident light. We also study the detail dynamics of an Au@Ag nanorod dimer with the incident light computationally using finite difference time domain (FDTD) technique when core-shell ratio, relative position of the nanorods, and angle of incidence of light change. We find that the results obtained using the developed analytical model match well with that obtained using FDTD simulations. Additionally, we investigate the sensitivity of the Au@Ag nanorod dimer, i.e., shift in the resonance wavelength, when a target biomolecule such as lysozyme (Lys), human serum albumin (HSA), anti-biotin (Abn), human catalase (CAT), and human fibrinogen (Fb) protein molecules are attached to the tips of the nanorods.

Glycolaldehyde Formation via the Dimerization of the Formyl Radical

NASA Astrophysics Data System (ADS)

Woods, Paul M.; Slater, Ben; Raza, Zamaan; Viti, Serena; Brown, Wendy A.; Burke, Daren J.

2013-11-01

Glycolaldehyde, the simplest monosaccharide sugar, has recently been detected in low- and high-mass star-forming cores. Following our previous investigation into glycolaldehyde formation, we now consider a further mechanism for the formation of glycolaldehyde that involves the dimerization of the formyl radical, HCO. Quantum mechanical investigation of the HCO dimerization process upon an ice surface is predicted to be barrierless and therefore fast. In an astrophysical context, we show that this mechanism can be very efficient in star-forming cores. It is limited by the availability of the formyl radical, but models suggest that only very small amounts of CO are required to be converted to HCO to meet the observational constraints.

Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein.

PubMed

Wang, Yue-Dan; Chen, Wei Feng

2004-11-01

To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.

Restoration of glycoprotein Erns dimerization via pseudoreversion partially restores virulence of classical swine fever virus.

PubMed

Tucakov, Anna Katharina; Yavuz, Sabine; Schürmann, Eva-Maria; Mischler, Manjula; Klingebeil, Anne; Meyers, Gregor

2018-01-01

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein E rns is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell. A further important feature with regard to its function as a virulence factor is the formation of disulfide-linked E rns homodimers that are found in virus-infected cells and virions. Mutant CSFV lacking cysteine (Cys) 171, the residue responsible for intermolecular disulfide bond formation, were found to be attenuated in pigs (Tews BA, Schürmann EM, Meyers G. J Virol 2009;83:4823-4834). In the course of an animal experiment with such a dimerization-negative CSFV mutant, viruses were reisolated from pigs that contained a mutation of serine (Ser) 209 to Cys. This mutation restored the ability to form disulphide-linked E rns homodimers. In transient expression studies E rns mutants carrying the S209C change were found to form homodimers with about wt efficiency. Also the secretion level of the mutated proteins was equivalent to that of wt E rns . Virus mutants containing the Cys171Ser/Ser209Cys configuration exhibited wt growth rates and increased virulence when compared with the Cys171Ser mutant. These results provide further support for the connection between CSFV virulence and E rns dimerization.

Computational design of d-peptide inhibitors of hepatitis delta antigen dimerization

NASA Astrophysics Data System (ADS)

Elkin, Carl D.; Zuccola, Harmon J.; Hogle, James M.; Joseph-McCarthy, Diane

2000-11-01

Hepatitis delta virus (HDV) encodes a single polypeptide called hepatitis delta antigen (DAg). Dimerization of DAg is required for viral replication. The structure of the dimerization region, residues 12 to 60, consists of an anti-parallel coiled coil [Zuccola et al., Structure, 6 (1998) 821]. Multiple Copy Simultaneous Searches (MCSS) of the hydrophobic core region formed by the bend in the helix of one monomer of this structure were carried out for many diverse functional groups. Six critical interaction sites were identified. The Protein Data Bank was searched for backbone templates to use in the subsequent design process by matching to these sites. A 14 residue helix expected to bind to the d-isomer of the target structure was selected as the template. Over 200 000 mutant sequences of this peptide were generated based on the MCSS results. A secondary structure prediction algorithm was used to screen all sequences, and in general only those that were predicted to be highly helical were retained. Approximately 100 of these 14-mers were model built as d-peptides and docked with the l-isomer of the target monomer. Based on calculated interaction energies, predicted helicity, and intrahelical salt bridge patterns, a small number of peptides were selected as the most promising candidates. The ligand design approach presented here is the computational analogue of mirror image phage display. The results have been used to characterize the interactions responsible for formation of this model anti-parallel coiled coil and to suggest potential ligands to disrupt it.

Gating of the designed trimeric/tetrameric voltage-gated H+ channel

PubMed Central

Fujiwara, Yuichiro; Kurokawa, Tatsuki; Takeshita, Kohei; Nakagawa, Atsushi; Larsson, H Peter; Okamura, Yasushi

2013-01-01

The voltage-gated H+ channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels. Each channel protomer has its own permeation pathway. The C-terminal coiled-coil domain has been shown to be necessary for both dimerization and cooperative gating in the two channel protomers. Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain. Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H+ permeation than dimeric channels, suggesting that some channel protomers in trimers and tetramers failed to produce gating cooperativity observed in wild-type dimers. Multimerization of trimer and tetramer channels were confirmed by the biochemical analysis of proteins, including crystallography. These findings indicate that the voltage-gated H+ channel is optimally designed as a dimeric channel on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative gating that ensures sustained and steep voltage-dependent H+ conductance in blood cells. PMID:23165764

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus.

PubMed

Bonaldo, Myrna C; Garratt, Richard C; Caufour, Philippe S; Freire, Marcos S; Rodrigues, Mauricio M; Nussenzweig, Ruth S; Galler, Ricardo

2002-01-25

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between beta-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses. Copyright 2002 Academic Press.

First-principles study of atomic and electronic structures of 60° perfect and 30°/90° partial glide dislocations in CdTe

DOE PAGES

Kweon, Kyoung E.; Aberg, Daniel; Lordi, Vincenzo

2016-05-16

The atomic and electronic structures of 60° glide perfect and 30°/90° glide partial dislocations in CdTe are studied using combined semi-empirical and density functional theory calculations. The calculations predict that the dislocation cores tend to undergo significant reconstructions along the dislocation lines from the singly-periodic (SP) structures, yielding either doubly-periodic (DP) ordering by forming a dimer or quadruply-periodic (QP) ordering by alternating a dimer and a missing dimer. Charge modulation along the dislocation line, accompanied by the QP reconstruction for the Cd-/Te-core 60° perfect and 30° partials or the DP reconstruction for the Cd-core 90° partial, results in semiconducting character,more » as opposed to the metallic character of the SP dislocation cores. Dislocation-induced defect states for the 60° Cd-/Te-core are located relatively close to the band edges, whereas the defect states lie in the middle of the band gap for the 30° Cd-/Te-core partial dislocations. In addition to the intracore charge modulation within each QP core, the possibility of intercore charge transfer between two different dislocation cores when they are paired together in the same system is discussed. As a result, the analysis of the electronic structures reveals the potential role of the dislocations on charge transport in CdTe, particularly in terms of charge trapping and recombination.« less

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

PubMed

Messer, William B; Yount, Boyd L; Royal, Scott R; de Alwis, Ruklanthi; Widman, Douglas G; Smith, Scott A; Crowe, James E; Pfaff, Jennifer M; Kahle, Kristen M; Doranz, Benjamin J; Ibarra, Kristie D; Harris, Eva; de Silva, Aravinda M; Baric, Ralph S

2016-05-15

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus.

PubMed

Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Supasa, Sunpetchuda; Zhang, Xiaokang; Dai, Xinghong; Rouvinski, Alexander; Jumnainsong, Amonrat; Edwards, Carolyn; Quyen, Nguyen Than Ha; Duangchinda, Thaneeya; Grimes, Jonathan M; Tsai, Wen-Yang; Lai, Chih-Yun; Wang, Wei-Kung; Malasit, Prida; Farrar, Jeremy; Simmons, Cameron P; Zhou, Z Hong; Rey, Felix A; Mongkolsapaya, Juthathip; Screaton, Gavin R

2015-02-01

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed Central

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-01-01

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. Images PMID:1645868

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

PubMed

Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P; Takeda, Makoto

2016-08-02

Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.

Crystal structure of the Japanese encephalitis virus envelope protein.

PubMed

Luca, Vincent C; AbiMansour, Jad; Nelson, Christopher A; Fremont, Daved H

2012-02-01

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE PAGES

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.; ...

2018-03-27

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Period-doubling reconstructions of semiconductor partial dislocations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Ji -Sang; Huang, Bing; Wei, Su -Huai

2015-09-18

Atomic-scale understanding and control of dislocation cores is of great technological importance, because they act as recombination centers for charge carriers in optoelectronic devices. Using hybrid density-functional calculations, we present period-doubling reconstructions of a 90 degrees partial dislocation in GaAs, for which the periodicity of like-atom dimers along the dislocation line varies from one to two, to four dimers. The electronic properties of a dislocation change drastically with each period doubling. The dimers in the single-period dislocation are able to interact, to form a dispersive one-dimensional band with deep-gap states. However, the inter-dimer interaction for the double-period dislocation becomes significantlymore » reduced; hence, it is free of mid-gap states. The Ga core undergoes a further period-doubling transition to a quadruple-period reconstruction induced by the formation of small hole polarons. In conclusion, the competition between these dislocation phases suggests a new passivation strategy via population manipulation of the detrimental single-period phase.« less

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

PubMed

van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

2014-01-01

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Topology and Dynamics of the Zebrafish Segmentation Clock Core Circuit

PubMed Central

Schröter, Christian; Isakova, Alina; Hens, Korneel; Soroldoni, Daniele; Gajewski, Martin; Jülicher, Frank; Maerkl, Sebastian J.; Deplancke, Bart; Oates, Andrew C.

2012-01-01

During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a “dimer cloud” that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks. PMID:22911291

Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

PubMed Central

Counihan, Natalie A.; Rawlinson, Stephen M.; Lindenbach, Brett D.

2011-01-01

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly. PMID:22028650

Spectral and kinetic effects accompanying the assembly of core complexes of Rhodobacter sphaeroides.

PubMed

Freiberg, Arvi; Chenchiliyan, Manoop; Rätsep, Margus; Timpmann, Kõu

2016-11-01

In the present work, spectral and kinetic changes accompanying the assembly of the light-harvesting 1 (LH1) complex with the reaction center (RC) complex into monomeric RC-LH1 and dimeric RC-LH1-PufX core complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides are systematically studied over the temperature range of 4.5-300K. The samples were interrogated with a combination of optical absorption, hole burning, fluorescence excitation, steady state and picosecond time resolved fluorescence spectroscopy. Fair additivity of the LH1 and RC absorption spectra suggests rather weak electronic coupling between them. A low-energy tail revealed at cryogenic temperatures in the absorption spectra of both monomeric and dimeric core complexes is proved to be due to the special pair of the RC. At selected excitation intensity and temperature, the fluorescence decay time of core complexes is shown to be a function of multiple factors, most importantly of the presence/absence of RCs, the supramolecular architecture (monomeric or dimeric) of the complexes, and whether the complexes were studied in a native membrane environment or in a detergent - purified state. Copyright © 2016 Elsevier B.V. All rights reserved.

Special Features of Light Absorption by the Dimer of Bilayer Microparticles

NASA Astrophysics Data System (ADS)

Geints, Yu. É.; Panina, E. K.; Zemlyanov, A. A.

2018-05-01

Results of numerical simulation of light absorption by the dimer of bilayer spherical particles consisting of a water core and a polymer shell absorbing radiation are presented. The spatial distribution and the amplitude characteristics of the volume density of the absorbed power are investigated. It is shown that for a certain spatial dimer configuration, the maximal achievable density of the absorbed power is realized. It is also established that for closely spaced microcapsules with high shell absorption indices, the total power absorbed in the dimer volume can increase in comparison with the radiation absorption by two insulated microparticles.

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

PubMed

Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J; Halfmann, Peter; Bornholdt, Zachary A; Abelson, Dafna M; Armbrust, Tammy; Stahelin, Robert V; Kawaoka, Yoshihiro; Saphire, Erica Ollmann

2016-02-15

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

PubMed

Bonnard, Damien; Le Rouzic, Erwann; Eiler, Sylvia; Amadori, Céline; Orlov, Igor; Bruneau, Jean-Michel; Brias, Julie; Barbion, Julien; Chevreuil, Francis; Spehner, Danièle; Chasset, Sophie; Ledoussal, Benoit; Moreau, François; Saïb, Ali; Klaholz, Bruno P; Emiliani, Stéphane; Ruff, Marc; Zamborlini, Alessia; Benarous, Richard

2018-04-20

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.

2014-10-02

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalkmore » domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.« less

Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion▿

PubMed Central

Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

2011-01-01

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion. PMID:21994464

Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korasick, David A.; Singh, Harkewal; Pemberton, Travis A.

2017-08-01

Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significancemore » of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs.« less

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein.

PubMed

del Alamo, Marta; Mateu, Mauricio G

2005-01-28

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

PubMed

Wong, Joyce J W; Young, Tracy A; Zhang, Jiayan; Liu, Shiheng; Leser, George P; Komives, Elizabeth A; Lamb, Robert A; Zhou, Z Hong; Salafsky, Joshua; Jardetzky, Theodore S

2017-10-03

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

PubMed

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

PubMed Central

Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

2016-01-01

Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions

DOE PAGES

Xu, Ling; Wang, Lijun; Peng, Junhui; ...

2017-12-05

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. For this study, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first timemore » that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex.« less

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ling; Wang, Lijun; Peng, Junhui

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. For this study, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first timemore » that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex.« less

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

PubMed

Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

2017-12-05

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface

PubMed Central

Bonaldo, Myrna C.; Garratt, Richard C.; Marchevsky, Renato S.; Coutinho, Evandro S. F.; Jabor, Alfredo V.; Almeida, Luís F. C.; Yamamura, Anna M. Y.; Duarte, Adriana S.; Oliveira, Prisciliana J.; Lizeu, Jackeline O. P.; Camacho, Luiz A. B.; Freire, Marcos S.; Galler, Ricardo

2005-01-01

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines. PMID:15956601

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain.

PubMed

Patel, Disha; Antwi, Janet; Koneru, Pratibha C; Serrao, Erik; Forli, Stefano; Kessl, Jacques J; Feng, Lei; Deng, Nanjie; Levy, Ronald M; Fuchs, James R; Olson, Arthur J; Engelman, Alan N; Bauman, Joseph D; Kvaratskhelia, Mamuka; Arnold, Eddy

2016-11-04

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC 50 of 72 μm and impaired HIV-1 infection of T cells at an EC 50 of 36 μm The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

PubMed

Johnson, Britney; Li, Jing; Adhikari, Jagat; Edwards, Megan R; Zhang, Hao; Schwarz, Toni; Leung, Daisy W; Basler, Christopher F; Gross, Michael L; Amarasinghe, Gaya K

2016-08-28

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Britney; Li, Jing; Adhikari, Jagat

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulatemore » nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.« less

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

PubMed

Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

2016-04-01

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Fano-like resonance in symmetry-broken gold nanotube dimer.

PubMed

Wu, DaJian; Jiang, ShuMin; Cheng, Ying; Liu, XiaoJun

2012-11-19

The influences of the symmetry-breaking on the plasmon resonance couplings in the isolated gold nanotube and the gold nanotube dimer have been investigated by means of the finite element method. It is found that the core offset of gold nanotubes leads to the red-shifts of the low energy modes and the enhanced near-field on the thin shell side of the symmetry-broken gold nanotube (SBGNT). In the weak coupling model of the SBGNT dimer, the interference of the bonding octupole mode of the dimer with the dipole modes causes a strong Fano-like resonance in scattering spectrum. The Fano dip shows a red-shift and becomes deep with the increase of the offset-value. In the strong coupling model of the SBGNT dimer, the coupling between two SBGNTs induces giant electric field enhancement at the gap of the dimer, which is much larger than that in the symmetry gold nanotube dimer. The SBGNT with larger offset-value exhibits stronger near-field at the "hot spot".

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

PubMed Central

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

1972-01-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax.

PubMed

Baranger, A M; Palmer, C R; Hamm, M K; Giebler, H A; Brauweiler, A; Nyborg, J K; Schepartz, A

1995-08-17

Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.

Structural insights into the stabilization of the human immunodeficiency virus type 1 capsid protein by the cyclophilin-binding domain and implications on the virus cycle.

PubMed

Cortines, Juliana R; Lima, Luís Mauricio T R; Mohana-Borges, Ronaldo; Millen, Thiago de A; Gaspar, Luciane Pinto; Lanman, Jason K; Prevelige, Peter E; Silva, Jerson L

2015-05-01

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385. Copyright © 2014. Published by Elsevier B.V.

Interaction of structural core protein of Classical Swine Fever Virus with endoplasmic reticulum-associated degradation pathway protein OS9

USDA-ARS?s Scientific Manuscript database

Classical Swine Fever Virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, t...

Factors Associated with D-Dimer Levels in HIV-Infected Individuals

PubMed Central

Borges, Álvaro H.; O’Connor, Jemma L.; Phillips, Andrew N.; Baker, Jason V.; Vjecha, Michael J.; Losso, Marcelo H.; Klinker, Hartwig; Lopardo, Gustavo; Williams, Ian; Lundgren, Jens D.

2014-01-01

Background Higher plasma D-dimer levels are strong predictors of mortality in HIV+ individuals. The factors associated with D-dimer levels during HIV infection, however, remain poorly understood. Methods In this cross-sectional study, participants in three randomized controlled trials with measured D-dimer levels were included (N = 9,848). Factors associated with D-dimer were identified by linear regression. Covariates investigated were: age, gender, race, body mass index, nadir and baseline CD4+ count, plasma HIV RNA levels, markers of inflammation (C-reactive protein [CRP], interleukin-6 [IL-6]), antiretroviral therapy (ART) use, ART regimens, co-morbidities (hepatitis B/C, diabetes mellitus, prior cardiovascular disease), smoking, renal function (estimated glomerular filtration rate [eGFR] and cystatin C) and cholesterol. Results Women from all age groups had higher D-dimer levels than men, though a steeper increase of D-dimer with age occurred in men. Hepatitis B/C co-infection was the only co-morbidity associated with higher D-dimer levels. In this subgroup, the degree of hepatic fibrosis, as demonstrated by higher hyaluronic acid levels, but not viral load of hepatitis viruses, was positively correlated with D-dimer. Other factors independently associated with higher D-dimer levels were black race, higher plasma HIV RNA levels, being off ART at baseline, and increased levels of CRP, IL-6 and cystatin C. In contrast, higher baseline CD4+ counts and higher high-density lipoprotein cholesterol were negatively correlated with D-dimer levels. Conclusions D-dimer levels increase with age in HIV+ men, but are already elevated in women at an early age due to reasons other than a higher burden of concomitant diseases. In hepatitis B/C co-infected individuals, hepatic fibrosis, but not hepatitis viral load, was associated with higher D-dimer levels. PMID:24626096

Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

PubMed

López-Lastra, M; Gabus, C; Darlix, J L

1997-11-01

The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

PubMed Central

Lartigue, Audrey; Burlat, Bénédicte; Coutard, Bruno; Chaspoul, Florence; Claverie, Jean-Michel

2014-01-01

ABSTRACT Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. IMPORTANCE Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs. PMID:25355875

Study of plasmonics in hybrids made from a quantum emitter and double metallic nanoshell dimer

NASA Astrophysics Data System (ADS)

Guo, Jiaohan; Black, Kevin; Hu, Jiawen; Singh, Mahi

2018-05-01

We developed a theory for the fluorescence (FL) for quantum emitter and double metallic nanoshell dimer hybrids using the density matrix method. The dimer is made from two identical double metallic nanoshells, which are made of a dielectric core, a gold metallic shell and a dielectric spacer layer. The quantum emitters are deposited on the surface of the spacer layers of the dimers due to the electrostatic absorptions. We consider that dimer hybrids are surrounded by biological cells. This can be achieved by injecting them into human or animal cells. The surface plasmon polaritons (SPP) are calculated for the dimer using Maxwell’s equations in the static wave approximation. The calculated SPP energy agrees with experimental data from Zhai et al (2017 Plasmonics 12 263) for the dimer made from a silica core, a gold metallic nanoshell and a silica spacer layer. We have also obtained an analytical expression of the FL using the density matrix method. We compare our theory with FL experimental data from Zhai et al (2017 Plasmonics 12 263) where the FL spectrum was measured by varying the thickness of the spacer layer from 9 nm to 40 nm. A good agreement between theory and experiment is found. We have shown that the enhancement of the FL increases as the thickness of the spacer layer decreases. We have also found that the enhancement of the FL increases as the distance between the double metallic nanoshells in the dimer decreases. These are interesting findings which are consistent with the experiments of Zhai et al (2017 Plasmonics 12 263) and can be used to control the FL enhancement in the FL-based biomedical imaging and cancer treatment. These interesting findings may also be useful in the fabrication of nanosensors and nanoswitches for applications in medicine.

Protein chainmail variants in dsDNA viruses

PubMed Central

Zhou, Z. Hong; Chiou, Joshua

2017-01-01

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses. PMID:29177192

Xylopiana A, a Dimeric Guaiane with a Case-Shaped Core from Xylopia vielana: Structural Elucidation and Biomimetic Conversion.

PubMed

Zhang, Ya-Long; Zhou, Xu-Wei; Wang, Xiao-Bing; Wu, Lin; Yang, Ming-Hua; Luo, Jun; Yin, Yong; Luo, Jian-Guang; Kong, Ling-Yi

2017-06-02

Xylopiana A (1), a dimeric guaiane with an unprecedented pentacyclo[5.2.1.0 1,2 .0 4,5' .0 5,4' ]decane-3,2'-dione core, and three biosynthetically related intermediates, compounds 2-4, were isolated from the leaves of Xylopia vielana. Their structures and absolute configurations were determined by a combination of spectroscopic data, X-ray crystallography, electronic circular dichroism calculations, and chemical conversion. The structure of known vielanin A was revised to be compound 3. Compound 4 exerted a 3.7-fold potentiation effect on doxorubicin susceptibility at the tested concentration of 10 μM.

Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain.

PubMed

Lu, Zhongyan; Yokoyama, Masaru; Chen, Ning; Oka, Tomoichiro; Jung, Kwonil; Chang, Kyeong-Ok; Annamalai, Thavamathi; Wang, Qiuhong; Saif, Linda J

2016-02-01

The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses. The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

PubMed Central

Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong

2014-01-01

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

X-ray induced dimerization of cinnamic acid: Time-resolved inelastic X-ray scattering study

NASA Astrophysics Data System (ADS)

Inkinen, Juho; Niskanen, Johannes; Talka, Tuomas; Sahle, Christoph J.; Müller, Harald; Khriachtchev, Leonid; Hashemi, Javad; Akbari, Ali; Hakala, Mikko; Huotari, Simo

2015-11-01

A classic example of solid-state topochemical reactions is the ultraviolet-light induced photodimerization of α-trans-cinnamic acid (CA). Here, we report the first observation of an X-ray-induced dimerization of CA and monitor it in situ using nonresonant inelastic X-ray scattering spectroscopy (NRIXS). The time-evolution of the carbon core-electron excitation spectra shows the effects of two X-ray induced reactions: dimerization on a short time-scale and disintegration on a long time-scale. We used spectrum simulations of CA and its dimerization product, α-truxillic acid (TA), to gain insight into the dimerization effects. From the time-resolved spectra, we extracted component spectra and time-dependent weights corresponding to CA and TA. The results suggest that the X-ray induced dimerization proceeds homogeneously in contrast to the dimerization induced by ultraviolet light. We also utilized the ability of NRIXS for direct tomography with chemical-bond contrast to image the spatial progress of the reactions in the sample crystal. Our work paves the way for other time-resolved studies on chemical reactions using inelastic X-ray scattering.

X-ray induced dimerization of cinnamic acid: Time-resolved inelastic X-ray scattering study

PubMed Central

Inkinen, Juho; Niskanen, Johannes; Talka, Tuomas; Sahle, Christoph J.; Müller, Harald; Khriachtchev, Leonid; Hashemi, Javad; Akbari, Ali; Hakala, Mikko; Huotari, Simo

2015-01-01

A classic example of solid-state topochemical reactions is the ultraviolet-light induced photodimerization of α-trans-cinnamic acid (CA). Here, we report the first observation of an X-ray-induced dimerization of CA and monitor it in situ using nonresonant inelastic X-ray scattering spectroscopy (NRIXS). The time-evolution of the carbon core-electron excitation spectra shows the effects of two X-ray induced reactions: dimerization on a short time-scale and disintegration on a long time-scale. We used spectrum simulations of CA and its dimerization product, α-truxillic acid (TA), to gain insight into the dimerization effects. From the time-resolved spectra, we extracted component spectra and time-dependent weights corresponding to CA and TA. The results suggest that the X-ray induced dimerization proceeds homogeneously in contrast to the dimerization induced by ultraviolet light. We also utilized the ability of NRIXS for direct tomography with chemical-bond contrast to image the spatial progress of the reactions in the sample crystal. Our work paves the way for other time-resolved studies on chemical reactions using inelastic X-ray scattering. PMID:26568420

Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope.

PubMed

Metz, Stefan W; Thomas, Ashlie; White, Laura; Stoops, Mark; Corten, Markus; Hannemann, Holger; de Silva, Aravinda M

2018-04-02

The 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form "rafts" that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion. A large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics. VLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1-4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations. This study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization.

PubMed

Donderis, Jorge; Bowring, Janine; Maiques, Elisa; Ciges-Tomas, J Rafael; Alite, Christian; Mehmedov, Iltyar; Tormo-Mas, María Angeles; Penadés, José R; Marina, Alberto

2017-09-01

The dUTPase (Dut) enzymes, encoded by almost all free-living organisms and some viruses, prevent the misincorporation of uracil into DNA. We previously proposed that trimeric Duts are regulatory proteins involved in different cellular processes; including the phage-mediated transfer of the Staphylococcus aureus pathogenicity island SaPIbov1. Recently, it has been shown that the structurally unrelated dimeric Dut encoded by phage ϕNM1 is similarly able to mobilize SaPIbov1, suggesting dimeric Duts could also be regulatory proteins. How this is accomplished remains unsolved. Here, using in vivo, biochemical and structural approaches, we provide insights into the signaling mechanism used by the dimeric Duts to induce the SaPIbov1 cycle. As reported for the trimeric Duts, dimeric Duts contain an extremely variable region, here named domain VI, which is involved in the regulatory capacity of these enzymes. Remarkably, our results also show that the dimeric Dut signaling mechanism is modulated by dUTP, as with the trimeric Duts. Overall, our results demonstrate that although unrelated both in sequence and structure, dimeric and trimeric Duts control SaPI transfer by analogous mechanisms, representing a fascinating example of convergent evolution. This conserved mode of action highlights the biological significance of Duts as regulatory molecules.

Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

PubMed Central

Ciges-Tomas, J. Rafael; Mehmedov, Iltyar; Tormo-Mas, María Angeles; Penadés, José R.

2017-01-01

The dUTPase (Dut) enzymes, encoded by almost all free-living organisms and some viruses, prevent the misincorporation of uracil into DNA. We previously proposed that trimeric Duts are regulatory proteins involved in different cellular processes; including the phage-mediated transfer of the Staphylococcus aureus pathogenicity island SaPIbov1. Recently, it has been shown that the structurally unrelated dimeric Dut encoded by phage ϕNM1 is similarly able to mobilize SaPIbov1, suggesting dimeric Duts could also be regulatory proteins. How this is accomplished remains unsolved. Here, using in vivo, biochemical and structural approaches, we provide insights into the signaling mechanism used by the dimeric Duts to induce the SaPIbov1 cycle. As reported for the trimeric Duts, dimeric Duts contain an extremely variable region, here named domain VI, which is involved in the regulatory capacity of these enzymes. Remarkably, our results also show that the dimeric Dut signaling mechanism is modulated by dUTP, as with the trimeric Duts. Overall, our results demonstrate that although unrelated both in sequence and structure, dimeric and trimeric Duts control SaPI transfer by analogous mechanisms, representing a fascinating example of convergent evolution. This conserved mode of action highlights the biological significance of Duts as regulatory molecules. PMID:28892519

Simplified methods for the construction of RNA and DNA virus infectious clones.

PubMed

Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

2015-01-01

Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Ping; Paterson, Reay G.; Leser, George P.

2012-09-06

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Heremore » we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase {beta}-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN{sub 0} and associated reduced virulence.« less

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

Crystal Structure of the Japanese Encephalitis Virus Envelope Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luca, Vincent C.; AbiMansour, Jad; Nelson, Christopher A.

2012-03-13

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-{angstrom} resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimermore » in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.« less

MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease.

PubMed

Baril, Martin; Racine, Marie-Eve; Penin, François; Lamarre, Daniel

2009-02-01

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-kappaB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-kappaB in antiviral innate immunity.

Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun Young; Song, Kyung-A; Samsung Biomedical Research Institute

Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation.more » In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.« less

Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

PubMed

Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

2018-05-01

Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

PubMed

Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

2016-10-18

The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

NSs protein of severe fever with thrombocytopenia syndrome virus suppresses interferon production through different mechanism than Rift Valley fever virus.

PubMed

Zhang, S; Zheng, B; Wang, T; Li, A; Wan, J; Qu, J; Li, C H; Li, D; Liang, M

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified Phlebovirus that causes severe fever with thrombocytopenia syndrome. Our study demonstrated that SFTSV NSs functioned as IFN antagonist mainly by suppressing TBK1/IKKε-IRF3 signaling pathway. NSs interacted with and relocalized TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures and this interaction could effectively inhibit downstream phosphorylation and dimerization of interferon regulatory factor 3 (IRF3), resulting in the suppression of antiviral signaling and IFN induction. Functional sites of SFTSV NSs binding with TBK1 were then studied and results showed that NSs had lost their IFN-inhibiting activity after deleting the 25 amino acids in N-terminal. Furthermore, the mechanism of Rift Valley fever virus (RVFV) NSs blocking IFN-β response were also investigated. Preliminary results showed that RVFV NSs proteins could neither interact nor co-localize with TBK1 in cytoplasm, but suppressed its expression levels, phosphorylation and dimerization of IRF3 in the subsequent steps, resulting in inhibition of the IFN-β production. Altogether, our data demonstrated the probable mechanism used by SFTSV to inhibit IFN responses which was different from RVFV and pointed toward a novel mechanism for RVFV suppressing IFN responses.

Accurate potential energy, dipole moment curves, and lifetimes of vibrational states of heteronuclear alkali dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedorov, Dmitry A.; Varganov, Sergey A., E-mail: svarganov@unr.edu; Derevianko, Andrei

2014-05-14

We calculate the potential energy curves, the permanent dipole moment curves, and the lifetimes of the ground and excited vibrational states of the heteronuclear alkali dimers XY (X, Y = Li, Na, K, Rb, Cs) in the X{sup 1}Σ{sup +} electronic state using the coupled cluster with singles doubles and triples method. All-electron quadruple-ζ basis sets with additional core functions are used for Li and Na, and small-core relativistic effective core potentials with quadruple-ζ quality basis sets are used for K, Rb, and Cs. The inclusion of the coupled cluster non-perturbative triple excitations is shown to be crucial for obtainingmore » the accurate potential energy curves. A large one-electron basis set with additional core functions is needed for the accurate prediction of permanent dipole moments. The dissociation energies are overestimated by only 14 cm{sup −1} for LiNa and by no more than 114 cm{sup −1} for the other molecules. The discrepancies between the experimental and calculated harmonic vibrational frequencies are less than 1.7 cm{sup −1}, and the discrepancies for the anharmonic correction are less than 0.1 cm{sup −1}. We show that correlation between atomic electronegativity differences and permanent dipole moment of heteronuclear alkali dimers is not perfect. To obtain the vibrational energies and wave functions the vibrational Schrödinger equation is solved with the B-spline basis set method. The transition dipole moments between all vibrational states, the Einstein coefficients, and the lifetimes of the vibrational states are calculated. We analyze the decay rates of the vibrational states in terms of spontaneous emission, and stimulated emission and absorption induced by black body radiation. In all studied heteronuclear alkali dimers the ground vibrational states have much longer lifetimes than any excited states.« less

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

A human antibody against Zika virus crosslinks the E protein to prevent infection

PubMed Central

Hasan, S. Saif; Miller, Andrew; Sapparapu, Gopal; Fernandez, Estefania; Klose, Thomas; Long, Feng; Fokine, Andrei; Porta, Jason C.; Jiang, Wen; Diamond, Michael S.; Crowe Jr., James E.; Kuhn, Richard J.; Rossmann, Michael G.

2017-01-01

The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes. PMID:28300075

A human antibody against Zika virus crosslinks the E protein to prevent infection.

PubMed

Hasan, S Saif; Miller, Andrew; Sapparapu, Gopal; Fernandez, Estefania; Klose, Thomas; Long, Feng; Fokine, Andrei; Porta, Jason C; Jiang, Wen; Diamond, Michael S; Crowe, James E; Kuhn, Richard J; Rossmann, Michael G

2017-03-16

The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes.

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer.

PubMed

Sanchez, Jacint G; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M; Sundquist, Wesley I; Pornillos, Owen

2014-02-18

Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

PubMed Central

Sanchez, Jacint G.; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M.; Sundquist, Wesley I.; Pornillos, Owen

2014-01-01

Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication. PMID:24550273

DNA Origami Directed Au Nanostar Dimers for Single-Molecule Surface-Enhanced Raman Scattering.

PubMed

Tanwar, Swati; Haldar, Krishna Kanta; Sen, Tapasi

2017-12-06

We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 10 10 and 8 × 10 9 , respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNA-directed assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing.

Protein-Protein Förster Resonance Energy Transfer Analysis of Nucleosome Core Particles Containing H2A and H2A.Z

PubMed Central

Hoch, Duane A.; Stratton, Jessica J.; Gloss, Lisa M.

2007-01-01

A protein-protein Förster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, in comparison to fluorophores used in previous NCP FRET studies, they: 1) are smaller and less hydrophobic which should minimize perturbations of histone and NCP structure; and 2) have an R0 of 20 Å, which is much less than the dimensions of the NCP (~50 Å width and ~100 Å diameter). CD and FL equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)2 tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 artificial positioning DNA sequence. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation as compared to previous studies using weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of each H2A-H2B dimer, confirming cooperativity in dimer dissociation. While cooperativity in the association/dissociation of the H2A-H2B dimers has been suggested previously, these data allow its quantitative description. The protein-protein FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. Comparison of the H2A and H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity for dimer dissociation from H2A.Z NCPs. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer association and dissociation from the NCP. PMID:17597150

Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus

PubMed Central

Mor, Sunil K.; Higgins, LeeAnn; Armien, Anibal; Youssef, Mohammed M.; Bruggeman, Peter J.; Goyal, Sagar M.

2018-01-01

Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O2 admixture were studied. We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid. PMID:29566061

Detection of Intermediates And Kinetic Control During Assembly of Bacteriophage P22 Procapsid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tuma, R.; Tsuruta, H.; French, K.H.

2009-05-26

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter {approx} 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibriummore » (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.« less

Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

PubMed Central

Holthauzen, Luis Marcelo F.; Ruggli, Nicolas

2016-01-01

ABSTRACT Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site. IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the Npro interaction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional cofactor, the CREB-binding protein. This is the first study to demonstrate that Npro is able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation. PMID:27334592

Dynamic and magneto-optic properties of bent-core liquid crystals

NASA Astrophysics Data System (ADS)

Salili, Seyyed Muhammad

In this work, we describe dynamic behavior of free-standing bent-core liquid crystal filaments under dilative and axial compressive stresses in the B7 phase. We found that such filaments demonstrate very complex structures depending on the filament's temperature relative to the isotropic phase, initial filament thickness, and velocity at which the filament is pulled or compressed. We also present our experimental methods, results and analysis of the rupture and recoil properties of several bent-core liquid crystal filaments, anticipating that they may serve as a model system for complex biological fibers. After that, we systematically describe rheological measurements for dimeric liquid crystal compounds. We studied the shear-induced alignment properties, measured the viscoelastic properties as a function of temperature, shear rate, stress and frequency, and compared the results with the rheological properties of conventional chiral nematic and smectic phases. Then we present results of chiral nematic liquid crystals composed of flexible dimer molecules subject to large DC magnetic fields between 0 and 31T. We observe that these fields lead to selective reflection of light depending on temperature and magnetic field. The band of reflected wavelengths can be tuned from ultraviolet to beyond the IR-C band. A similar effect induced by electric fields has been presented previously, and was explained by a field-induced oblique-heliconical director deformation in accordance with early theoretical predictions. Finally, we report an unprecedented magnetic field-induced shifts of the isotropic-nematic phase transition temperature observed in liquid crystal dimers where two rigid linear mesogens are linked by flexible chains of either even- or odd-numbered hydrocarbon groups. This effect is explained in terms of quenching of the thermal fluctuations and decrease of the average bend angle of molecules in the odd-numbered dimers.

Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity.

PubMed

Jamin, Augusta; Wicklund, April; Wiebe, Matthew S

2014-05-01

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

PubMed

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Bipolarons in one-dimensional extended Peierls-Hubbard models

NASA Astrophysics Data System (ADS)

Sous, John; Chakraborty, Monodeep; Krems, Roman; Berciu, Mona

2017-04-01

We study two particles in an infinite chain and coupled to phonons by interactions that modulate their hopping as described by the Peierls/Su-Schrieffer-Heeger (SSH) model. In the case of hard-core bare particles, we show that exchange of phonons generates effective nearest-neighbor repulsion between particles and also gives rise to interactions that move the pair as a whole. The two-polaron phase diagram exhibits two sharp transitions, leading to light dimers at strong coupling and the flattening of the dimer dispersion at some critical values of the parameters. This dimer (quasi)self-trapping occurs at coupling strengths where single polarons are mobile. On the other hand, in the case of soft-core particles/ spinfull fermions, we show that phonon-mediated interactions are attractive and result in strongly bound and mobile bipolarons in a wide region of parameter space. This illustrates that, depending on the strength of the phonon-mediated interactions and statistics of bare particles, the coupling to phonons may completely suppress or strongly enhance quantum transport of correlated particles. This work was supported by NSERC of Canada and the Stewart Blusson Quantum Matter Institute.

Dynamics and asymmetry in the dimer of the norovirus major capsid protein.

PubMed

Tubiana, Thibault; Boulard, Yves; Bressanelli, Stéphane

2017-01-01

Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

PubMed Central

Ramírez-Sarmiento, César A.; Baez, Mauricio; Wilson, Christian A.M.; Babul, Jorge; Komives, Elizabeth A.; Guixé, Victoria

2013-01-01

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. PMID:23708365

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

PubMed Central

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.

PubMed

Ramírez-Sarmiento, César A; Baez, Mauricio; Wilson, Christian A M; Babul, Jorge; Komives, Elizabeth A; Guixé, Victoria

2013-05-21

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Reactions of Laser-Ablated Aluminum Atoms with Cyanogen: Matrix Infrared Spectra and Electronic Structure Calculations for Aluminum Isocyanides Al(NC)1,2,3 and Their Novel Dimers.

PubMed

Andrews, Lester; Cho, Han-Gook; Gong, Yu

2018-05-15

Laser-ablated Al atoms react with (CN)2 in excess argon during condensation at 4 K to produce AlNC, Al(NC)2 and Al(NC)3,which were computed (B3LYP) to be 27, 16 and 28 kJ/mol lower in energy, respectively, than their cyanide counterparts. Irradiation at 220-580 nm increased absorptions for the above molecules and the very stable Al(NC)4- anion. Annealing to 30, 35, and 40 K allowed for diffusion and reaction of trapped species and produced new bands for the Al(NC)1,2,3 dimers including a rhombic ring core (C)(AlN)2(C) with C's attached to the N's, a (NC)2Al(II)-Al(II)(NC)2 dimer with computed Al-Al length 2.557 Å, and the dibridged Al2(NC)6 molecule with a calculated D2h structure and rhombic ring core like Al2H6. In contrast the Al(NC)4- anion was destroyed on annealing presumably due to neutralization by Al+. B3LYP calculations also show that aluminum chlorides form the analogous molecules and dimers. In our search for possible new products, we calculated Al(NC)4 and found it to be a stable molecule, but it was not detected here.

Mechanism of DNA binding enhancement by hepatitis B virus protein pX.

PubMed

Palmer, C R; Gegnas, L D; Schepartz, A

1997-12-09

At least three hundred million people worldwide are infected with the hepatitis B virus (HBV), and epidemiological studies show a clear correlation between chronic HBV infection and the development of hepatocellular carcinoma. HBV encodes a protein, pX, which abducts the cellular transcriptional machinery in several ways including direct interactions with bZIP transcription factors. These interactions increase the DNA affinities of target bZIP proteins in a DNA sequence-dependent manner. Here we use a series of bZIP peptide models to explore the mechanism by which pX interacts with bZIP proteins. Our results suggest that pX increases bZIP.DNA stability by increasing the stability of the bZIP dimer as well as the affinity of the dimer for DNA. Additional experiments provide evidence for a mechanism in which pX recognizes the composite structure of the peptide.DNA complex, not simply the primary peptide sequence. These experiments provide a framework for understanding how pX alters the patterns of transcription within the nucleus. The similarities between the mechanism proposed for pX and the mechanism previously proposed for the human T-cell leukemia virus protein Tax are discussed.

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels.

PubMed

Gloss, L M; Simler, B R; Matthews, C R

2001-10-05

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts. Copyright 2001 Academic Press.

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

PubMed Central

Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

2011-01-01

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly. PMID:21814513

Phase transitions in nanocomposites of hydrogen-bonded dimeric liquid crystals with mesogenic and non-mesogenic components

NASA Astrophysics Data System (ADS)

Katranchev, Boyko; Petrov, Minko

2016-02-01

Microtextural polarization, phase transitions, and electro-optical effects are studied in a series of nanocomposites, grown by mixing alkyloxybenzoic acids (nOBAs), displaying hydrogen-bonded dimeric liquid crystal (LC) state, with non-mesogens (single-walled carbon nanotubes (SWCNTs), perfluorooctanoic acid) or mesogens (bent-core LC compound D14F3). Each of the studied nanocomposites, in which the nOBA serves as a matrix, forms complexes with bent-shaped dimeric, caused by the interaction between the dopant structural units and the dimer rings. This feature, coordinated with the surface anchoring, bulk and electrical effects, leads to drastic reduction of the LC system symmetry. As a result, transitions from achiral (characteristic for the pristine nOBA) to chiral states (including ferroelectric smectic C with C2 symmetry and ferroelectric smectic CG with the lowest C1 triclinic one) take place. The functionalization of the SWCNTs causes drastic increase of the ferroelectricity.

Protein-protein Förster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z.

PubMed

Hoch, Duane A; Stratton, Jessica J; Gloss, Lisa M

2007-08-24

A protein-protein Förster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, compared to previous NCP FRET fluorophores, they: (1) are smaller and less hydrophobic, which should minimize perturbations of histone and NCP structure; and (2) have an R0 of 20 A, which is much less than the dimensions of the NCP (approximately 50 A width and approximately 100 A diameter). Equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)2 tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 DNA positioning element. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation relative to weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of individual H2A-H2B dimers, confirming cooperativity as suggested previously; these data allow quantitative description of the cooperativity. The FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity of the dimer dissociations. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer association and dissociation from the NCP.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.

PubMed

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-02-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

PubMed Central

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-01-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. PMID:18033802

Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

PubMed Central

Huang, Yao-Wei; Patterson, Abby R.; Opriessnig, Tanja; Dryman, Barbara A.; Gallei, Andreas; Harrall, Kylie K.; Vaughn, Eric M.; Roof, Michael B.

2012-01-01

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology. PMID:22491450

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

PubMed

Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

Design of Broad-Spectrum Inhibitors of Influenza A Virus M2 Proton Channels: A Molecular Modeling Approach.

PubMed

Klimochkin, Yuri N; Shiryaev, Vadim A; Petrov, Pavel V; Radchenko, Eugene V; Palyulin, Vladimir A; Zefirov, Nikolay S

2016-01-01

The influenza A virus M2 proton channel plays a critical role in its life cycle. However, known M2 inhibitors have lost their clinical efficacy due to the spread of resistant mutant channels. Thus, the search for broad-spectrum M2 channel inhibitors is of great importance. The goal of the present work was to develop a general approach supporting the design of ligands interacting with multiple labile targets and to propose on its basis the potential broad-spectrum inhibitors of the M2 proton channel. The dynamic dimer-of-dimers structures of the three primary M2 target variants, wild-type, S31N and V27A, were modeled by molecular dynamics and thoroughly analyzed in order to define the inhibitor binding sites. The potential inhibitor structures were identified by molecular docking and their binding was verified by molecular dynamics simulation. The binding sites of the M2 proton channel inhibitors were analyzed, a number of potential broad-spectrum inhibitors were identified and the binding modes and probable mechanisms of action of one promising compound were clarified. Using the molecular dynamics and molecular docking techniques, we have refined the dynamic dimer-ofdimers structures of the WT, S31N and V27A variants of the M2 proton channel of the influenza A virus, analyzed the inhibitor binding sites, identified a number of potential broad-spectrum inhibitor structures targeting them, and clarified the binding modes and probable mechanisms of action of one promising compound. The proposed approach is also suitable for the design of ligands interacting with other multiple labile targets.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Metal-organic chemical vapour deposition of polycrystalline tetragonal indium sulphide (InS) thin films

NASA Technical Reports Server (NTRS)

Macinnes, Andrew N.; Cleaver, William M.; Barron, Andrew R.; Power, Michael B.; Hepp, Aloysius F.

1992-01-01

The dimeric indium thiolate /(t Bu)2In(mu-S sup t Bu)/2 has been used as a single-source precursor for the MOCVD of InS thin films. The dimeric In2S2 core is proposed to account for the formation of the nonequilibrium high-pressure tetragonal phase in the deposited films. Analysis of the deposited films has been obtained by TEM, with associated energy-dispersive X-ray analysis and X-ray photoelectron spectroscopy.

Biomarkers and electrocardiographic evidence of myocardial ischemia in patients with human immunodeficiency virus infection.

PubMed

Gupta, Mihir; Miller, Christopher J; Baker, Jason V; Lazar, Jason; Bogner, Johannes R; Calmy, Alexandra; Soliman, Elsayed Z; Neaton, James D

2013-03-01

We assessed the relation of inflammatory and coagulation biomarkers with electrocardiographic (ECG) evidence of myocardial ischemia. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer levels were measured at study entry for 3,085 human immunodeficiency virus-infected participants (mean age 44 years; 26.4% women; 24.6% black) in the Strategies for Management of Antiretroviral Therapy trial. Logistic regression models were used to examine the associations of these biomarkers with prevalent and incident myocardial ischemia. The latter analyses were performed for 1,411 participants who were randomly assigned to receive continuous antiretroviral therapy during follow-up to suppress the human immunodeficiency virus viral load and had ≥1 ECG reading during the follow-up period. The median hsCRP, IL-6, and D-dimer level was 1.65 μg/ml (interquartile range 0.69 to 4.11), 1.60 pg/ml (interquartile range 1.00 to 2.75), and 0.18 μg/ml (interquartile range 0.11 to 0.32), respectively. At baseline, the prevalence of major or minor Q-QS or ST-T ECG abnormalities was 18.6%. The biomarker levels were associated with prevalent major or minor ischemic abnormalities on the univariate analyses; however, adjustment for traditional risk factors attenuated these associations. The adjusted odds ratio for major or minor ischemic abnormalities and 95% confidence intervals for the greatest versus lowest quartiles was 1.3 (95% confidence interval 0.9 to 1.7) for hsCRP, 1.0 (95% confidence interval 0.7 to 1.3) for IL-6, and 1.1 (95% confidence interval 0.9 to 1.5) for D-dimer. During a median follow-up of 2.3 years, new definite or probable ischemic ECG abnormalities developed in 11.7% of participants receiving continuous antiretroviral therapy. Biomarker levels were not associated with incident abnormalities on unadjusted or adjusted analyses. In conclusion, higher levels of hsCRP, IL-6, and D-dimer were not associated with ischemic ECG abnormalities. Elevated biomarker levels and ECG abnormalities indicating myocardial ischemia might reflect different risk pathways for cardiovascular disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

Adsorption of ethylene on Sn and In terminated Si(001) surface studied by photoelectron spectroscopy and scanning tunneling microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zimmermann, Petr, E-mail: petr.zimmermann@mff.cuni.cz; Sobotík, Pavel; Kocán, Pavel

2016-09-07

Interaction of ethylene (C{sub 2}H{sub 4}) with Si(001)-Sn-2 × 2 and Si(001)-In-2 × 2 at room temperature has been studied using core level (C 1s) X-ray photoelectron spectroscopy with synchrotron radiation and scanning tunneling microscopy. Sn and In form similar dimer chains on Si(001)2 × 1, but exhibit different interaction with ethylene. While ethylene adsorbs on top of Sn dimers of the Si(001)-Sn-2 × 2 surface, the Si(001)-In-2 × 2 surface turned out to be inert. Furthermore, the reactivity of the Sn terminated surface is found to be considerably decreased in comparison with Si(001)2 × 1. According to the proposedmore » adsorption model ethylene bonds to Sn dimers via [2 + 2] cycloaddition by interacting with their π dimer bonds. In contrast, indium dimers do not contain π bonds, which renders the In terminated Si(001) surface inert for ethylene adsorption.« less

Therapeutic and protective efficacy of a dengue antibody against Zika infection in rhesus monkeys.

PubMed

Abbink, Peter; Larocca, Rafael A; Dejnirattisai, Wanwisa; Peterson, Rebecca; Nkolola, Joseph P; Borducchi, Erica N; Supasa, Piyada; Mongkolsapaya, Juthathip; Screaton, Gavin R; Barouch, Dan H

2018-06-01

Strategies to treat Zika virus (ZIKV) infection in dengue virus (DENV)-endemic areas are urgently needed. Here we show that a DENV-specific antibody against the E-dimer epitope (EDE) potently cross-neutralizes ZIKV and provides robust therapeutic efficacy as well as prophylactic efficacy against ZIKV in rhesus monkeys. Viral escape was not detected, suggesting a relatively high bar to escape. These data demonstrate the potential for antibody-based therapy and prevention of ZIKV.

Monomeric and dimeric hydrolysable tannins of Tamarix nilotica.

PubMed

Orabi, Mohamed A A; Taniguchi, Shoko; Hatano, Tsutomu

2009-07-01

An ellagitannin monomer, nilotinin M1 (1), and three dimers, nilotinins D1 (2), D2 (3), and D3 (4), were isolated from leaves of Tamarix nilotica (Ehrenb.) Bunge. Structures were elucidated based on analysis of spectroscopic data and chemical correlations with known compounds. In addition, six known tannins, hirtellin A (5) (dimer), remurin A (6), remurin B (7), 1,3-di-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-beta-D-glucose (8), gemin D (9), and hippomanin A (10) (monomers), were isolated for the first time from this plant species. The reported (13)C NMR assignments of the dehydrodigalloyl moiety and glucose cores of 5 are revised, and the (13)C NMR spectroscopic data for 6 and 7 are also reported for the first time.

Template Dimerization Promotes an Acceptor Invasion-Induced Transfer Mechanism during Human Immunodeficiency Virus Type 1 Minus-Strand Synthesis

PubMed Central

Balakrishnan, Mini; Roques, Bernard P.; Fay, Philip J.; Bambara, Robert A.

2003-01-01

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism. PMID:12663778

Effect of core twisting on self-assembly and optical properties of perylene bisimide dyes in solution and columnar liquid crystalline phases.

PubMed

Chen, Zhijian; Baumeister, Ute; Tschierske, Carsten; Würthner, Frank

2007-01-01

A series of highly soluble and fluorescent core-twisted perylene bisimide dyes (PBIs) 3 a-f with different substituents at the bay area (1,6,7,12 positions of the perylene core) were synthesized and fully characterized by (1)H NMR, UV/Vis spectroscopy, MS spectrometry, and elemental analysis. The pi-pi aggregation properties of these new functional dyes were investigated in detail both in solution and in condensed phase by UV/Vis and fluorescence spectroscopy, vapor pressure osmometry (VPO), differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffraction. Concentration-dependent UV/Vis measurements and VPO analysis revealed that these core-twisted pi-conjugated systems show distinct self-dimerization equilibria in apolar solvent methylcyclohexane (MCH) with dimerization constants between 1.3x10(4) and 30 M(-1). The photoluminescence spectra of the dimers of PBIs 3 a-f exhibit bathochromic shifts of quite different magnitude which could be attributed to different longitudinal or rotational offsets between the dyes as well as differences in the respective pi-pi stacking distance. In condensed state, quite a few of these PBIs form luminescent rectangular or hexagonal columnar liquid crystalline phases with low isotropization temperatures. The effects of the distortion of the pi systems on their pi-pi stacking and the optical properties of the resultant stacks in solution and in LC phases have been explored in detail. In one case (3 a) a particularly interesting phase change from crystalline into liquid crystalline could be observed upon annealing that was accompanied by a transformation from non-fluorescent H-type into strongly fluorescent J-type packing of the dyes.

Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

PubMed Central

Lee, Choongho

2013-01-01

Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif (79PGYPWP84). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif (79AGYAWP84) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy. PMID:24009866

Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding

PubMed Central

Lutzke, Ramon A. Puras; Plasterk, Ronald H. A.

1998-01-01

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN220–270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN220–270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN220–270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN. PMID:9573250

Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome?

PubMed Central

Han, Weining; Li, Yuejin; Bagaya, Bernard S.; Tian, Meijuan; Chamanian, Mastooreh; Zhu, Chuanwu; Shen, Jie; Gao, Yong

2016-01-01

Although the process of reverse transcription is well elucidated, it remains unclear if viral core disruption provides a more cellular or viral milieu for HIV-1 reverse transcription. We have devised a method to require mixing of viral cores or core constituents to produce infectious progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each other and when together can complete viral reverse transcription. Only the heterodiploid virus containing both the nfl and cplt_R/U5/gag/Δpol sgRNAs can complete reverse transcription and propagate infectious virus upon de novo infection. Dual exposure of U87.CD4.CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/Δpol virus particles did not result in productive virus infection. On the other hand, in early endosomes, the HIV-1 sgRNAs released from viral cores can retain function and complete the reverse transcription and result in productive infection. These findings confirm the assumptions that, in natural infection, HIV-1 cores, and likely other retrovirus cores, remain largely intact and do not mix/fuse in the cytoplasm during the reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA (produced through transfection) could not help the complementary sgRNA in the viral core to complement the reverse transcription process. PMID:27239643

Ultraviolet irradiation of herpes simplex virus (type 1): delayed transcription and comparative sensitivites of virus functions.

PubMed

Eglin, R P; Gugerli, P; Wildy, P

1980-07-01

The delay in the replication of herpes simplex virus surviving u.v. irradiation occurs after the uncoating of virus, as judged by sensitivity to DNase. It occurs before translation, judged by the kinetics of appearance of various virus-specific proteins, and before transcription, judged by the detection of virus-specific RNA by in situ hybridization. Since the delays in both transcription and translation are reversed by photoreactivation, the simplest hypothesis is that pyrimidine dimers directly obstruct transcription;unless these are broken by photoreactivating enzymes, there will be transcriptional delay until reactivating processes have repaired the lesion. The u.v. sensitivities of the abilities to induce various enzymes (thymidine kinase, DNase and DNA polymerase) were only about four times less than that of infectivity. The The ability to induce the three enzymes was three times less sensitive than that of the structural antigen (Band II).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrila, J.; Gabelli, S; Bacha, U

Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many ofmore » the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.« less

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

PubMed Central

Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

1975-01-01

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly.

PubMed

Kulshrestha, Saurabh; Hallan, Vipin; Sharma, Anshul; Seth, Chandrika Attri; Chauhan, Anjali; Zaidi, Aijaz Asghar

2013-09-01

Coat protein (CP) and RNA3 from Prunus necrotic ringspot virus (PNRSV-rose), the most prevalent virus infecting rose in India, were characterized and regions in the coat protein important for self-interaction, during dimer formation were identified. The sequence analysis of CP and partial RNA 3 revealed that the rose isolate of PNRSV in India belongs to PV-32 group of PNRSV isolates. Apart from the already established group specific features of PV-32 group member's additional group-specific and host specific features were also identified. Presence of methionine at position 90 in the amino acid sequence alignment of PNRSV CP gene (belonging to PV-32 group) was identified as the specific conserved feature for the rose isolates of PNRSV. As protein-protein interaction plays a vital role in the infection process, an attempt was made to identify the portions of PNRSV CP responsible for self-interaction using yeast two-hybrid system. It was found (after analysis of the deletion clones) that the C-terminal region of PNRSV CP (amino acids 153-226) plays a vital role in this interaction during dimer formation. N-terminal of PNRSV CP is previously known to be involved in CP-RNA interactions, but our results also suggested that N-terminal of PNRSV CP represented by amino acids 1-77 also interacts with C-terminal (amino acids 153-226) in yeast two-hybrid system, suggesting its probable involvement in the CP-CP interaction.

A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity.

PubMed

Wang, Junyong; Zeng, Yan; Xu, Shuai; Yang, Jiayun; Wang, Wanbing; Zhong, Bo; Ge, Jinying; Yin, Lei; Bu, Zhigao; Shu, Hong-Bing; Chen, Hualan; Lei, Cao-Qi; Zhu, Qiyun

2018-06-01

Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals. IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines. Copyright © 2018 American Society for Microbiology.

Viruslike Nanoparticles with Maghemite Cores Allow for Enhanced MRI Contrast Agents

DOE PAGES

Malyutin, Andrey G.; Easterday, Rosemary; Lozovyy, Yaroslav; ...

2014-12-15

Here, for the first time, we demonstrate formation of virus-like nanoparticles (VNPs) utilizing gold-coated iron oxide nanoparticles as cores and capsidprotein of brome mosaic virus (BMV) or hepatitis B virus (HBV) as shells. Further, utilizing cryo-electron microscopy and single particle methods, we are able to show that the BMV coat on VNPs assembles into a structure very close to that of a native virion. This is a consequence of an optimal iron oxide NP size (~11 nm) fitting the virus cavity and an ultrathin gold layer on the maghemite cores, which allows for utilization of SH-(CH 2) 11-(CH 2-CH 2-O)more » 4-OCH 2-COOH as capping molecules to provide sufficient stability, charge density, and small form factor. MRI studies show unique relaxivity ratios that diminish only slightly with gold coating. In conclusion, a virus protein coating of a magnetic core mimicking the wild-type virus makes these VNPs a versatile platform for biomedical applications.« less

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA.

PubMed

Sakuragi, Sayuri; Yokoyama, Masaru; Shioda, Tatsuo; Sato, Hironori; Sakuragi, Jun-Ichi

2016-11-11

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5' end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell). In this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G-C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle. We discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.

Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

PubMed

Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

2013-06-24

Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins. Crystallographic interfaces revealed several interactions that might be involved in FIV replication. Further studies are needed to better understand their biological relevance in the function of FIV Gag during viral replication.

Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles*

PubMed Central

Nagpal, Latika; Haque, Mohammad M.; Saha, Amit; Mukherjee, Nirmalya; Ghosh, Arnab; Ranu, Brindaban C.; Stuehr, Dennis J.; Panda, Koustubh

2013-01-01

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases. PMID:23696643

A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

PubMed Central

Leonard, John D.; Narlikar, Geeta J.

2015-01-01

SUMMARY The ATP-dependent chromatin assembly factor (ACF) is a dimeric motor that spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes. PMID:25684208

Virulent Poxviruses Inhibit DNA Sensing by Preventing STING Activation

PubMed Central

Georgana, Iliana; Sumner, Rebecca P.; Towers, Greg J.

2018-01-01

ABSTRACT Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence. IMPORTANCE Poxviruses are double-stranded DNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as efficient oncolytics in virotherapy. The successful therapeutic use of VACV depends on a detailed understanding of its ability to modulate host innate immune responses. DNA sensing is a critical cellular mechanism for pathogen detection and activation of innate immunity that is centrally coordinated by the endoplasmic reticulum-resident protein STING. Here, STING is shown to mediate immune activation in response to MVA, but not in response to virulent VACV strains or other virulent poxviruses, which prevent STING activation and DNA sensing during infection and after DNA transfection. These results provide new insights into poxvirus immune evasion and have implications in the rational design of VACV-based therapeutics. PMID:29491158

Functional Characterization of the Putative Hepatitis B Virus Core Protein Late Domain Using Retrovirus Chimeras

PubMed Central

Garcia, Mayra L.; Reynolds, Tracy D.; Mothes, Walther; Robek, Michael D.

2013-01-01

The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP138) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells. PMID:24009707

(±)-Uncarilins A and B, Dimeric Isoechinulin-Type Alkaloids from Uncaria rhynchophylla.

PubMed

Geng, Chang-An; Huang, Xiao-Yan; Ma, Yun-Bao; Hou, Bo; Li, Tian-Ze; Zhang, Xue-Mei; Chen, Ji-Jun

2017-04-28

(±)-Uncarilins A and B (1a/1b and 2a/2b), two pairs of unusual dimeric isoechinulin-type enantiomers with a symmetric four-membered core, were isolated from Uncaria rhynchophylla driven by LCMS-IT-TOF analyses. Their structures were elucidated by extensive 1D and 2D NMR spectra, X-ray diffraction, and ECD spectroscopic data. (-)-Uncarilin B (2a) showed activities on MT 1 and MT 2 receptors with agonistic rates of 11.26% and 52.44% at a concentration of 0.25 mM.

ent-Kaurane-Based Regio- and Stereoselective Inverse Electron Demand Hetero-Diels-Alder Reactions: Synthesis of Dihydropyran-Fused Diterpenoids†

PubMed Central

Ding, Chunyong; Wang, Lili; Chen, Haijun; Wild, Christopher; Ye, Na; Ding, Ye; Wang, Tianzhi; White, Mark A.; Shen, Qiang; Zhou, Jia

2014-01-01

A mild and concise approach for the construction of 3,4-dihydro-2H-pyran ring integrated into the A-ring of the natural product oridonin using an optimized inverse electron demand hetero-Diels-Alder (IED HDA) reaction is reported herein. A self-dimerization of the exocyclic enone installed in the A-ring through a homo-HDA reaction was identified to exclusively give a dimeric ent-kaurane diterpenoid with the spirochroman core. Moreover, the efficient cross-HDA cycloadditions of this enone with various vinyl ethers or vinyl sulfides, instead of its own homo-HDA dimerization, were achieved in regio- and stereoselective manners, thus providing the access to novel dihydropyran-fused diterpenoids as potential anticancer agents to overcome chemoresistance. PMID:25225052

Shearing of the CENP-A dimerization interface mediates plasticity in the octameric centromeric nucleosome

PubMed Central

Winogradoff, David; Zhao, Haiqing; Dalal, Yamini; Papoian, Garegin A.

2015-01-01

The centromeric nucleosome is a key epigenetic determinant of centromere identity and function. Consequently, deciphering how CENP-A containing nucleosomes contribute structurally to centromere function is a fundamental question in chromosome biology. Here, we performed microsecond timescale all-atom molecular dynamics (MD) simulations of CENP-A and H3 nucleosomes, and report that the octameric CENP-A core particles and nucleosomes display different dynamics from their canonical H3-containing counterparts. The most significant motion observed is within key interactions at the heart of the CENP-A octameric core, wherein shearing of contacts within the CENP-A:CENP-A’ dimerization interface results in a weaker four helix bundle, and an extrusion of 10–30 bp of DNA near the pseudo-dyad. Coupled to other local and global fluctuations, the CENP-A nucleosome occupies a more rugged free energy landscape than the canonical H3 nucleosome. Taken together, our data suggest that CENP-A encodes enhanced distortability to the octameric nucleosome, which may allow for enhanced flexing of the histone core in vivo. PMID:26602160

Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

Cancer.gov

During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the HIV-1 core, and NC packages the viral RNA within the core. Current models of the development of HIV-1 suggest that when CA is cleaved from Gag it dissociates from the membrane and moves into the virus interior before nucleating, in a concentration-dependent manner, into the core, which is the last step in virus maturation. The core is thought to grow from its narrow end stopping only when it reaches the opposite side of the virus membrane. Since blocking the formation of infectious viral particles is an important therapeutic strategy, it is critical to understand the detailed mechanism of core maturation.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

Molecular dynamics simulations and structure-based network analysis reveal structural and functional aspects of G-protein coupled receptor dimer interactions.

PubMed

Baltoumas, Fotis A; Theodoropoulou, Margarita C; Hamodrakas, Stavros J

2016-06-01

A significant amount of experimental evidence suggests that G-protein coupled receptors (GPCRs) do not act exclusively as monomers but also form biologically relevant dimers and oligomers. However, the structural determinants, stoichiometry and functional importance of GPCR oligomerization remain topics of intense speculation. In this study we attempted to evaluate the nature and dynamics of GPCR oligomeric interactions. A representative set of GPCR homodimers were studied through Coarse-Grained Molecular Dynamics simulations, combined with interface analysis and concepts from network theory for the construction and analysis of dynamic structural networks. Our results highlight important structural determinants that seem to govern receptor dimer interactions. A conserved dynamic behavior was observed among different GPCRs, including receptors belonging in different GPCR classes. Specific GPCR regions were highlighted as the core of the interfaces. Finally, correlations of motion were observed between parts of the dimer interface and GPCR segments participating in ligand binding and receptor activation, suggesting the existence of mechanisms through which dimer formation may affect GPCR function. The results of this study can be used to drive experiments aimed at exploring GPCR oligomerization, as well as in the study of transmembrane protein-protein interactions in general.

Molecular dynamics simulations and structure-based network analysis reveal structural and functional aspects of G-protein coupled receptor dimer interactions

NASA Astrophysics Data System (ADS)

Baltoumas, Fotis A.; Theodoropoulou, Margarita C.; Hamodrakas, Stavros J.

2016-06-01

A significant amount of experimental evidence suggests that G-protein coupled receptors (GPCRs) do not act exclusively as monomers but also form biologically relevant dimers and oligomers. However, the structural determinants, stoichiometry and functional importance of GPCR oligomerization remain topics of intense speculation. In this study we attempted to evaluate the nature and dynamics of GPCR oligomeric interactions. A representative set of GPCR homodimers were studied through Coarse-Grained Molecular Dynamics simulations, combined with interface analysis and concepts from network theory for the construction and analysis of dynamic structural networks. Our results highlight important structural determinants that seem to govern receptor dimer interactions. A conserved dynamic behavior was observed among different GPCRs, including receptors belonging in different GPCR classes. Specific GPCR regions were highlighted as the core of the interfaces. Finally, correlations of motion were observed between parts of the dimer interface and GPCR segments participating in ligand binding and receptor activation, suggesting the existence of mechanisms through which dimer formation may affect GPCR function. The results of this study can be used to drive experiments aimed at exploring GPCR oligomerization, as well as in the study of transmembrane protein-protein interactions in general.

Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5.

PubMed

Suyama, Keitaro; Taniguchi, Suguru; Tatsubo, Daiki; Maeda, Iori; Nose, Takeru

2016-04-01

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n>40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

PubMed

Pumpens, P; Grens, E

2016-01-01

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

A Recombinant HAV Expressing a Neutralization Epitope of HEV Induces Immune Response against HAV and HEV in Mice.

PubMed

Xiang, Kui; Kusov, Yuri; Ying, Guan; Yan, Wang; Shan, Yi; Jinyuan, Wu; Na, Yin; Yan, Zhou; Hongjun, Li; Maosheng, Sun

2017-09-15

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are causative agents of acute viral hepatitis transmitted via the fecal-oral route. Both viruses place a heavy burden on the public health and economy of developing countries. To test the possibility that HAV could be used as an expression vector for the development of a combination vaccine against hepatitis A and E infections, recombinant HAV-HEp148 was created as a vector to express an HEV neutralization epitope (HEp148) located at aa 459-606 of the HEV capsid protein. The recombinant virus expressed the HEp148 protein in a partially dimerized state in HAV-susceptible cells. Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice. Thus, the present study demonstrates a novel approach to the development of a combined hepatitis A and E vaccine.

A Recombinant HAV Expressing a Neutralization Epitope of HEV Induces Immune Response against HAV and HEV in Mice

PubMed Central

Kui, Xiang; Yuri, Kusov; Guan, Ying; Wang, Yan; Yi, Shan; Wu, Jinyuan; Yin, Na; Zhou, Yan; Li, Hongjun; Sun, Maosheng

2017-01-01

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are causative agents of acute viral hepatitis transmitted via the fecal–oral route. Both viruses place a heavy burden on the public health and economy of developing countries. To test the possibility that HAV could be used as an expression vector for the development of a combination vaccine against hepatitis A and E infections, recombinant HAV-HEp148 was created as a vector to express an HEV neutralization epitope (HEp148) located at aa 459–606 of the HEV capsid protein. The recombinant virus expressed the HEp148 protein in a partially dimerized state in HAV-susceptible cells. Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice. Thus, the present study demonstrates a novel approach to the development of a combined hepatitis A and E vaccine. PMID:28914805

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Development of massive multilevel molecular dynamics simulation program, Platypus (PLATform for dYnamic Protein Unified Simulation), for the elucidation of protein functions.

PubMed

Takano, Yu; Nakata, Kazuto; Yonezawa, Yasushige; Nakamura, Haruki

2016-05-05

A massively parallel program for quantum mechanical-molecular mechanical (QM/MM) molecular dynamics simulation, called Platypus (PLATform for dYnamic Protein Unified Simulation), was developed to elucidate protein functions. The speedup and the parallelization ratio of Platypus in the QM and QM/MM calculations were assessed for a bacteriochlorophyll dimer in the photosynthetic reaction center (DIMER) on the K computer, a massively parallel computer achieving 10 PetaFLOPs with 705,024 cores. Platypus exhibited the increase in speedup up to 20,000 core processors at the HF/cc-pVDZ and B3LYP/cc-pVDZ, and up to 10,000 core processors by the CASCI(16,16)/6-31G** calculations. We also performed excited QM/MM-MD simulations on the chromophore of Sirius (SIRIUS) in water. Sirius is a pH-insensitive and photo-stable ultramarine fluorescent protein. Platypus accelerated on-the-fly excited-state QM/MM-MD simulations for SIRIUS in water, using over 4000 core processors. In addition, it also succeeded in 50-ps (200,000-step) on-the-fly excited-state QM/MM-MD simulations for the SIRIUS in water. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

PubMed Central

Suomalainen, Maarit; Zheng, Yueting; Boucke, Karin

2017-01-01

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection. PMID:28628648

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand.

PubMed

Wang, Jianjun; Kim, Young-Seung; Liu, Shuang

2008-03-01

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.

Progress on New Hepatitis C Virus Targets: NS2 and NS5A

NASA Astrophysics Data System (ADS)

Marcotrigiano, Joseph

Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

C-terminal interactions mediate the quaternary dynamics of αB-crystallin

PubMed Central

Hilton, Gillian R.; Hochberg, Georg K. A.; Laganowsky, Arthur; McGinnigle, Scott I.; Baldwin, Andrew J.; Benesch, Justin L. P.

2013-01-01

αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins. PMID:23530258

Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Cynthia L., E-mail: c-rowe@northwestern.edu; Matsuura, Hisae, E-mail: hisaem@stanford.edu; Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208

The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLAmore » class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.« less

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

PubMed Central

Wang, Chao-Wen; Cheng, Yun-Hsin; Irokawa, Hayato; Hwang, Gi-Wook; Naganuma, Akira; Kuge, Shusuke

2016-01-01

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins. PMID:27459103

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions

PubMed Central

Joseph, Prem Raj B.; Mosier, Philip D.; Desai, Umesh R.; Rajarathnam, Krishna

2015-01-01

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8–GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer–GAG interactions and function. PMID:26371375

A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.

PubMed

Liu, Q; Astell, C R

1996-10-01

During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

Structural characterization of Mumps virus fusion protein core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Yueyong; Xu Yanhui; Lou Zhiyong

2006-09-29

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus,more » forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.« less

Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA ▿ †

PubMed Central

Baig, Tayyba T.; Lanchy, Jean-Marc; Lodmell, J. Stephen

2009-01-01

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging. PMID:18971263

Initial Stage of Aerosol Formation from Oversaturated Vapors

NASA Astrophysics Data System (ADS)

Lushnikov, A. A.; Zagainov, V. A.; Lyubovtseva, Yu. S.

2018-03-01

The formation of aerosol particles from oversaturated vapor was considered assuming that the stable nuclei of the new phase contain two (dimers) or three (trimers) condensing vapor molecules. Exact expressions were derived and analyzed for the partition functions of the dimer and trimer suspended in a carrier gas for the rectangular well and repulsive core intermolecular potentials. The equilibrium properties of these clusters and the nucleation rate of aerosol particles were discussed. The bound states of clusters were introduced using a limitation on their total energy: molecular clusters with a negative total energy were considered to exclude configurations with noninteracting fragments.

A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus▿†

PubMed Central

Jones, Daniel M.; Atoom, Ali M.; Zhang, Xiaozhen; Kottilil, Shyamasundaran; Russell, Rodney S.

2011-01-01

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1T-64–66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1T-64–66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1T-64–66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly. PMID:21957313

Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

NASA Astrophysics Data System (ADS)

Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

2016-10-01

We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

Discovery, Total Synthesis and Key Structural Elements for the Immunosuppressive Activity of Cocosolide, a Symmetrical Glycosylated Macrolide Dimer from Marine Cyanobacteria.

PubMed

Gunasekera, Sarath P; Li, Yang; Ratnayake, Ranjala; Luo, Danmeng; Lo, Jeannette; Reibenspies, Joseph H; Xu, Zhengshuang; Clare-Salzler, Michael J; Ye, Tao; Paul, Valerie J; Luesch, Hendrik

2016-06-06

A new dimeric macrolide xylopyranoside, cocosolide (1), was isolated from the marine cyanobacterium preliminarily identified as Symploca sp. from Guam. The structure was determined by a combination of NMR spectroscopy, HRMS, X-ray diffraction studies and Mosher's analysis of the base hydrolysis product. Its carbon skeleton closely resembles that of clavosolides A-D isolated from the sponge Myriastra clavosa, for which no bioactivity is known. We performed the first total synthesis of cocosolide (1) along with its [α,α]-anomer (26) and macrocyclic core (28), thus leading to the confirmation of the structure of natural 1. The convergent synthesis featured Wadsworth-Emmons cyclopropanation, Sakurai annulation, Yamaguchi macrocyclization/dimerization reaction, α-selective glycosidation and β-selective glycosidation. Compounds 1 and 26 potently inhibited IL-2 production in both T-cell receptor dependent and independent manners. Full activity requires the presence of the sugar moiety as well as the intact dimeric structure. Cocosolide also suppressed the proliferation of anti-CD3-stimulated T-cells in a dose-dependent manner. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-step synthesis of Er3+ and Yb3+ ions doped molybdate/Gd2O3 core-shell nanoparticles for biomedical imaging

NASA Astrophysics Data System (ADS)

Kamińska, Izabela; Elbaum, Danek; Sikora, Bożena; Kowalik, Przemysław; Mikulski, Jakub; Felcyn, Zofia; Samol, Piotr; Wojciechowski, Tomasz; Minikayev, Roman; Paszkowicz, Wojciech; Zaleszczyk, Wojciech; Szewczyk, Maciej; Konopka, Anna; Gruzeł, Grzegorz; Pawlyta, Mirosława; Donten, Mikołaj; Ciszak, Kamil; Zajdel, Karolina; Frontczak-Baniewicz, Małgorzata; Stępień, Piotr; Łapiński, Mariusz; Wilczyński, Grzegorz; Fronc, Krzysztof

2018-01-01

Nanostructures as color-tunable luminescent markers have become major, promising tools for bioimaging and biosensing. In this paper separated molybdate/Gd2O3 doped rare earth ions (erbium, Er3+ and ytterbium, Yb3+) core-shell nanoparticles (NPs), were fabricated by a one-step homogeneous precipitation process. Emission properties were studied by cathodo- and photoluminescence. Scanning electron and transmission electron microscopes were used to visualize and determine the size and shape of the NPs. Spherical NPs were obtained. Their core-shell structures were confirmed by x-ray diffraction and energy-dispersive x-ray spectroscopy measurements. We postulated that the molybdate rich core is formed due to high segregation coefficient of the Mo ion during the precipitation. The calcination process resulted in crystallization of δ/ξ (core/shell) NP doped Er and Yb ions, where δ—gadolinium molybdates and ξ—molybdates or gadolinium oxide. We confirmed two different upconversion mechanisms. In the presence of molybdenum ions, in the core of the NPs, Yb3+-{{{{MoO}}}4}2- (∣2F7/2, 3T2〉) dimers were formed. As a result of a two 980 nm photon absorption by the dimer, we observed enhanced green luminescence in the upconversion process. However, for the shell formed by the Gd2O3:Er, Yb NPs (without the Mo ions), the typical energy transfer upconversion takes place, which results in red luminescence. We demonstrated that the NPs were transported into cytosol of the HeLa and astrocytes cells by endocytosis. The core-shell NPs are sensitive sensors for the environment prevailing inside (shorter luminescence decay) and outside (longer luminescence decay) of the tested cells. The toxicity of the NPs was examined using MTT assay.

Interaction between core protein of classical swine fever virus with cellular IQGAP1 proetin appears essential for virulence in swine

USDA-ARS?s Scientific Manuscript database

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton affecting cell adhesion, polarization and migration, interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified a defined set of residues within CSFV Core prote...

Fibrillar dimer formation of islet amyloid polypeptides

DOE PAGES

Chiu, Chi -cheng; de Pablo, Juan J.

2015-05-08

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

Fibrillar dimer formation of islet amyloid polypeptides

NASA Astrophysics Data System (ADS)

Chiu, Chi-cheng; de Pablo, Juan J.

2015-09-01

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

Synthesis of partially and fully fused polyaromatics by annulative chlorophenylene dimerization.

PubMed

Koga, Yoshito; Kaneda, Takeshi; Saito, Yutaro; Murakami, Kei; Itami, Kenichiro

2018-01-26

Since the discovery by Ullmann and Bielecki in 1901, reductive dimerization (or homocoupling) of aryl halides has been extensively exploited for the generation of a range of biaryl-based functional molecules. In contrast to the single-point connection in these products, edge-sharing fused aromatic systems have not generally been accessible from simple aryl halides via annulation cascades. Here we report a single-step synthesis of fused aromatics with a triphenylene core by the palladium-catalyzed annulative dimerization of structurally and functionally diverse chlorophenylenes through double carbon-hydrogen bond activation. The partially fused polyaromatics can be transformed into fully fused, small graphene nanoribbons, which are otherwise difficult to synthesize. This simple, yet powerful, method allows access to functional π-systems of interest in optoelectronics research. Copyright © 2018, The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Quantitative imaging of carbon dimer precursor for nanomaterial synthesis in the carbon arc

DOE PAGES

Vekselman, V.; Khrabry, A.; Kaganovich, I.; ...

2018-02-06

Delineating the dominant processes responsible for nanomaterial synthesis in a plasma environment requires measurements of the precursor species contributing to the growth of nanostructures. Here, we performed comprehensive measurements of spatial and temporal profiles of carbon dimers (C 2) in sub-atmospheric-pressure carbon arc by laser-induced fluorescence. Measured spatial profiles of C 2 coincide with the growth region of carbon nanotubes (Fang et al 2016 Carbon 107 273–80) and vary depending on the arc operation mode, which is determined by the discharge current and the ablation rate of the graphite anode. The C 2 density profile exhibits large spatial and timemore » variations due to motion of the arc core. A comparison of the experimental data with the 2D simulation results of self-consistent arc modeling shows good agreement. The model predicts well the main processes determining spatial profiles of carbon dimers (C 2).« less

Quantitative imaging of carbon dimer precursor for nanomaterial synthesis in the carbon arc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vekselman, V.; Khrabry, A.; Kaganovich, I.

Delineating the dominant processes responsible for nanomaterial synthesis in a plasma environment requires measurements of the precursor species contributing to the growth of nanostructures. Here, we performed comprehensive measurements of spatial and temporal profiles of carbon dimers (C 2) in sub-atmospheric-pressure carbon arc by laser-induced fluorescence. Measured spatial profiles of C 2 coincide with the growth region of carbon nanotubes (Fang et al 2016 Carbon 107 273–80) and vary depending on the arc operation mode, which is determined by the discharge current and the ablation rate of the graphite anode. The C 2 density profile exhibits large spatial and timemore » variations due to motion of the arc core. A comparison of the experimental data with the 2D simulation results of self-consistent arc modeling shows good agreement. The model predicts well the main processes determining spatial profiles of carbon dimers (C 2).« less

Atom-Economical Dimerization Strategy by the Rhodium-Catalyzed Addition of Carboxylic Acids to Allenes: Protecting-Group-Free Synthesis of Clavosolide A and Late-Stage Modification.

PubMed

Haydl, Alexander M; Breit, Bernhard

2015-12-14

Natural products of polyketide origin with a high level of symmetry, in particular C2 -symmetric diolides as a special macrolactone-based product class, often possess a broad spectrum of biological activity. An efficient route to this important structural motif was developed as part of a concise and highly convergent synthesis of clavosolide A. This strategy features an atom-economic "head-to-tail" dimerization by the stereoselective rhodium-catalyzed addition of carboxylic acids to terminal allenes with the simultaneous construction of two new stereocenters. The excellent efficiency and selectivity with which the C2 -symmetric core structures were obtained are remarkable considering the outcome under classical dimerization conditions. Furthermore, this approach facilitates late-stage modification and provides ready access to potential new lead structures. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phonon-mediated repulsion, sharp transitions and (quasi)self-trapping in the extended Peierls-Hubbard model

NASA Astrophysics Data System (ADS)

Sous, John; Chakraborty, Monodeep; Krems, Roman; Berciu, Mona

We study two identical fermions, or two hard-core bosons, in an infinite chain and coupled to phonons by interactions that modulate their hopping as described by the Peierls/Su-Schrieffer- Heeger (SSH) model. We show that exchange of phonons generates effective nearest-neighbor repulsion between particles and also gives rise to interactions that move the pair as a whole. The two-polaron phase diagram exhibits two sharp transitions, leading to light dimers at strong coupling and the flattening of the dimer dispersion at some critical values of the parameters. This dimer (quasi)self-trapping occurs at coupling strengths where single polarons are mobile. This illustrates that, depending on the strength of the phonon-mediated interactions, the coupling to phonons may completely suppress or strongly enhance quantum transport of correlated particles. NSERC, Stewart Blusson Quantum Matter Institute.

Hepatitis Virus Capsid Polymorphs Respond Differently to Changes in Encapsulated Cargo Size

PubMed Central

He, Li; Porterfield, J. Zachary; van der Schoot, Paul; Zlotnick, Adam; Dragnea, Bogdan

2017-01-01

A templated assembly approach for Hepatitis B virus-like particles was employed to determine how the T = 3 and T = 4 polymorphs of the Hepatitis B virus (HBV) icosahedral cores respond to a systematic, gradual change in the encapsulated cargo size. It was found that assembly into complete virus-like particles occurs cooperatively around a variety of core diameters, albeit the degree of cooperativity varies. Among these virus-like particles, it was found that those of an outer diameter similar to T = 4 are able to accommodate the widest range of cargo sizes. PMID:24010404

Optoelectronic properties and structural effects of the incremental addition of pyridyl moieties on a rhodium dimer.

PubMed

Chartrand, Daniel; Hanan, Garry S

2014-11-13

The synthesis and characterization of five C-C coupling products obtained from the reaction of a paddlewheel tetrakis 4-bromo-N,N'-diphenylbenzamidinate dirhodium dimer with 4-pyridineboronic acid pinacol ester are reported. The coupling reactions occur on one to four amidinate ligands, leading to rhodium dimers containing [tetrakis, tris, cis-bis, trans-bis, or mono]-N,N'-diphenyl-4-(pyridin-4-yl)benzamidinate ligands, effectively creating new binding sites on the metal complexes. The new compounds were isolated by column chromatography, and the exact conformations were verified by X-ray crystallography. Redox processes showed only a small variation within the coupling products and included two oxidations (1.30 ± 0.02 V, 0.27 ± 0.01 V vs SCE) and one reduction (-1.55 ± 0.02 V vs SCE), all centered on the Rh-Rh core. Time-dependent density functional theory (TD-DFT) was used to analyze this series with four other fully characterized N,N'-diphenyl-aryl-amidinate rhodium dimers that were found in the literature. The two main absorption bands of these nine rhodium dimers were compared to TD-DFT calculations, both giving excellent correlation. The first, a metal-to-metal (MM) transition around 11800 cm(-1) (845 nm) was blue-shifted in the calculation, with an average difference of 1378 cm(-1) but had only a 15 cm(-1) standard deviation, showing a strong correlation despite the energy difference. The second, a metal-to-ligand charge transfer (MLCT) transition around 18900 cm(-1) (530 nm) was a near perfect match with only a 64 cm(-1) average difference and a 35 cm(-1) standard deviation. The electronic transition, redox potentials, and HOMO and LUMO energies of all dimers were plotted versus the Hammett parameter (σ) of the aryl group and Taft's model with 2 components: field effects (σF) and resonance (σR). The properties involving only the Rh-Rh core (MM band, all oxidation potentials, HOMO and LUMO) were fit with a single set of σF and σR contributions (73% and 27%), with a goodness-of-fit (R(2)) value ranging from 90% to 99.7%. The metal-dimer to ligand charge-transfer band, involving the amidinate ligand, displayed different values of contribution with 45% and 55% for the σF and σR, respectively, with a fit of 94.8%. The accuracy of these fits enables the designed modification of amidinate-based dirhodium complexes to achieve desirable redox and spectroscopic properties.

Human Pyruvate Dehydrogenase Complex E2 and E3BP Core Subunits: New Models and Insights from Molecular Dynamics Simulations.

PubMed

Hezaveh, Samira; Zeng, An-Ping; Jandt, Uwe

2016-05-19

Targeted manipulation and exploitation of beneficial properties of multienzyme complexes, especially for the design of novel and efficiently structured enzymatic reaction cascades, require a solid model understanding of mechanistic principles governing the structure and functionality of the complexes. This type of system-level and quantitative knowledge has been very scarce thus far. We utilize the human pyruvate dehydrogenase complex (hPDC) as a versatile template to conduct corresponding studies. Here we present new homology models of the core subunits of the hPDC, namely E2 and E3BP, as the first time effort to elucidate the assembly of hPDC core based on molecular dynamic simulation. New models of E2 and E3BP were generated and validated at atomistic level for different properties of the proteins. The results of the wild type dimer simulations showed a strong hydrophobic interaction between the C-terminal and the hydrophobic pocket which is the main driving force in the intertrimer binding and the core self-assembly. On the contrary, the C-terminal truncated versions exhibited a drastic loss of hydrophobic interaction leading to a dimeric separation. This study represents a significant step toward a model-based understanding of structure and function of large multienzyme systems like PDC for developing highly efficient biocatalyst or bioreaction cascades.

Structure and assembly of a paramyxovirus matrix protein

PubMed Central

Battisti, Anthony J.; Meng, Geng; Winkler, Dennis C.; McGinnes, Lori W.; Plevka, Pavel; Steven, Alasdair C.; Morrison, Trudy G.; Rossmann, Michael G.

2012-01-01

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host’s cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly. PMID:22891297

Structure and assembly of a paramyxovirus matrix protein.

PubMed

Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

2012-08-28

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

Molecular architecture of the TRAPPII complex and implications for vesicle tethering.

PubMed

Yip, Calvin K; Berscheminski, Julia; Walz, Thomas

2010-11-01

Multisubunit tethering complexes participate in the process of vesicle tethering--the initial interaction between transport vesicles and their acceptor compartments. TRAPPII (named for transport protein particle II) is a highly conserved tethering complex that functions in the late Golgi apparatus and consists of all of the subunits of TRAPPI and three additional, specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle EM. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer, which is responsible for dimerization. TRAPPII binds the Ypt1 GTPase and probably uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss the implications of the structure of TRAPPII for coat interaction and TRAPPII-associated human pathologies.

Comprehensive investigation of core-shell dimer nanoparticles size, distance and thicknesses on performance of a hybrid organic-inorganic halide perovskite solar cell

NASA Astrophysics Data System (ADS)

Heidarzadeh, Hamid

2018-03-01

Significant performance enhancement in an ultrathin perovskite (CH3NH3PbI3) solar cell is done using plasmonic embedded core–shell dimer nanoparticles. Three-dimensional finite difference time-domain (FDTD) method is used. A perovskite absorber with a volume of 400 × 400 × 200 nm3 is considered. At first, a cell with one embedded nanoparticle is simulated. Absorptance of CH3NH3PbI3 absorber and gold nanoparticle are obtained. An optimization is done. Then a cell with embedded dimer nanoparticles is evaluated. The results show higher photocurrent enhancement for that in compared to a cell with one embedded nanoparticle. To further photocurrent enhancement, gold-SiO2 core–shell nanoparticles are used. Photocurrents of 23.37 mA cm‑2, 23.3 mA cm‑2, 22.5 mA cm‑2 and 21.47 mA cm‑2 are obtained for a cell with two embedded core–shell nanoparticles with core radius of 60 nm and shell thickness of 2 nm, 5 nm, 10 nm and 20 nm, respectively. It is important to mention that the photocurrent is 17.9 mA cm‑2 for reference cell and 19.8 mA cm‑2 for a cell with one embedded nanoparticle. Higher photocurrent is due to the near-field plasmonic effect.

Jozilebomines A and B, Naphthylisoquinoline Dimers from the Congolese Liana Ancistrocladus ileboensis, with Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Cell Line.

PubMed

Li, Jun; Seupel, Raina; Bruhn, Torsten; Feineis, Doris; Kaiser, Marcel; Brun, Reto; Mudogo, Virima; Awale, Suresh; Bringmann, Gerhard

2017-10-27

Two new naphthylisoquinoline dimers, jozilebomines A (1a) and B (1b), were isolated from the roots of the Congolese plant Ancistrocladus ileboensis, along with the known dimer jozimine A 2 (2). These compounds are Dioncophyllaceae-type metabolites, i.e., lacking oxygen functions at C-6 and with an R-configuration at C-3 in their tetrahydroisoquinoline moieties. The dimers 1a and 1b consist of two 7,1'-coupled naphthylisoquinoline monomers linked through an unprecedented 3',6″-coupling in the binaphthalene core and not, as in 2, via the C-3-positions of the two naphthalene units. Thus, different from the C 2 -symmetric jozimine A 2 (2), the new jozilebomines are constitutionally unsymmetric. The central biaryl axis of each of the three dimers is rotationally hindered, so that 1a, 1b, and 2 possess three consecutive chiral axes. The two jozilebomines have identical constitutions and the same absolute configurations at all four stereogenic centers, but differ from each other in their axial chirality. Their structural elucidation was achieved by HRESIMS, 1D and 2D NMR, oxidative degradation, and experimental and calculated ECD data. They exhibited distinct and specific antiplasmodial activities. All dimers showed potent cytotoxicity against HeLa human cervical cancer cells and preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions. Furthermore, these dimers significantly inhibited the colony formation of PANC-1 cells, even when exposed to noncytotoxic concentration for a short time. Jozilebomines A (1a) and B (1b) and jozimine A 2 (2) represent novel potential candidates for future drug development against pancreatic cancer.

Formation of high-order oligomers is required for functional bioactivity of an African bat henipavirus surface glycoprotein.

PubMed

Behner, Laura; Zimmermann, Louisa; Ringel, Marc; Weis, Michael; Maisner, Andrea

2018-05-01

Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic henipaviruses originating from fruit bats in Australia and Asia that can cause severe infections in livestock and humans. In recent years, also African bat henipaviruses were identified at the nucleic acid level. To assess their potential to replicate in non-bat species, several studies were performed to characterize the two surface glycoproteins required for virus entry and spread by cell-cell fusion. It has been shown that surface expression and fusion-helper function of the receptor-binding G protein of Kumasi virus (KV), the prototypic Ghanaian bat henipavirus, is reduced compared to other non-African henipavirus G proteins. Immunostainings and pulse-chase analysis revealed a delayed export of KV G from the ER. As defects in oligomerization of viral glycoproteins can be responsible for limited surface transport thereby restricting the bioactivity, we analyzed the oligomerization pattern of KV G. In contrast to HeV and NiV whose G proteins are known to be expressed at a dimer-tetramer ratio of 1:1, KV G almost exclusively formed stable tetramers or higher oligomers. KV G also showed less stringent requirements for defined stalk cysteines to form dimers and tetramers. Interestingly, any changes in the oligomeric forms negatively affected the fusion-helper activity although surface expression and receptor binding was unchanged. This clearly indicates that the formation of mostly higher oligomeric KV G forms is not a deficiency responsible for ER retention, but is rather a basic structural feature essential for the bioactivity of this African bat henipavirus glycoprotein. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

PubMed Central

Wijesinghe, Kaveesha J.; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E.; Gerstman, Bernard S.; Chapagain, Prem P.; Li, Sheng; Stahelin, Robert V.

2017-01-01

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. PMID:28167534

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

PubMed

Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

2017-04-14

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Enantioselective Rhodium-Catalyzed Dimerization of ω-Allenyl Carboxylic Acids: Straightforward Synthesis of C2 -Symmetric Macrodiolides.

PubMed

Steib, Philip; Breit, Bernhard

2018-04-19

Herein, we report on the first enantioselective and atom-efficient catalytic one-step dimerization method to selectively transform ω-allenyl carboxylic acids into C 2 -symmetric 14- to 28-membered bismacrolactones (macrodiolides). This convenient asymmetric access serves as an attractive route towards multiple naturally occuring homodimeric macrocyclic scaffolds and demonstrates excellent efficiency to construct the complex, symmetric core structures. By utilizing a rhodium catalyst with a modified chiral cyclopentylidene-diop ligand, the desired diolides were obtained in good to high yields, high diastereoselectivity, and excellent enantioselectivity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlated Optical Spectroscopy and Transmission Electron Microscopy of Individual Hollow Nanoparticles and their Dimers

PubMed Central

Yang, Linglu; Yan, Bo; Reinhard, Björn M.

2009-01-01

The optical spectra of individual Ag-Au alloy hollow particles were correlated with the particles’ structures obtained by transmission electron microscopy (TEM). The TEM provided direct experimental access to the dimension of the cavity, thickness of the metal shell, and the interparticle distance of hollow particle dimers with high spatial resolution. The analysis of correlated spectral and structural information enabled the quantification of the influence of the core-shell structure on the resonance energy, plasmon lifetime, and plasmon coupling efficiency. Electron beam exposure during TEM inspection was observed to affect plasmon wavelength and lifetime, making optical inspection prior to structural characterization mandatory. PMID:19768108

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops. Copyright 2001 Academic Press.

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

PubMed

Zhu, Tengfei; Song, Hao; Peng, Ruchao; Shi, Yi; Qi, Jianxun; Gao, George F

2017-09-15

Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

Anti-hepatitis B virus activities and absolute configurations of sesquiterpenoid glycosides from Phyllanthus emblica.

PubMed

Lv, Jun-Jiang; Wang, Ya-Feng; Zhang, Jing-Min; Yu, Shan; Wang, Dong; Zhu, Hong-Tao; Cheng, Rong-Rong; Yang, Chong-Ren; Xu, Min; Zhang, Ying-Jun

2014-11-21

During the process exploring anti-viral compounds from Phyllanthus species, eight new highly oxygenated bisabolane sesquiterpenoid glycoside phyllaemblicins G1–G8 (1–8) were isolated from Phyllanthus emblica, along with three known compounds, phyllaemblicin F (9), phyllaemblic acid (10) and glochicoccin D (11). Phyllaemblicin G2 (2), bearing a tricyclo [3.1.1.1] oxygen bridge ring system, is an unusual sesquiterpenoid glycoside, while phyllaemblicins G6–G8 (6–8) are dimeric sesquiterpenoid glycosides with two norbisabolane units connecting through a disaccharide. All the structures were elucidated by the extensive analysis of HRMS and NMR data. The relative configuration of phyllaemblicin G2 was constructed based on heteronuclear coupling constants measurement, and the absolute configurations for all new compounds were established by calculated electronic circular dichroism (ECD) using time dependent density functional theory. The sesquiterpenoid glycoside dimers 6–9 displayed potential anti-hepatitis B virus (HBV) activities, especially for the new compound 6 with IC50 of 8.53 ± 0.97 and 5.68 ± 1.75 μM towards the HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) secretion, respectively.

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

PubMed

Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

2011-09-01

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability.

PubMed

Nonin, S; Phan, A T; Leroy, J L

1997-09-15

Repetitive cytosine-rich DNA sequences have been identified in telomeres and centromeres of eukaryotic chromosomes. These sequences play a role in maintaining chromosome stability during replication and may be involved in chromosome pairing during meiosis. The C-rich repeats can fold into an 'i-motif' structure, in which two parallel-stranded duplexes with hemiprotonated C.C+ pairs are intercalated. Previous NMR studies of naturally occurring repeats have produced poor NMR spectra. This led us to investigate oligonucleotides, based on natural sequences, to produce higher quality spectra and thus provide further information as to the structure and possible biological function of the i-motif. NMR spectroscopy has shown that d(5mCCTTTACC) forms an i-motif dimer of symmetry-related and intercalated folded strands. The high-definition structure is computed on the basis of the build-up rates of 29 intraresidue and 35 interresidue nuclear Overhauser effect (NOE) connectivities. The i-motif core includes intercalated interstrand C.C+ pairs stacked in the order 2*.8/1.7*/1*.7/2.8* (where one strand is distinguished by an asterisk and the numbers relate to the base positions within the repeat). The TTTA sequences form two loops which span the two wide grooves on opposite sides of the i-motif core; the i-motif core is extended at both ends by the stacking of A6 onto C2.C8+. The lifetimes of pairs C2.C8+ and 5mC1.C7+ are 1 ms and 1 s, respectively, at 15 degrees C. Anomalous exchange properties of the T3 imino proton indicate hydrogen bonding to A6 N7 via a water bridge. The d(5mCCTTTTCC) deoxyoligonucleotide, in which position 6 is occupied by a thymidine instead of an adenine, also forms a symmetric i-motif dimer. However, in this structure the two TTTT loops are located on the same side of the i-motif core and the C.C+ pairs are formed by equivalent cytidines stacked in the order 8*.8/1.1*/7*.7/2.2*. Oligodeoxynucleotides containing two C-rich repeats can fold and dimerize into an i-motif. The change of folding topology resulting from the substitution of a single nucleoside emphasizes the influence of the loop residues on the i-motif structure formed by two folded strands.

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

PubMed Central

Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

2006-01-01

SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

Maturation of the Gag core decreases the stability of retroviral lipid membranes.

PubMed

Davidoff, Candice; Payne, Riley J; Willis, Sharon H; Doranz, Benjamin J; Rucker, Joseph B

2012-11-25

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. Copyright © 2012 Elsevier Inc. All rights reserved.

Maturation of the Gag core decreases the stability of retroviral lipid membranes

PubMed Central

Davidoff, Candice; Payne, Riley; Willis, Sharon H.; Doranz, Benjamin J.; Rucker, Joseph B.

2012-01-01

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. PMID:22995186

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Designer interface peptide grafts target estrogen receptor alpha dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, S.; Asare, B.K.; Biswas, P.K., E-mail: pbiswas@tougaloo.edu

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization.more » Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable complexes with estrogen receptor in silico. • Inhibitor peptides significantly decrease estrogen induced cell proliferation of ER positive breast cancer cells in vitro.« less

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

Synthesis, structure, and reactivity of N-benzoyl iminophosphoranes ortho lithiated at the benzoyl group.

PubMed

Aguilar, David; Fernández, Ignacio; Cuesta, Luciano; Yañez-Rodríguez, Víctor; Soler, Tatiana; Navarro, Rafael; Urriolabeitia, Esteban P; López Ortiz, Fernando

2010-10-01

Ortho lithiation of N-benzamido-P,P,P-triaryliminophosphoranes through deprotonation with alkyllithium bases was achieved with ortho-C═O and ortho-P═N chemoselectivity. However, the synthetic scope of these processes was rather limited. Ortho-lithiated N-benzamido-P,P,P-triphenyliminophosphorane 8 was efficiently prepared via lithium/halogen exchange of the corresponding ortho-brominated precursor with s-BuLi in THF at -90 °C. The reaction of 8 with a variety of electrophiles provides an easy and mild method for the regioselective synthesis of ortho-modified iminophosphoranes via C-C (alkylation and hydroxyalkylation) and C-X (X = I, Si, P, Sn, and Hg) bond-forming reactions. NMR characterization of 8 in THF solution showed that 8 exists as an equilibrium mixture of one monomer and two dimers. The Li atoms of these species become members of five-membered rings through chelation by the ortho-metalated carbon and the carbonyl oxygen. The dimers differ in the relative orientation of the two chelates with respect to the plane defined by the C(2)Li(2) core. The equilibrium between all species is established by splitting the dimers into monomers and subsequent recombination with formation of a different dimer.

Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes*

PubMed Central

Gupta, Kushol; Martin, Renee; Sharp, Robert; Sarachan, Kathryn L.; Ninan, Nisha S.; Van Duyne, Gregory D.

2015-01-01

The survival motor neuron (SMN) protein forms the oligomeric core of a multiprotein complex required for the assembly of spliceosomal small nuclear ribonucleoproteins. Deletions and mutations in the SMN1 gene are associated with spinal muscular atrophy (SMA), a devastating neurodegenerative disease that is the leading heritable cause of infant mortality. Oligomerization of SMN is required for its function, and some SMA patient mutations disrupt the ability of SMN to self-associate. Here, we investigate the oligomeric nature of the SMN·Gemin2 complexes from humans and fission yeast (hSMN·Gemin2 and ySMN·Gemin2). We find that hSMN·Gemin2 forms oligomers spanning the dimer to octamer range. The YG box oligomerization domain of SMN is both necessary and sufficient to form these oligomers. ySMN·Gemin2 exists as a dimer-tetramer equilibrium with Kd = 1.0 ± 0.9 μm. A 1.9 Å crystal structure of the ySMN YG box confirms a high level of structural conservation with the human ortholog in this important region of SMN. Disulfide cross-linking experiments indicate that SMN tetramers are formed by self-association of stable, non-dissociating dimers. Thus, SMN tetramers do not form symmetric helical bundles such as those found in glycine zipper transmembrane oligomers. The dimer-tetramer nature of SMN complexes and the dimer of dimers organization of the SMN tetramer provide an important foundation for ongoing studies to understand the mechanism of SMN-assisted small nuclear ribonucleoprotein assembly and the underlying causes of SMA. PMID:26092730

High-speed atomic force microscopy reveals structural dynamics of α -synuclein monomers and dimers

NASA Astrophysics Data System (ADS)

Zhang, Yuliang; Hashemi, Mohtadin; Lv, Zhengjian; Williams, Benfeard; Popov, Konstantin I.; Dokholyan, Nikolay V.; Lyubchenko, Yuri L.

2018-03-01

α-Synuclein (α-syn) is the major component of the intraneuronal inclusions called Lewy bodies, which are the pathological hallmark of Parkinson's disease. α-Syn is capable of self-assembly into many different species, such as soluble oligomers and fibrils. Even though attempts to resolve the structures of the protein have been made, detailed understanding about the structures and their relationship with the different aggregation steps is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. Here we report the structural flexibility of α-syn monomers and dimers in an aqueous solution environment as probed by single-molecule time-lapse high-speed AFM. In addition, we present the molecular basis for the structural transitions using discrete molecular dynamics (DMD) simulations. α-Syn monomers assume a globular conformation, which is capable of forming tail-like protrusions over dozens of seconds. Importantly, a globular monomer can adopt fully extended conformations. Dimers, on the other hand, are less dynamic and show a dumbbell conformation that experiences morphological changes over time. DMD simulations revealed that the α-syn monomer consists of several tightly packed small helices. The tail-like protrusions are also helical with a small β-sheet, acting as a "hinge". Monomers within dimers have a large interfacial interaction area and are stabilized by interactions in the non-amyloid central (NAC) regions. Furthermore, the dimer NAC-region of each α-syn monomer forms a β-rich segment. Moreover, NAC-regions are located in the hydrophobic core of the dimer.

Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran.

PubMed

Zhand, Sareh; Tabarraei, Alijan; Nazari, Amineh; Moradi, Abdolvahab

2017-07-01

Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran. In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559). The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes' (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions. CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

PubMed Central

Yamada, Kazunori; Kondoh, Yasumitsu; Hikono, Hirokazu; Osada, Hiroyuki; Tomii, Kentaro; Saito, Takehiko; Aida, Yoko

2015-01-01

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP. PMID:26222066

Pressure-induced structural transition of mature HIV-1 protease from a combined NMR/MD simulation approach.

PubMed

Roche, Julien; Louis, John M; Bax, Ad; Best, Robert B

2015-12-01

We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Mechanism of Inhibition of Hsp90 Dimerization by Gyrase B Inhibitor Coumermycin A1 (C-A1) Revealed by Molecular Dynamics Simulations and Thermodynamic Calculations.

PubMed

Cele, Favourite N; Kumalo, Hezekiel; Soliman, Mahmoud E S

2016-09-01

Heat shock protein (Hsp) 90 an emerging and attracting target in the anti-HIV drug discovery process due to the key role it plays in the pathogenicity of HIV-1 virus. In this research study, long-range all-atom molecular dynamics simulations were engaged for the bound and the unbound proteins to enhance the understanding of the molecular mechanisms of the Hsp90 dimerization and inhibition. Results evidently showed that coumermycin A1 (C-A1), a recently discovered Hsp90 inhibitor, binds at the dimer's active site of the Hsp90 protein and leads to a substantial parting between dimeric opposed residues, which include Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669.A. Significant differences in magnitudes were observed in radius of gyration, root-mean-square deviation and root-mean-square fluctuation, which confirms a reasonably more flexible state in the apo conformation associated with it dimerization. In contrast, the bound conformer of Hsp90 showed less flexibility. This visibly highpoints the inhibition process resulting from the binding of the ligand. These findings were further validated by principal component analysis. We believe that the detailed dynamic analyses of Hsp90 presented in this study, would give an imperative insight and better understanding to the function and mechanisms of inhibition. Furthermore, information obtained from the binding mode of the inhibitor would be of great assistance in the design of more potent inhibitors against the HIV target Hsp90.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Hepatitis C Virus Core Mutations Associated with False-Negative Serological Results for Genotype 3a Core Antigen

PubMed Central

Dunford, Linda; Freitas, Ines; Holder, Paul; Nguyen, Lan Anh; O'Gorman, Joanne; Connell, Jeff; Carr, Michael; Hall, William; De Gascun, Cillian

2015-01-01

Genetic characterization of the genotype 3a (GT3a) hepatitis C virus (HCV) core region from HCV core antigen (HCVcAg)-negative/RNA-positive cases and HCVcAg-positive/RNA-positive controls identified significant associations between the substitutions A48T and T49A/P and failure to detect HCVcAg (P < 0.05). Polymorphisms at residues 48 and 49 in the core protein are present across all major epidemic and endemic GTs. These findings have implications for HCV diagnosis, particularly in low-income regions in which GT3a HCV is endemic. PMID:25994168

Impact of molecular flexibility on binding strength and self-sorting of chiral π-surfaces.

PubMed

Safont-Sempere, Marina M; Osswald, Peter; Stolte, Matthias; Grüne, Matthias; Renz, Manuel; Kaupp, Martin; Radacki, Krzysztof; Braunschweig, Holger; Würthner, Frank

2011-06-22

In this work, we have explored for the first time the influence of conformational flexibility of π-core on chiral self-sorting properties of perylene bisimides (PBIs) that are currently one of the most prominent classes of functional dyes. For this purpose, two series of chiral macrocyclic PBIs 3a-c and 4a-c comprising oligoethylene glycol bridges of different lengths at the 1,7 bay positions were synthesized and their atropo-enantiomers (P and M enantiomers) were resolved. Single crystal analysis of atropo-enantiomerically pure (P)-3a not only confirmed the structural integrity of the ethylene glycol bridged macrocycle but also illustrated the formation of π-stacked dimers with left-handed supramolecular helicity. Our detailed studies with the series of highly soluble chiral PBIs 4a-c by 1- and 2-D (1)H NMR techniques, and temperature- and concentration-dependent UV/vis absorption and circular dichroism (CD) spectroscopy revealed that in π-π-stacking dimerization of these PBIs chiral self-recognition (i.e., PP and MM homodimer formation) prevails over self-discrimination (i.e., PM heterodimer formation). Our studies clearly showed that with increasing conformational flexibility of PBI cores imparted by longer bridging units, the binding strength for the dimerization process increases, however, the efficiency for chiral self-recognition decreases. These results are rationalized in terms of an induced-fit mechanism facilitating more planarized π-scaffolds of PBIs containing longer bridging units upon π-π-stacking.

A converged calculation of the energy barrier to internal rotation in the ethylene-sulfur dioxide dimer

NASA Astrophysics Data System (ADS)

Resende, Stella M.; De Almeida, Wagner B.; van Duijneveldt-van de Rijdt, Jeanne G. C. M.; van Duijneveldt, Frans B.

2001-08-01

Geometrical parameters for the equilibrium (MIN) and lowest saddle-point (TS) geometries of the C2H4⋯SO2 dimer, and the corresponding binding energies, were calculated using the Hartree-Fock and correlated levels of ab initio theory, in basis sets ranging from the D95(d,p) double-zeta basis set to the aug-cc-pVQZ correlation consistent basis set. An assessment of the effect of the basis set superposition error (BSSE) on these results was made. The dissociation energy from the lowest vibrational state was estimated to be 705±100 cm-1 at the basis set limit, which is well within the range expected from experiment. The barrier to internal rotation was found to be 53±5 cm-1, slightly higher than the (revised) experimental result of 43 cm-1, probably due to zero-point vibrational effects. Our results clearly show that, in direct contrast with recent ideas, the BSSE correction affects differentially the MIN and TS binding energies and so has to be included in the calculation of small energy barriers such as that in the C2H4⋯SO2 dimer. Previous reports of positive MP2 frozen-core binding energies for this complex in basis D95(d,p) are confirmed. The anomalies are shown to be an artifact arising from an incorrect removal of virtual orbitals by the default frozen-core option in the GAUSSIAN program.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

PubMed Central

Gamache, Eric R.; Doh, Jung H.; Ritz, Justin; Laederach, Alain; Bellaousov, Stanislav; Mathews, David H.; Curcio, M. Joan

2017-01-01

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging. PMID:28445416

Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

PubMed

Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

2017-06-21

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

PubMed Central

Etienne, Loïc; Blanchard, Emmanuelle; Boyer, Audrey; Desvignes, Virginie; Gaillard, Julien; Meunier, Jean-Christophe; Roingeard, Philippe; Hourioux, Christophe

2015-01-01

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. PMID:26339783

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed Central

Ni, C. Z.; White, C. A.; Mitchell, R. S.; Wickersham, J.; Kodandapani, R.; Peabody, D. S.; Ely, K. R.

1996-01-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine. PMID:8976557

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed

Ni, C Z; White, C A; Mitchell, R S; Wickersham, J; Kodandapani, R; Peabody, D S; Ely, K R

1996-12-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

PubMed

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-10-17

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells

PubMed Central

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-01-01

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. PMID:29156827

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

PubMed Central

Tellinghuisen, Timothy L.; Hamburger, Agnes E.; Fisher, Bonnie R.; Ostendorp, Ralf; Kuhn, Richard J.

1999-01-01

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly. PMID:10364277

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shang Liang; Hunter, Eric, E-mail: eric.hunter2@emory.ed

2010-09-01

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusionmore » mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.« less

Study of spectroscopic properties of nanosized particles of core-shell morphology

NASA Astrophysics Data System (ADS)

Bzhalava, T. N.; Kervalishvili, P. J.

2018-03-01

Method of studying spectroscopic properties of nanosized particles and estimation of resonance wavelength range for determination of specific and unique “spectral” signatures in purpose of sensing, identification of nanobioparticles, viruses is proposed. Elaboration of relevant models of viruses, estimation of spectral response on interaction of electromagnetic (EM) field and viral nanoparticle is the goal of proposed methodology. Core-shell physical model is used as the first approximation of shape-structure of virion. Theoretical solution of EM wave scattering on single spherical virus-like particle (VLP) is applied for determination of EM fields in the areas of core, shell and surrounding medium of (VLP), as well as scattering and absorption characteristics. Numerical results obtained by computer simulation for estimation of EM “spectra” of bacteriophage T7 demonstrate the strong dependence of spectroscopic characteristics on core-shell related electric and geometric parameters of VLP in resonance wavelengths range. Expected spectral response is observable on far-field characterizations. Obtained analytical EM field expressions, modelling technique in complement with experimental spectroscopic methods should be the way of providing the virus spectral signatures, important in bioparticles characterization.

Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guu, Tom S.Y.; Liu, Zheng; Ye, Qiaozhen

Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-{angstrom} structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt {beta}-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsidmore » protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.« less

Efficient numerical technique for calculating the properties of interacting dimers in the Peierls-Hubbard model

NASA Astrophysics Data System (ADS)

Sous, John; Chakraborty, Monodeep; Krems, Roman; Berciu, Mona

2017-04-01

We develop a method to compute the Green's function for two particles in an infinite chain and coupled to phonons by interactions that modulate their hopping as described by the Peierls/Su-Schrieffer-Heeger (SSH) model. The method is based on a variational approximation to the Bogoliubov-Born-Green-Kirkwood-Yvon (BBGKY) hierarchy and is shown to agree with exact digaonalization calculations. We show that the properties of bipolarons arising in such models is qualitatively different from those of the well-studied Holstein bipolarons. In particular, we show that depending on the particle statistics, strongly bound bipolarons may or may not form. In the case of hard-core bosons, we demonstrate novel effects for dimers such as sharp transitions and self-trapping. In the case of soft-core particles/ spinfull fermions, we show that the mediated interactions lead to overscreeing of the bare Hubbard U repulsion resulting in the formation of strongly bound bipolarons. This work was supported by NSERC of Canada and the Stewart Blusson Quantum Matter Institute.

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, Peter G.; Mothersole, David J.; Ng, Irene W.

2011-01-01

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre–light-harvesting 1–PufX (RC–LH1–PufX) ‘core’ complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX -). Lower rates of LH2more » assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX - mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC–LH1–PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX - membranes, resulting in locally ordered clusters of monomeric RC–LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.« less

Production and pathogenicity of hepatitis C virus core gene products

PubMed Central

Li, Hui-Chun; Ma, Hsin-Chieh; Yang, Chee-Hing; Lo, Shih-Yen

2014-01-01

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and its infection is also associated with insulin resistance and type 2 diabetes mellitus. HCV, belonging to the Flaviviridae family, is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein. The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein (a.a. 1-177) and the cleaved peptide (a.a. 178-191). Core protein could induce insulin resistance, steatosis and even hepatocellular carcinoma through various mechanisms. The peptide (a.a. 178-191) may play a role in the immune response. The polymorphism of this peptide is associated with the cellular lipid drop accumulation, contributing to steatosis development. In addition to the conventional open reading frame (ORF), in the +1 frame, an ORF overlaps with the core protein-coding sequence and encodes the alternative reading frame proteins (ARFP or core+1). ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism. In this review, we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis. PMID:24966583

Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

PubMed Central

Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

2009-01-01

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis. PMID:19605478

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion.

PubMed

Kacprzak, Sylwia; Njimona, Ibrahim; Renz, Anja; Feng, Juan; Reijerse, Edward; Lubitz, Wolfgang; Krauss, Norbert; Scheerer, Patrick; Nagano, Soshichiro; Lamparter, Tilman; Weber, Stefan

2017-05-05

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of the host defense activity of the barrier to autointegration factor against vaccinia virus.

PubMed

Ibrahim, Nouhou; Wicklund, April; Wiebe, Matthew S

2011-11-01

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

PubMed Central

Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

2018-01-01

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

IR photodissociation spectroscopy of (OCS){sub n}{sup +} and (OCS){sub n}{sup −} cluster ions: Similarity and dissimilarity in the structure of CO{sub 2}, OCS, and CS{sub 2} cluster ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inokuchi, Yoshiya, E-mail: y-inokuchi@hiroshima-u.ac.jp; Ebata, Takayuki

2015-06-07

Infrared photodissociation (IRPD) spectra of (OCS){sub n}{sup +} and (OCS){sub n}{sup −} (n = 2–6) cluster ions are measured in the 1000–2300 cm{sup −1} region; these clusters show strong CO stretching vibrations in this region. For (OCS){sub 2}{sup +} and (OCS){sub 2}{sup −}, we utilize the messenger technique by attaching an Ar atom to measure their IR spectra. The IRPD spectrum of (OCS){sub 2}{sup +}Ar shows two bands at 2095 and 2120 cm{sup −1}. On the basis of quantum chemical calculations, these bands are assigned to a C{sub 2} isomer of (OCS){sub 2}{sup +}, in which an intermolecular semi-covalent bondmore » is formed between the sulfur ends of the two OCS components by the charge resonance interaction, and the positive charge is delocalized over the dimer. The (OCS){sub n}{sup +} (n = 3–6) cluster ions show a few bands assignable to “solvent” OCS molecules in the 2000–2080 cm{sup −1} region, in addition to the bands due to the (OCS){sub 2}{sup +} ion core at ∼2090 and ∼2120 cm{sup −1}, suggesting that the dimer ion core is kept in (OCS){sub 3–6}{sup +}. For the (OCS){sub n}{sup −} cluster anions, the IRPD spectra indicate the coexistence of a few isomers with an OCS{sup −} or (OCS){sub 2}{sup −} anion core over the cluster range of n = 2–6. The (OCS){sub 2}{sup −}Ar anion displays two strong bands at 1674 and 1994 cm{sup −1}. These bands can be assigned to a C{sub s} isomer with an OCS{sup −} anion core. For the n = 2–4 anions, this OCS{sup −} anion core form is dominant. In addition to the bands of the OCS{sup −} core isomer, we found another band at ∼1740 cm{sup −1}, which can be assigned to isomers having an (OCS){sub 2}{sup −} ion core; this dimer core has C{sub 2} symmetry and {sup 2}A electronic state. The IRPD spectra of the n = 3–6 anions show two IR bands at ∼1660 and ∼2020 cm{sup −1}. The intensity of the latter component relative to that of the former one becomes stronger and stronger with increasing the size from n = 2 to 4, which corresponds to the increase of “solvent” OCS molecules attached to the OCS{sup −} ion core, but it suddenly decreases at n = 5 and 6. These IR spectral features of the n = 5 and 6 anions are ascribed to the formation of another (OCS){sub 2}{sup −} ion core having C{sub 2v} symmetry with {sup 2}B{sub 2} electronic state.« less

SOCS1 and SOCS3 Are Targeted by Hepatitis C Virus Core/gC1qR Ligation To Inhibit T-Cell Function

PubMed Central

Yao, Zhi Qiang; Waggoner, Stephen N.; Cruise, Michael W.; Hall, Caroline; Xie, Xuefang; Oldach, David W.; Hahn, Young S.

2005-01-01

T cells play an important role in the control of hepatitis C virus (HCV) infection. We have previously demonstrated that the HCV core inhibits T-cell responses through interaction with gC1qR. We show here that core proteins from chronic and resolved HCV patients differ in sequence, gC1qR-binding ability, and T-cell inhibition. Specifically, chronic core isolates bind to gC1qR more efficiently and inhibit T-cell proliferation as well as gamma interferon (IFN-γ) production more profoundly than resolved core isolates. This inhibition is mediated by the disruption of STAT phosphorylation through the induction of SOCS molecules. Silencing either SOCS1 or SOCS3 by small interfering RNA dramatically augments the production of IFN-γ in T cells, thereby abrogating the inhibitory effect of core. Additionally, the ability of core proteins from patients with chronic infections to induce SOCS proteins and suppress STAT activation greatly exceeds that of core proteins from patients with resolved infections. These results suggest that the HCV core/gC1qR-induced T-cell dysfunction involves the induction of SOCS, a powerful inhibitor of cytokine signaling, which represents a novel mechanism by which a virus usurps the host machinery for persistence. PMID:16306613

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

PubMed Central

Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

1998-01-01

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

Protective V127 prion variant prevents prion disease by interrupting the formation of dimer and fibril from molecular dynamics simulations.

PubMed

Zhou, Shuangyan; Shi, Danfeng; Liu, Xuewei; Liu, Huanxiang; Yao, Xiaojun

2016-02-24

Recent studies uncovered a novel protective prion protein variant: V127 variant, which was reported intrinsically resistant to prion conversion and propagation. However, the structural basis of its protective effect is still unknown. To uncover the origin of the protective role of V127 variant, molecular dynamics simulations were performed to explore the influence of G127V mutation on two key processes of prion propagation: dimerization and fibril formation. The simulation results indicate V127 variant is unfavorable to form dimer by reducing the main-chain H-bond interactions. The simulations of formed fibrils consisting of β1 strand prove V127 variant will make the formed fibril become unstable and disorder. The weaker interaction energies between layers and reduced H-bonds number for V127 variant reveal this mutation is unfavorable to the formation of stable fibril. Consequently, we find V127 variant is not only unfavorable to the formation of dimer but also unfavorable to the formation of stable core and fibril, which can explain the mechanism on the protective role of V127 variant from the molecular level. Our findings can deepen the understanding of prion disease and may guide the design of peptide mimetics or small molecule to mimic the protective effect of V127 variant.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Kailang; Li, Weikai; Peng, Guiqing

NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homologymore » in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.« less

The effect of bovine BST2A1 on the release and cell-to-cell transmission of retroviruses.

PubMed

Liang, Zhibin; Zhang, Yang; Song, Jie; Zhang, Hui; Zhang, Suzhen; Li, Yue; Tan, Juan; Qiao, Wentao

2017-09-06

Human BST2 (hBST2, also called Tetherin) is a host restriction factor that blocks the release of various enveloped viruses. BST2s from different mammals also possess antiviral activity. Bovine BST2s (bBST2s), bBST2A1 and bBST2A2, reduce production of cell-free bovine leukemia virus (BLV) and vesicular stomatitis virus (VSV). However, the effect of bBST2 on other retroviruses remains unstudied. Here, we studied the antiviral activity of wildtype and mutant bBST2A1 proteins on retroviruses including human immunodeficiency virus type 1 (HIV-1), prototypic foamy virus (PFV), bovine foamy virus (BFV) and bovine immunodeficiency virus (BIV). The results showed that wildtype bBST2A1 suppressed the release of HIV-1, PFV and BFV. We also generated bBST2A1 mutants, and found that GPI anchor and dimerization, but not glycosylation, are essential for antiviral activity of bBST2A1. Moreover, unlike hBST2, bBST2A1 displayed no inhibitory effect on cell-to-cell transmission of PFV, BFV and BIV. Our data suggested that bBST2A1 inhibited retrovirus release, however, had no effect on cell-to-cell transmission of retroviruses.

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

PubMed

Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

2015-10-29

HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

Structural insights into the multifunctional protein VP3 of birnaviruses.

PubMed

Casañas, Arnau; Navarro, Aitor; Ferrer-Orta, Cristina; González, Dolores; Rodríguez, José F; Verdaguer, Núria

2008-01-01

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two alpha-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morand, Patrice; Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble; Budayova-Spano, Monika

A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiationmore » (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.« less

Conformational plasticity of the Ebola virus matrix protein.

PubMed

Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

2014-11-01

Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

PubMed Central

Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan

2016-01-01

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

PubMed

Datta, Siddhartha A K; Clark, Patrick K; Fan, Lixin; Ma, Buyong; Harvin, Demetria P; Sowder, Raymond C; Nussinov, Ruth; Wang, Yun-Xing; Rein, Alan

2016-02-15

HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

PubMed Central

Clark, Patrick K.; Fan, Lixin; Ma, Buyong; Harvin, Demetria P.; Sowder, Raymond C.; Nussinov, Ruth; Wang, Yun-Xing

2015-01-01

ABSTRACT HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a “spacer” between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it “association competent.” Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. PMID:26637452

Hard X-ray photoemission study of the Fabre salts (TMTTF)2X (X = SbF6 and PF6)

NASA Astrophysics Data System (ADS)

Medjanik, Katerina; de Souza, Mariano; Kutnyakhov, Dmytro; Gloskovskii, Andrei; Müller, Jens; Lang, Michael; Pouget, Jean-Paul; Foury-Leylekian, Pascale; Moradpour, Alec; Elmers, Hans-Joachim; Schönhense, Gerd

2014-11-01

Core-level photoemission spectra of the Fabre salts with X = SbF6 and PF6 were taken using hard X-rays from PETRA III, Hamburg. In these salts TMTTF layers show a significant stack dimerization with a charge transfer of 1 e per dimer to the anion SbF6 or PF6. At room temperature and slightly below the core-level spectra exhibit single lines, characteristic for a well-screened metallic state. At reduced temperatures progressive charge localization sets in, followed by a 2nd order phase transition into a charge-ordered ground state. In both salts groups of new core-level signals occur, shifted towards lower kinetic energies. This is indicative of a reduced transverse-conductivity across the anion layers, visible as layer-dependent charge depletion for both samples. The surface potential was traced via shifts of core-level signals of an adsorbate. A well-defined potential could be established by a conducting cap layer of 5 nm aluminum which appears "transparent" due to the large probing depth of HAXPES (8-10 nm). At the transition into the charge-ordered phase the fluorine 1 s line of (TMTTF)2SbF6 shifts by 2.8 eV to higher binding energy. This is a spectroscopic fingerprint of the loss of inversion symmetry accompanied by a cooperative shift of the SbF6 anions towards the more positively charged TMTTF donors. This shift does not occur for the X = PF6 compound, most likely due to smaller charge disproportion or due to the presence of charge disorder.

Marek’s Disease Virus influences the core gut microbiome of the chicken during the early and late phases of viral replication

USDA-ARS?s Scientific Manuscript database

Marek’s disease (MD) is an important neoplastic disease of chickens caused by the Marek’s disease virus (MDV), an oncogenic alphaherpesvirus. In this study, dysbiosis induced by MDV on the core gut flora of chicken was assessed using next generation sequence (NGS) analysis. Total fecal and cecum-der...

VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

PubMed

Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

1997-07-01

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

PubMed Central

Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

2016-01-01

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.

2014-08-21

Calpains are Ca 2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca 2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3'smore » PEF domain and its crystal structure in the presence of Ca 2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca 2+ bound at the EF5-hand used for homodimer association. Three of the four Ca 2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca 2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.« less

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

PubMed

Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

2013-10-01

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR. Copyright © 2013 Elsevier B.V. All rights reserved.

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

PubMed Central

Stahl, S; MacKay, P; Magazin, M; Bruce, S A; Murray, K

1982-01-01

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples. Images PMID:7041126

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease.

PubMed

Jochmans, Dirk; Anders, Maria; Keuleers, Inge; Smeulders, Liesbeth; Kräusslich, Hans-Georg; Kraus, Günter; Müller, Barbara

2010-10-15

Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action.

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

PubMed Central

2010-01-01

Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. Conclusion These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action. PMID:20950436

1-[(Anthracen-9-yl)carbon­yl]-2,7-di­meth­oxy­naphthalene: a chain-like structure composed of face-to-face type dimeric mol­ecular aggregates

PubMed Central

Siqingaowa; Tsumuki, Takehiro; Ogata, Kazuki; Yonezawa, Noriyuki; Okamoto, Akiko

2016-01-01

The asymmetric unit of the title compound, C27H20O3, contains two independent mol­ecules (A and B). The anthracene ring system is connected to the 2,7-di­meth­oxy­naphthalene core in a twisted manner, with dihedral angles of 86.38 (5) and 79.36 (8)°, respectively, for conformers A and B. In the crystal, face-to-face type dimeric mol­ecular aggregates of each conformer are observed. The dimer of conformer A is formed by two pairs of C—H⋯π inter­actions, and that of conformer B by a pair of (sp 2)C—H⋯O hydrogen bonds. The dimers of conformer A are linked to each other via a π–π stacking inter­action between the anthracene rings to form a chain along the b axis and the chains are aligned along the c axis, forming a sheet structure. The dimers of conformer B are connected to each other via a couple of C—H⋯π inter­actions to form a chain along the b axis. The chains are aligned along the c axis through (sp 2)C—H⋯O=C hydrogen bonds, forming a sheet parallel to the bc plane. The sheets of conformers A and B are alternately stacked along the a axis via two kinds of inter­molecular (sp 2)C—H⋯O=C hydrogen bonds. PMID:27980839

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus.

PubMed

Mandl, C W; Holzmann, H; Meixner, T; Rauscher, S; Stadler, P F; Allison, S L; Heinz, F X

1998-03-01

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

PubMed Central

Moussatche, Nissin; Condit, Richard C.

2014-01-01

ABSTRACT Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCE The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid. PMID:25253347

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.

PubMed

Jurado, Kellie A; Wang, Hao; Slaughter, Alison; Feng, Lei; Kessl, Jacques J; Koh, Yasuhiro; Wang, Weifeng; Ballandras-Colas, Allison; Patel, Pratiq A; Fuchs, James R; Kvaratskhelia, Mamuka; Engelman, Alan

2013-05-21

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities*

PubMed Central

Lim, Siew Pheng; Koh, Jolene Hong Kiew; Seh, Cheah Chen; Liew, Chong Wai; Davidson, Andrew D.; Chua, Leng Shiew; Chandrasekaran, Ramya; Cornvik, Tobias C.; Shi, Pei-Yong; Lescar, Julien

2013-01-01

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp. PMID:24025331

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guan, Rongjin; Aiyer, Sriram; Cote, Marie L.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structuresmore » of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.« less

Surface damage in cystine, an amino acid dimer, induced by keV ions.

PubMed

Salles, R C M; Coutinho, L H; da Veiga, A G; Sant'Anna, M M; de Souza, G G B

2018-01-28

We have studied the interaction of an ion beam (17.6 keV F - ) with cystine, a dimer formed by the binding of two cysteine residues. Cystine can be considered as an ideal prototype for the study of the relevance of the disulfide (-S-S-) chemical bond in biomolecules. For the sake of comparison, the amino acid cysteine has also been subjected to the same experimental conditions. Characterization of the samples by XPS and NEXAFS shows that both pristine cystine and pristine cysteine are found as a dipolar ion (zwitterion). Following irradiation, the dimer and the amino acid show a tendency to change from the dipole ion form to the normal uncharged form. The largest spectral modification was observed in the high resolution XPS spectra obtained at around the N 1s core level for the two biomolecules. The 2p sulfur edge spectra of cysteine and cystine were much less sensitive to radiation effects. We suggest that the disulfide bond (-S-S-) remains stable before and after irradiation, contributing to the larger radiation stability of cystine as compared to the amino acid cysteine.

On the scattering directionality of a dielectric particle dimer of High Refractive Index.

PubMed

Barreda, Ángela I; Saleh, Hassan; Litman, Amélie; González, Francisco; Geffrin, Jean-Michel; Moreno, Fernando

2018-05-22

Low-losses and directionality effects exhibited by High Refractive Index Dielectric particles make them attractive for applications where radiation direction control is relevant. For instance, isolated metallo-dielectric core-shell particles or aggregates (dimers) of High Refractive Index Dielectric particles have been proposed for building operational switching devices. Also, the possibility of using isolated High Refractive Index Dielectric particles for optimizing solar cells performance has been explored. Here, we present experimental evidence in the microwave range, that a High Refractive Index Dielectric dimer of spherical particles is more efficient for redirecting the incident radiation in the forward direction than the isolated case. In fact, we report two spectral regions in the dipolar spectral range where the incident intensity is mostly scattered in the forward direction. They correspond to the Zero-Backward condition (also observed for isolated particles) and to a new condition, denoted as "near Zero-Backward" condition, which comes from the interaction effects between the particles. The proposed configuration has implications in solar energy harvesting devices and in radiation guiding.

A theoretical study of the photoinduced desorption of I — from a CF3I dimer

NASA Astrophysics Data System (ADS)

Tossell, J. A.

1997-04-01

Ab initio SCF-MO calculations using effective core-potential basis sets are employed to evaluate ionization potentials and electron affinities for CF3I and the geometries and energies of the singlet and triplet states of the CF3I dimer. The calculated geometry of the single state of the dimer is in qualitative agreement with the experimental geometry for condensed phase CF3I. The calculated energy for vertical excitation from the singlet to the triplet state is 4.1 eV at the Hartree-Fock level and 4.5 eV after incorporation of correlation at the Moller-Plesset 2nd-order level, consistent with excitation by 193 nm (6.4 eV) light. The equilibrium geometry of the triplet consists essentially of a CF3I+, CF3I- ion pair, in which the Csbnd I bond distance in the anionic component has increased to 5.5Å, compared with 2.1Åin the neutral molecule. The calculated binding energy of the triplet ion pair is about 4 eV.

A unique chromatin complex occupies young α-satellite arrays of human centromeres

PubMed Central

Henikoff, Jorja G.; Thakur, Jitendra; Kasinathan, Sivakanthan; Henikoff, Steven

2015-01-01

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes. PMID:25927077

Enantioselective Total Syntheses of (−)-Palau’amine, (−)- Axinellamines, and (−)-Massadines

PubMed Central

Seiple, Ian B.; Su, Shun; Young, Ian S.; Nakamura, Akifumi; Yamaguchi, Junichiro; Jørgensen, Lars; Rodriguez, Rodrigo A.; O’Malley, Daniel P.; Gaich, Tanja; Köck, Matthias; Baran, Phil S.

2011-01-01

Dimeric pyrrole-imidazole alkaloids represent a rich and topologically unique class of marine natural products. This full account will follow the progression of efforts that culminated in the enantioselective total syntheses of the most structurally ornate members of this family: the axinellamines, the massadines, and palau’amine. A bio-inspired approach capitalizing on the pseudo-symmetry of the members of this class is recounted, delivering a deschloro derivative of the natural product core. Next, the enantioselective synthesis of the chlorocyclopentane core featuring a scalable, catalytic, enantioselective Diels–Alder reaction of a 1-siloxydiene is outlined in detail. Finally, the successful divergent conversion of this core to each of the aforementioned natural products, and the ensuing methodological developments are described. PMID:21861522

Evaluation of Hepatitis B Virus Photoinactivation in Serum and Cellular Blood Components by the Polymerase Chain Reaction.

DTIC Science & Technology

1992-08-01

and there is no test for the disease that has yet to be discovered. This situation occurred with the human immunodeficiency virus (HIV). This virus...antibodies to human immunodeficiency virus I (anti-HIV-1), antibodies to hepatitis C virus (anti-HCV), antibodies to hepatitis B core (anti-HBc) and a...photoinactivation have used model virus systems to measure the effectiveness of the inactivation procedure. These viruses include feline leukemia

Comparative activities of several nucleoside analogs against duck hepatitis B virus in vitro.

PubMed Central

Yokota, T; Konno, K; Chonan, E; Mochizuki, S; Kojima, K; Shigeta, S; de Clercq, E

1990-01-01

Duck hepatitis B virus (DHBV) replication in primary duck hepatocytes was monitored by examining the synthesis of both DHBV DNA and DHBV core antigen. Several nucleoside analogs which were previously shown to inhibit the replication of DNA viruses (i.e., herpesviruses) and retroviruses were examined for their inhibitory effects on the synthesis of DHBV core antigen in primary duck hepatocytes. (S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine, 2',3'-dideoxyadenosine, and 2',3'-dideoxycytidine inhibited DHBV core antigen synthesis at concentrations that were significantly lower than those found to be toxic to the primary hepatocytes. Of all the compounds tested, (S)-HPMPA showed the lowest 50% effective concentration (0.5 micrograms/ml). The selectivity index or ratio of the 50% cytotoxic concentration to the 50% effective concentration of (S)-HPMPA was greater than 300. (S)-HPMPA not only inhibited DHBV core antigen but also DHBV DNA synthesis in DHBV-infected hepatocytes. PMID:2201250

Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

PubMed Central

Zhao, Bingchun; Zhang, Xiao; Krummenacher, Claude; Song, Shuo; Gao, Ling; Zhang, Haojiong; Xu, Miao; Feng, Lin; Feng, Qisheng; Zeng, Musheng; Xu, Yuting; Zeng, Yixin

2018-01-01

Epstein–Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb) and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications. PMID:29765376

The herpesvirus proteases as targets for antiviral chemotherapy.

PubMed

Waxman, L; Darke, P L

2000-01-01

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.

PubMed

Del Vecchio, Kathryn; Frick, Cary T; Gc, Jeevan B; Oda, Shun-Ichiro; Gerstman, Bernard S; Saphire, Erica Ollmann; Chapagain, Prem P; Stahelin, Robert V

2018-03-02

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures ( i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys 224, 225 and Lys 274, 275 ). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

PubMed Central

Hansen, M S; Barklis, E

1995-01-01

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

African Swine Fever Virus Gets Undressed: New Insights on the Entry Pathway.

PubMed

Andrés, Germán

2017-02-15

African swine fever virus (ASFV) is a large, multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid, and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm. Copyright © 2017 American Society for Microbiology.

The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

PubMed Central

Zheng, Wenjie; Olson, John; Vakharia, Vikram; Tao, Yizhi Jane

2013-01-01

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. PMID:24068932

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site

PubMed Central

Liang, Chen; Rong, Liwei; Quan, Yudong; Laughrea, Michael; Kleiman, Lawrence; Wainberg, Mark A.

1999-01-01

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard. PMID:10400801

Variation of the Specificity of the Human Antibody Responses after Tick-Borne Encephalitis Virus Infection and Vaccination

PubMed Central

Jarmer, Johanna; Zlatkovic, Jürgen; Tsouchnikas, Georgios; Vratskikh, Oksana; Strauß, Judith; Aberle, Judith H.; Chmelik, Vaclav; Kundi, Michael; Stiasny, Karin

2014-01-01

ABSTRACT Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCE Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines. PMID:25253341

Variation of the specificity of the human antibody responses after tick-borne encephalitis virus infection and vaccination.

PubMed

Jarmer, Johanna; Zlatkovic, Jürgen; Tsouchnikas, Georgios; Vratskikh, Oksana; Strauß, Judith; Aberle, Judith H; Chmelik, Vaclav; Kundi, Michael; Stiasny, Karin; Heinz, Franz X

2014-12-01

Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

Cancer.gov

The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of

Transmission and pathogenesis of vesicular stomatitis viruses

USDA-ARS?s Scientific Manuscript database

Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.

PubMed

Chek, Min Fey; Kim, Sun-Yong; Mori, Tomoyuki; Arsad, Hasni; Samian, Mohammed Razip; Sudesh, Kumar; Hakoshima, Toshio

2017-07-13

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

Direct Visualization of an Impurity Depletion Zone

NASA Technical Reports Server (NTRS)

Chernov, Alex A.; Garcia-Ruiz, Juan Ma; Thomas, Bill R.

2000-01-01

When a crystal incorporates more impurity per unit of its volume than the impurity concentration in solution, the solution in vicinity of the growing crystal is depleted with respect to the impurity I,2. With a stagnant solution, e. g. in microgravity or gels, an impurity depletion zone expands as the crystal grows and results in greater purity in most of the outer portion of the crystal than in the core. Crystallization in gel provides an opportunity to mimic microgravity conditions and visualize the impurity depletion zone. Colorless, transparent apoferritin (M congruent to 450 KDa) crystals were grown in the presence of red holoferritin dimer as a microheterogeneous impurity (M congruent to 900 KDa) within agarose gel by counterdiffusion with Cd(2+) precipitant. Preferential trapping of dimers, (distribution coefficient K = 4 (exp 1,2)) results in weaker red color around the crystals grown in the left tube in the figure as compared to the control middle tube without crystals. The left and the middle tubes contain colored ferritin dimers, the right tube contains colored trimers. The meniscus in the left tube separate gel (below) and liquid solution containing Cd(2+) (above). Similar solutions, though without precipitants, were present on top of the middle and right tube allowing diffusion of dimers and trimers. The area of weaker color intensity around crystals directly demonstrates overlapped impurity depletion zones.

Development of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus.

PubMed

Rao, Xueqin; Sun, Jie

2015-09-01

Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus , causes significant loss in Cucurbitaceae plants. Development of a highly sensitive and reliable detection method for WSMoV. Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27 watermelon samples showing WSMoV infection symptoms. The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and melting curves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Capsicum chlorosis virus (genus Tospovirus ) and Cucumber mosaic virus (genus Cucumovirus). Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%. A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detection of WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

PubMed Central

Ziv-Av, Amotz; Gerber, Doron; Jacob, Yves; Einav, Shirit

2012-01-01

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development. PMID:22916011

Stepwise Priming by Acidic pH and a High K+ Concentration Is Required for Efficient Uncoating of Influenza A Virus Cores after Penetration

PubMed Central

Feng, Yuehan; Nebioglu, Firat; Heilig, Rosalie; Picotti, Paola

2014-01-01

ABSTRACT Influenza A virus (IAV) uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the prefusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to a pH below 6.5. The second step is triggered by a pH of <6.0 and by the influx of K+ ions. It causes additional changes in M1 as well as a loss of stability in the viral ribonucleoprotein bundle. Our results indicate that both the switch from Na+ to K+ in maturing endosomes and the decreasing pH are needed to prime IAV cores for efficient uncoating and infection of the host cell. IMPORTANCE The entry of IAV involves several steps, including endocytosis and fusion at late endosomes. Entry also includes disassembly of the viral core, which is composed of the viral ribonucleoproteins and the RNA genome. We have found that the uncoating process of IAV is initiated long before the core is delivered into the cytosol. M2, an ion channel in the viral membrane, is activated when the virus passes through early endosomes. Here, we show that protons entering the virus through M2 cause a conformational change in the matrix protein, M1. This weakens interactions between M1 and the viral ribonucleoproteins. A second change was found to occur when the virus enters late endosomes. The preacidified core is then exposed to a high concentration of K+, which affects the interactions between the ribonucleoproteins. Thus, when cores are finally delivered to the cytosol, they are already partially destabilized and, therefore, uncoating competent and infectious. PMID:25165113

A mechanism for histone chaperoning activity of nucleoplasmin: thermodynamic and structural models.

PubMed

Taneva, Stefka G; Bañuelos, Sonia; Falces, Jorge; Arregi, Igor; Muga, Arturo; Konarev, Petr V; Svergun, Dmitri I; Velázquez-Campoy, Adrián; Urbaneja, María A

2009-10-23

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different "affinity windows" for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.

X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bornholdt, Zachary A.; Prasad, B.V. Venkataram

2009-04-08

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains - a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker - is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60%more » of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-{angstrom}-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.« less

High-resolution structure of a retroviral protease folded as a monomer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilski, Miroslaw; Polish Academy of Sciences, 61-704 Poznan; Kazmierczyk, Maciej

2011-11-01

The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerizationmore » of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C{sup α} deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.« less

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

NASA Astrophysics Data System (ADS)

Xie, Bo; Luo, Xiaoyang; Zhao, Cheng; Priest, Christina Marie; Chan, Shiu-Yung; O'Connor, Peter B.; Kirschner, Daniel A.; Costello, Catherine E.

2007-12-01

Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS compact myelin.

Attenuation of Marek's disease virus by codon pair deoptimization of a core gene

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus of Gallus gallus, the domesticated chicken. Control strategies rely upon comprehensive vaccination in ovo with live attenuated virus vaccines consisting of antigenically similar avian herpesviruses or attenuated strains of MDV. Recent stud...

In vitro replication of poliovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

Dimerization of a Viral SET Protein Endows its Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Wei; M Zhou

Histone modifications are regarded as the most indispensible phenomena in epigenetics. Of these modifications, lysine methylation is of the greatest complexity and importance as site- and state-specific lysine methylation exerts a plethora of effects on chromatin structure and gene transcription. Notably, paramecium bursaria chlorella viruses encode a conserved SET domain methyltransferase, termed vSET, that functions to suppress host transcription by methylating histone H3 at lysine 27 (H3K27), a mark for eukaryotic gene silencing. Unlike mammalian lysine methyltransferases (KMTs), vSET functions only as a dimer, but the underlying mechanism has remained elusive. In this study, we demonstrate that dimeric vSET operatesmore » with negative cooperativity between the two active sites and engages in H3K27 methylation one site at a time. New atomic structures of vSET in the free form and a ternary complex with S-adenosyl homocysteine and a histone H3 peptide and biochemical analyses reveal the molecular origin for the negative cooperativity and explain the substrate specificity of H3K27 methyltransferases. Our study suggests a 'walking' mechanism, by which vSET acts all by itself to globally methylate host H3K27, which is accomplished by the mammalian EZH2 KMT only in the context of the Polycomb repressive complex.« less

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

PubMed Central

Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

2016-01-01

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

Conformational dynamics of cancer-associated MyD88-TIR domain mutant L252P (L265P) allosterically tilts the landscape toward homo-dimerization

PubMed Central

Zhan, Chendi; Qi, Ruxi; Wei, Guanghong; Guven-Maiorov, Emine; Nussinov, Ruth; Ma, Buyong

2016-01-01

MyD88 is an essential adaptor protein, which mediates the signaling of the toll-like and interleukin-1 receptors’ superfamily. The MyD88 L252P (L265P) mutation has been identified in diffuse large B-cell lymphoma. The identification of this mutation has been a major advance in the diagnosis of patients with aldenstrom macroglobulinemia and related lymphoid neoplasms. Here we used computational methods to characterize the conformational effects of the mutation. Our molecular dynamics simulations revealed that the mutation allosterically quenched the global conformational dynamics of the toll/IL-1R (TIR) domain, and readjusted its salt bridges and dynamic community network. Specifically, the mutation changed the orientation and reduced the fluctuation of α-helix 3, possibly through eliminating/weakening ~8 salt bridges and enhancing the salt bridge D225-K258. Using the energy landscape of the TIR domains of MyD88, we identified two dynamic conformational basins, which correspond to the binding sites used in homo- and hetero-oligomerization, respectively. Our results indicate that the mutation stabilizes the core of the homo-dimer interface of the MyD88-TIR domain, and increases the population of homo-dimer-compatible conformational states in MyD88 family proteins. However, the dampened motion restricts its ability to heterodimerize with other TIR domains, thereby curtailing physiological signaling. In conclusion, the L252P both shifts the landscape toward homo-dimerization and restrains the dynamics of the MyD88-TIR domain, which disfavors its hetero-dimerization with other TIR domains. We further put these observations within the framework of MyD88-mediated cell signaling. PMID:27503954

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

PubMed Central

Stewart, H.; Bingham, R.J.; White, S. J.; Dykeman, E. C.; Zothner, C.; Tuplin, A. K.; Stockley, P. G.; Twarock, R.; Harris, M.

2016-01-01

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly. PMID:26972799

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

PubMed

Tharakaraman, Kannan; Watanabe, Satoru; Chan, Kuan Rong; Huan, Jia; Subramanian, Vidya; Chionh, Yok Hian; Raguram, Aditya; Quinlan, Devin; McBee, Megan; Ong, Eugenia Z; Gan, Esther S; Tan, Hwee Cheng; Tyagi, Anu; Bhushan, Shashi; Lescar, Julien; Vasudevan, Subhash G; Ooi, Eng Eong; Sasisekharan, Ram

2018-05-09

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks. Copyright © 2018. Published by Elsevier Inc.

Integrated oxide graphene based device for laser inactivation of pathogenic microorganisms

NASA Astrophysics Data System (ADS)

Grishkanich, Alexsandr; Ruzankina, Julia; Afanasyev, Mikhail; Paklinov, Nikita; Hafizov, Nail

2018-02-01

We develop device for virus disinfection of pathogenic microorganisms. Viral decontamination can be carried out due to hard ultraviolet irradiation and singlet oxygen destroying the genetic material of a virus capsid. UV rays can destroy DNA, leading to the formation of dimers of nucleic acids. This practically does not occur in tissues, tk. UV rays penetrate badly through them, however, the viral particles are small and UV can destroy their genetic material, RNA / DNA and the virus can not replicate. It is with the construction of the ultraviolet laser water disinfection system (UFLOV) based on the continuous and periodic pulsed ultraviolet laser sources (pump) binds to solve sterility and depyrogenation of water. It has been established that small doses of UV irradiation stimulate reproduction, and large doses cause the death of pathogenic microorganisms. The effect of a dose of ultraviolet is the result of photochemical action on the substance of a living bacterial cell or virion. Also complex photodynamic laser inactivation on graphene oxide is realized.

Mechanism of Fusion Triggering by Human Parainfluenza Virus Type III

PubMed Central

Porotto, Matteo; Palmer, Samantha G.; Palermo, Laura M.; Moscona, Anne

2012-01-01

Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection. PMID:22110138

Crystal structure of PAV1-137: a protein from the virus PAV1 that infects Pyrococcus abyssi.

PubMed

Leulliot, N; Quevillon-Cheruel, S; Graille, M; Geslin, C; Flament, D; Le Romancer, M; van Tilbeurgh, H

2013-01-01

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four- α -helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Structural basis of GM-CSF and IL-2 sequestration by the viral decoy receptor GIF

PubMed Central

Felix, Jan; Kandiah, Eaazhisai; De Munck, Steven; Bloch, Yehudi; van Zundert, Gydo C.P.; Pauwels, Kris; Dansercoer, Ann; Novanska, Katka; Read, Randy J.; Bonvin, Alexandre M.J.J.; Vergauwen, Bjorn; Verstraete, Kenneth; Gutsche, Irina; Savvides, Savvas N.

2016-01-01

Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses. PMID:27819269

Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules.

PubMed

Paximadis, M; Rey, M E

2001-12-01

The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

Genome-Scale Phylogeny of the Alphavirus Genus Suggests a Marine Origin

PubMed Central

Palacios, G.; Tesh, R. B.; Savji, N.; Guzman, H.; Sherman, M.; Weaver, S. C.; Lipkin, W. I.

2012-01-01

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers. PMID:22190718

Proteins of Vasicular Stomatitis Virus

PubMed Central

Kang, C. Y.; Prevec, L.

1969-01-01

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with 14C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2. PMID:4306195

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

PubMed Central

Hernáez, Bruno; Guerra, Milagros; Salas, María L.

2016-01-01

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

Genotype diversity of hepatitis C virus (HCV) in HCV-associated liver disease patients in Indonesia.

PubMed

Utama, Andi; Tania, Navessa Padma; Dhenni, Rama; Gani, Rino Alvani; Hasan, Irsan; Sanityoso, Andri; Lelosutan, Syafruddin A R; Martamala, Ruswhandi; Lesmana, Laurentius Adrianus; Sulaiman, Ali; Tai, Susan

2010-09-01

Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry

NASA Astrophysics Data System (ADS)

Pang, Xueqin; Jia, Chenxi; Chen, Zhengwei; Li, Lingjun

2017-01-01

The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.

Diphenylphenoxy-Thiophene-PDI Dimers as Acceptors for OPV Applications with Open Circuit Voltage Approaching 1 Volt.

PubMed

Stenta, Caterina; Molina, Desiré; Viterisi, Aurélien; Montero-Rama, María Pilar; Pla, Sara; Cambarau, Werther; Fernández-Lázaro, Fernando; Palomares, Emilio; Marsal, Lluis F; Sastre-Santos, Ángela

2018-03-30

Two new perylenediimides (PDIs) have been developed for use as electron acceptors in solution-processed bulk heterojunction solar cells. The compounds were designed to exhibit maximal solubility in organic solvents, and reduced aggregation in the solid state. In order to achieve this, diphenylphenoxy groups were used to functionalize a monomeric PDI core, and two PDI dimers were bridged with either one or two thiophene units. In photovoltaic devices prepared using PDI dimers and a monomer in conjunction with PTB7, it was found that the formation of crystalline domains in either the acceptor or donor was completely suppressed. Atomic force microscopy, X-ray diffraction, charge carrier mobility measurements and recombination kinetics studies all suggest that the lack of crystallinity in the active layer induces a significant drop in electron mobility. Significant surface recombination losses associated with a lack of segregation in the material were also identified as a significant loss mechanism. Finally, the monomeric PDI was found to have sub-optimum LUMO energy matching the cathode contact, thus limiting charge carrier extraction. Despite these setbacks, all PDIs produced high open circuit voltages, reaching almost 1 V in one particular case.

Pseudopotentials for quantum Monte Carlo studies of transition metal oxides

DOE PAGES

Krogel, Jaron T.; Santana Palacio, Juan A.; Reboredo, Fernando A.

2016-02-22

Quantum Monte Carlo (QMC) calculations of transition metal oxides are partially limited by the availability of high-quality pseudopotentials that are both accurate in QMC and compatible with major plane-wave electronic structure codes. We have generated a set of neon-core pseudopotentials with small cutoff radii for the early transition metal elements Sc to Zn within the local density approximation of density functional theory. The pseudopotentials have been directly tested for accuracy within QMC by calculating the first through fourth ionization potentials of the isolated transition metal (M) atoms and the binding curve of each M-O dimer. We find the ionization potentialsmore » to be accurate to 0.16(1) eV, on average, relative to experiment. The equilibrium bond lengths of the dimers are within 0.5(1)% of experimental values, on average, and the binding energies are also typically accurate to 0.18(3) eV. The level of accuracy we find for atoms and dimers is comparable to what has recently been observed for bulk metals and oxides using the same pseudopotentials. Our QMC pseudopotential results compare well with the findings of previous QMC studies and benchmark quantum chemical calculations.« less

Chemistry of chlorinated species in the Antarctic stratosphere

NASA Technical Reports Server (NTRS)

Molina, Mario J.; Tso, Tai-Ly; Wang, Frank C.-Y.

1988-01-01

The chemistry of Cl sub 2 O sub 2, the chlorine monoxide dimer, has been further investigated in order to better asses its potential role in catalytic ozone destruction cycles. The dimer has been generated in a flow system, in the 200 to 250 K temperature range, by using ozone and chlorine atoms as ClO precursors. The Cl-atoms are produced by a microwave discharge of either Cl sub 2, or of F sub 2 with subsequent addition of HCl. With this later scheme the dimer can be generated in the absence of Cl sub 2. The Fourier transform infrared spectra of the products clearly indicates the presence of two isomers, in agreement with earlier results (J. Phys. Shen., 91, 433, 1987). None of the observed IR bands can be attributed to a ClO-OClO adduct, since they all appear in the absence of any detectable amount of OCl. It is likely that the particles in the polar stratospheric clouds will have a relatively dilute nitric acid outer layer, even if the core is the nitric acid monohydrate, since the particles are in equilibrium with the ambient water vapor, which is present at levels of a few parts per million.

Adsorption equilibrium and kinetics of monomer-dimer monoclonal antibody mixtures on a cation exchange resin.

PubMed

Reck, Jason M; Pabst, Timothy M; Hunter, Alan K; Wang, Xiangyang; Carta, Giorgio

2015-07-10

Adsorption equilibrium and kinetics are determined for a monoclonal antibody (mAb) monomer and dimer species, individually and in mixtures, on a macroporous cation exchange resin both under the dilute limit of salt gradient elution chromatography and at high protein loads and low salt based on batch adsorption equilibrium and confocal laser scanning microscopy (CLSM) experiments. In the dilute limit and weak binding conditions, the dimer/monomer selectivity in 10mM phosphate at pH 7 varies between 8.7 and 2.3 decreasing with salt concentration in the range of 170-230mM NaCl. At high protein loads and strong binding conditions (0-60mM NaCl), the selectivity in the same buffer is near unity with no NaCl added, but increases gradually with salt concentration reaching high values between 2 and 15 with 60mM added NaCl. For these conditions, the two-component adsorption kinetics is controlled by pore diffusion and is predicted approximately by a dual shrinking core model using parameters based on single component equilibrium and kinetics measurements. Copyright © 2015 Elsevier B.V. All rights reserved.

Pandoraviruses are highly derived phycodnaviruses

PubMed Central

2013-01-01

The recently discovered Pandoraviruses are by far the largest viruses known, with their 2 megabase genomes exceeding in size the genomes of numerous bacteria and archaea. Pandoraviruses show a distant relationship with other nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes, lack some of the NCLDV core genes and in particular do not appear to be specifically related to the other, better characterized family of giant viruses, the Mimiviridae. Here we report phylogenetic analysis of 6 core NCLDV genes that confidently places Pandoraviruses within the family Phycodnaviridae, with an apparent specific affinity with Coccolithoviruses. We conclude that, despite their many unusual characteristics, Pandoraviruses are highly derived phycodnaviruses. These findings imply that giant viruses have independently evolved from smaller NCLDV on at least two occasions. This article was reviewed by Patrick Forterre and Lakshminarayan Iyer. For the full reviews, see the Reviewers’ reports section. PMID:24148757

RNA-dependent RNA polymerases of dsRNA bacteriophages.

PubMed

Makeyev, Eugene V; Grimes, Jonathan M

2004-04-01

Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, Ayako; Okamura, Hideyasu; Morishita, Ryo

2012-08-24

Highlights: Black-Right-Pointing-Pointer Protease (PR) of XMR virus (XMRV) was successfully synthesized with cell-free system. Black-Right-Pointing-Pointer Interface of XMRV PR with an inhibitor, amprenavir (APV), was identified with NMR. Black-Right-Pointing-Pointer Structural heterogeneity is induced for two PR protomers in the APV:PR = 1:2 complex. Black-Right-Pointing-Pointer Structural heterogeneity is transmitted even to distant regions from the interface. Black-Right-Pointing-Pointer Long-range transmission of structural change may be utilized for drug discovery. -- Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nudemore » mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially {sup 15}N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [{sup 1}H, {sup 15}N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) . A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.« less

A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior

PubMed Central

Estácio, Sílvia G.; Krobath, Heinrich; Vila-Viçosa, Diogo; Machuqueiro, Miguel; Shakhnovich, Eugene I.; Faísca, Patrícia F. N.

2014-01-01

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis. PMID:24809460

Delocalized and localized states of eg electrons in half-doped manganites.

PubMed

Winkler, E L; Tovar, M; Causa, M T

2013-07-24

We have studied the magnetic behaviour of half-doped manganite Y0.5Ca0.5MnO3 in an extended range of temperatures by means of magnetic susceptibility, χ(T), and electron spin resonance (ESR) experiments. At high temperature the system crystallizes in an orthorhombic structure. The resistivity value, ρ ≃ 0.05 Ω cm at 500 K, indicates a metallic behaviour, while the Curie-Weiss dependence of χ(T) and the thermal evolution of the ESR parameters are very well described by a model that considers a system conformed by localized Mn(4+) cores, [Formula: see text], and itinerant, eg, electrons. The strong coupling between t2g and eg electrons results in an enhanced Curie constant and an FM Curie-Weiss temperature that overcomes the AFM interactions between the [Formula: see text] cores. A transition to a more distorted phase is observed at T ≈ 500 K and signatures of localization of the eg electrons appear in the χ(T) behaviour below 300 K. A new Curie-Weiss regime is observed, where the Curie-constant value is consistent with dimer formation. Based on mean-field calculations, the dimer formation is predicted as a function of the interaction strength between the t2g and eg electrons.

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid.

PubMed

Benevides, James M; Juuti, Jarmo T; Tuma, Roman; Bamford, Dennis H; Thomas, George J

2002-10-08

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.

Gene repertoire of amoeba-associated giant viruses.

PubMed

Colson, Philippe; Raoult, Didier

2010-01-01

Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. Copyright 2010 S. Karger AG, Basel.

Unexpected and novel putative viruses in the sediments of a deep-dark permanently anoxic freshwater habitat.

PubMed

Borrel, Guillaume; Colombet, Jonathan; Robin, Agnès; Lehours, Anne-Catherine; Prangishvili, David; Sime-Ngando, Télesphore

2012-11-01

Morphological diversity, abundance and community structure of viruses were examined in the deep and anoxic sediments of the volcanic Lake Pavin (France). The sediment core, encompassing 130 years of sedimentation, was subsampled every centimeter. High viral abundances were recorded and correlated to prokaryotic densities. Abundances of viruses and prokaryotes decreased with the depth, contrasting the pattern of virus-to-prokaryote ratio. According to fingerprint analyses, the community structure of viruses, bacteria and archaea gradually changed, and communities of the surface (0-10 cm) could be discriminated from those of the intermediate (11-27 cm) and deep (28-40 cm) sediment layers. Viral morphotypes similar to virions of ubiquitous dsDNA viruses of bacteria were observed. Exceptional morphotypes, previously never reported in freshwater systems, were also detected. Some of these resembled dsDNA viruses of hyperthermophilic and hyperhalophilic archaea. Moreover, unusual types of spherical and cubic virus-like particles (VLPs) were observed. Infected prokaryotic cells were detected in the whole sediment core, and their vertical distribution correlated with both viral and prokaryotic abundances. Pleomorphic ellipsoid VLPs were visible in filamentous cells tentatively identified as representatives of the archaeal genus Methanosaeta, a major group of methane producers on earth.

Unexpected and novel putative viruses in the sediments of a deep-dark permanently anoxic freshwater habitat

PubMed Central

Borrel, Guillaume; Colombet, Jonathan; Robin, Agnès; Lehours, Anne-Catherine; Prangishvili, David; Sime-Ngando, Télesphore

2012-01-01

Morphological diversity, abundance and community structure of viruses were examined in the deep and anoxic sediments of the volcanic Lake Pavin (France). The sediment core, encompassing 130 years of sedimentation, was subsampled every centimeter. High viral abundances were recorded and correlated to prokaryotic densities. Abundances of viruses and prokaryotes decreased with the depth, contrasting the pattern of virus-to-prokaryote ratio. According to fingerprint analyses, the community structure of viruses, bacteria and archaea gradually changed, and communities of the surface (0–10 cm) could be discriminated from those of the intermediate (11–27 cm) and deep (28–40 cm) sediment layers. Viral morphotypes similar to virions of ubiquitous dsDNA viruses of bacteria were observed. Exceptional morphotypes, previously never reported in freshwater systems, were also detected. Some of these resembled dsDNA viruses of hyperthermophilic and hyperhalophilic archaea. Moreover, unusual types of spherical and cubic virus-like particles (VLPs) were observed. Infected prokaryotic cells were detected in the whole sediment core, and their vertical distribution correlated with both viral and prokaryotic abundances. Pleomorphic ellipsoid VLPs were visible in filamentous cells tentatively identified as representatives of the archaeal genus Methanosaeta, a major group of methane producers on earth. PMID:22648129

Structure of large dsDNA viruses

PubMed Central

Klose, Thomas; Rossmann, Michael G.

2015-01-01

Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host. PMID:25003382

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction.

PubMed

Chareza, Sarah; Slavkovic Lukic, Dragana; Liu, Yang; Räthe, Ann-Mareen; Münk, Carsten; Zabogli, Elisa; Pistello, Mauro; Löchelt, Martin

2012-03-15

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable. Copyright © 2012 Elsevier Inc. All rights reserved.

Crystal Structure of the Oligomeric Form of Lassa Virus Matrix Protein Z.

PubMed

Hastie, Kathryn M; Zandonatti, Michelle; Liu, Tong; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

2016-05-01

The arenavirus matrix protein Z is highly multifunctional and occurs in both monomeric and oligomeric forms. The crystal structure of a dodecamer of Z from Lassa virus, presented here, illustrates a ring-like structure with a highly basic center. Mutagenesis demonstrates that the dimeric interface within the dodecamer and a Lys-Trp-Lys triad at the center of the ring are important for oligomerization. This structure provides an additional template to explore the many functions of Z. The arenavirus Lassa virus causes hundreds of thousands of infections each year, many of which develop into fatal hemorrhagic fever. The arenavirus matrix protein Z is multifunctional, with at least four distinct roles. Z exists in both monomeric and oligomeric forms, each of which likely serves a specific function in the viral life cycle. Here we present the dodecameric form of Lassa virus Z and demonstrate that Z forms a "wreath" with a highly basic center. This structure and that of monomeric Z now provide a pair of critical templates by which the multiple roles of Z in the viral life cycle may be interpreted. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Non-active site mutations disturb the loop dynamics, dimerization, viral budding and egress of VP40 of the Ebola virus.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-02-28

The first account of the dynamic features of the loop region of VP40 of the Ebola virus (EboV) using accelerated molecular dynamics (aMD) simulations is reported herein. Due to its major role in the Ebola life cycle, VP40 is considered a promising therapeutic target. The available experimental data on the N-terminal domain (NTD) loop indicates that mutations K127A, T129A and N130A demonstrate an unrecognized role for NTD-plasma membrane (PM) interaction for efficient VP40-PM localization, oligomerization, matrix assembly and egress. Despite experimental results, the molecular description of VP40 and the information it can provide still remain vague. Therefore, to gain further molecular insight into the effect of mutations on the loop region of VP40 and its effects on the overall protein conformation and VP40 dimerization, aMD simulations and post-dynamic analyses were employed for wildtype (WT) and mutant systems. The results showed significant variations in the presence of mutations as per RMSF, RMSD, R g , PCA and distance calculations in comparison to the WT. These results could provide researchers with insight with regards to the conformational aspects concerning VP40 and its close relation to the experimental data. We believe that the results presented in this study will ultimately provide a useful understanding of the structural landscape of the loop region of VP40, which would contribute towards the discovery of novel EboV inhibitors.

Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.

PubMed

Pallesen, Jesper; Murin, Charles D; de Val, Natalia; Cottrell, Christopher A; Hastie, Kathryn M; Turner, Hannah L; Fusco, Marnie L; Flyak, Andrew I; Zeitlin, Larry; Crowe, James E; Andersen, Kristian G; Saphire, Erica Ollmann; Ward, Andrew B

2016-08-08

The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

Hepatitis C virus core protein triggers abnormal porphyrin metabolism in human hepatocellular carcinoma cells.

PubMed

Nakano, Takafumi; Moriya, Kyoji; Koike, Kazuhiko; Horie, Toshiharu

2018-01-01

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

PubMed Central

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells. Conclusions Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN. PMID:22889230

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Genome variations associated with viral susceptibility and calcification in Emiliania huxleyi.

PubMed

Kegel, Jessica U; John, Uwe; Valentin, Klaus; Frickenhaus, Stephan

2013-01-01

Emiliania huxleyi, a key player in the global carbon cycle is one of the best studied coccolithophores with respect to biogeochemical cycles, climatology, and host-virus interactions. Strains of E. huxleyi show phenotypic plasticity regarding growth behaviour, light-response, calcification, acidification, and virus susceptibility. This phenomenon is likely a consequence of genomic differences, or transcriptomic responses, to environmental conditions or threats such as viral infections. We used an E. huxleyi genome microarray based on the sequenced strain CCMP1516 (reference strain) to perform comparative genomic hybridizations (CGH) of 16 E. huxleyi strains of different geographic origin. We investigated the genomic diversity and plasticity and focused on the identification of genes related to virus susceptibility and coccolith production (calcification). Among the tested 31940 gene models a core genome of 14628 genes was identified by hybridization among 16 E. huxleyi strains. 224 probes were characterized as specific for the reference strain CCMP1516. Compared to the sequenced E. huxleyi strain CCMP1516 variation in gene content of up to 30 percent among strains was observed. Comparison of core and non-core transcripts sets in terms of annotated functions reveals a broad, almost equal functional coverage over all KOG-categories of both transcript sets within the whole annotated genome. Within the variable (non-core) genome we identified genes associated with virus susceptibility and calcification. Genes associated with virus susceptibility include a Bax inhibitor-1 protein, three LRR receptor-like protein kinases, and mitogen-activated protein kinase. Our list of transcripts associated with coccolith production will stimulate further research, e.g. by genetic manipulation. In particular, the V-type proton ATPase 16 kDa proteolipid subunit is proposed to be a plausible target gene for further calcification studies.

Genome Variations Associated with Viral Susceptibility and Calcification in Emiliania huxleyi

PubMed Central

Kegel, Jessica U.; John, Uwe; Valentin, Klaus; Frickenhaus, Stephan

2013-01-01

Emiliania huxleyi, a key player in the global carbon cycle is one of the best studied coccolithophores with respect to biogeochemical cycles, climatology, and host-virus interactions. Strains of E. huxleyi show phenotypic plasticity regarding growth behaviour, light-response, calcification, acidification, and virus susceptibility. This phenomenon is likely a consequence of genomic differences, or transcriptomic responses, to environmental conditions or threats such as viral infections. We used an E. huxleyi genome microarray based on the sequenced strain CCMP1516 (reference strain) to perform comparative genomic hybridizations (CGH) of 16 E. huxleyi strains of different geographic origin. We investigated the genomic diversity and plasticity and focused on the identification of genes related to virus susceptibility and coccolith production (calcification). Among the tested 31940 gene models a core genome of 14628 genes was identified by hybridization among 16 E. huxleyi strains. 224 probes were characterized as specific for the reference strain CCMP1516. Compared to the sequenced E. huxleyi strain CCMP1516 variation in gene content of up to 30 percent among strains was observed. Comparison of core and non-core transcripts sets in terms of annotated functions reveals a broad, almost equal functional coverage over all KOG-categories of both transcript sets within the whole annotated genome. Within the variable (non-core) genome we identified genes associated with virus susceptibility and calcification. Genes associated with virus susceptibility include a Bax inhibitor-1 protein, three LRR receptor-like protein kinases, and mitogen-activated protein kinase. Our list of transcripts associated with coccolith production will stimulate further research, e.g. by genetic manipulation. In particular, the V-type proton ATPase 16 kDa proteolipid subunit is proposed to be a plausible target gene for further calcification studies. PMID:24260453

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Fluorescence biosensor based on CdTe quantum dots for specific detection of H5N1 avian influenza virus

NASA Astrophysics Data System (ADS)

Hoa Nguyen, Thi; Dieu Thuy Ung, Thi; Hien Vu, Thi; Tran, Thi Kim Chi; Quyen Dong, Van; Khang Dinh, Duy; Liem Nguyen, Quang

2012-09-01

This report highlights the fabrication of fluorescence biosensors based on CdTe quantum dots (QDs) for specific detection of H5N1 avian influenza virus. The core biosensor was composed of (i) the highly luminescent CdTe/CdS QDs, (ii) chromatophores extracted from bacteria Rhodospirillum rubrum, and (iii) the antibody of β-subunit. This core part was linked to the peripheral part of the biosensor via a biotin-streptavidin-biotin bridge and finally connected to the H5N1 antibody to make it ready for detecting H5N1 avian influenza virus. Detailed studies of each constituent were performed showing the image of QDs-labeled chromatophores under optical microscope, proper photoluminescence (PL) spectra of CdTe/CdS QDs, chromatophores and the H5N1 avian influenza viruses.

[Inverse PCR amplification of the complete major capsid protein gene of lymphocystis disease virus isolated from Rachycentron canadum and the phylogenetic analysis of the virus].

PubMed

Fu, Xiao-Zhe; Shi, Cun-Bin; Li, Ning-Qiu; Pan, Hou-Jun; Chang, Ou-Qin; Wu, Shu-Qin

2007-09-01

The major capsid protein of lymphocystis disease virus isolated from Rachycentron canadum (LCDV-rc) was amplified and analysed. The 457bp DNA core fragment was amplified with the degenerate primers designed according to the conserved sequences of MCP gene of iridoviruses, then the flaking sequences adjacent to the core region were amplified by inverse PCR, and the complete sequence was obtained by combining all of them. The open reading frame of the gene is 1380bp in length, encoding a putative protein of 459 aa with molecular weight 51.12 kD and pI 6.87. Constructing the phylogenetic tree for comparing the MCP amino acid of iridoviruses, the results indicated that LCDV-rc is most homologous to the other Lymphocystis viruses and all of them constitute a branch. Accordingly LCDV-rc is identified as Lymphocystivirus.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

PubMed

Moustafa, Savvina; Karakasiliotis, Ioannis; Mavromara, Penelope

2018-05-01

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis. IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression. Copyright © 2018 American Society for Microbiology.

A simple and efficient method for agroinfection of Vernonia cinerea with infectious clones of Vernonia yellow vein virus.

PubMed

Packialakshmi, R M; Usha, R

2011-12-01

Vernonia yellow vein virus (VeYVV) is a distinct monopartite begomovirus associated with a satellite DNA β. After constructing dimers of both DNA A and DNA β in binary vectors, a number of infection methods were attempted. However, only a modified stem-prick method produced up to 83% infection in the natural host Vernonia cinerea, thus, fulfilling the Koch's postulate. The presence of the viral DNA in the agroinfected plants was confirmed by rolling circle amplification (RCA), followed by Southern hybridization. DNA β induces typical symptoms of Vernonia yellow vein disease (VeYVD) when co-agroinoculated with the begomovirus to Vernonia and also leads to the accumulation of DNA A systemically. VeYVV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.

Real-time PCR probe optimization using design of experiments approach.

PubMed

Wadle, S; Lehnert, M; Rubenwolf, S; Zengerle, R; von Stetten, F

2016-03-01

Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.

Prevalence of antibodies to hepatitis B, hepatitis C, and HIV and risk factors in entrants to Irish prisons: a national cross sectional survey

PubMed Central

Long, Jean; Allwright, Shane; Barry, Joseph; Reynolds, Sheilagh Reaper; Thornton, Lelia; Bradley, Fiona; Parry, John V

2001-01-01

Objectives To determine the prevalence of antibodies to hepatitis B core antigen, hepatitis C virus, and HIV in entrants to Irish prisons and to examine risk factors for infection. Design Cross sectional, anonymous survey, with self completed risk factor questionnaire and oral fluid specimen for antibody testing. Setting Five of seven committal prisons in the Republic of Ireland. Participants 607 of the 718 consecutive prison entrants from 6 April to 1 May 1999. Main outcome measures Prevalence of antibodies to hepatitis B core antigen, hepatitis C virus, and HIV in prison entrants, and self reported risk factor status. Results Prevalence of antibodies to hepatitis B core antigen was 37/596 (6%; 95% confidence interval 4% to 9%), to hepatitis C virus was 130/596 (22%; 19% to 25%), and to HIV was 12/596 (2%; 1% to 4%). A third of the respondents had never previously been in prison; these had the lowest prevalence of antibodies to hepatitis B core antigen (4/197, 2%), to hepatitis C (6/197, 3%), and to HIV (0/197). In total 29% of respondents (173/593) reported ever injecting drugs, but only 7% (14/197) of those entering prison for the first time reported doing so compared with 40% (157/394) of those previously in prison. Use of injected drugs was the most important predictor of antibodies to hepatitis B core antigen and hepatitis C virus. Conclusions Use of injected drugs and infection with hepatitis C virus are endemic in Irish prisons. A third of prison entrants were committed to prison for the first time. Only a small number of first time entrants were infected with one or more of the viruses. These findings confirm the need for increased infection control and harm reduction measures in Irish prisons. What is already known on this topicHigh rates of using injected drugs, initiation of use of injected drugs, and sharing injecting equipment occur in Irish prisonsInjecting drug users have high rates of infection with hepatitis B and C viruses, and hepatitis C is endemic in injecting drug users and in Irish prisonersWhat this study addsThe prevalence of antibodies to hepatitis B core antigen, to hepatitis C, and to HIV in prison entrants who had previously been imprisoned was similar to that found in the recent national survey of Irish prisoners, but the prevalence of these antibodies was much lower in the third of prison entrants who had never previously been in prisonTattooing in prison is an independent risk factor for hepatitis C infection in prisoners who have never used injected drugs PMID:11719410

Complete Genome Viral Phylogenies Suggests the Concerted Evolution of Regulatory Cores and Accessory Satellites

PubMed Central

Zanotto, Paolo Marinho de Andrade; Krakauer, David C.

2008-01-01

We consider the concerted evolution of viral genomes in four families of DNA viruses. Given the high rate of horizontal gene transfer among viruses and their hosts, it is an open question as to how representative particular genes are of the evolutionary history of the complete genome. To address the concerted evolution of viral genes, we compared genomic evolution across four distinct, extant viral families. For all four viral families we constructed DNA-dependent DNA polymerase-based (DdDp) phylogenies and in addition, whole genome sequence, as quantitative descriptions of inter-genome relationships. We found that the history of the polymerase gene was highly predictive of the history of the genome as a whole, which we explain in terms of repeated, co-divergence events of the core DdDp gene accompanied by a number of satellite, accessory genetic loci. We also found that the rate of gene gain in baculovirus and poxviruses proceeds significantly more quickly than the rate of gene loss and that there is convergent acquisition of satellite functions promoting contextual adaptation when distinct viral families infect related hosts. The congruence of the genome and polymerase trees suggests that a large set of viral genes, including polymerase, derive from a phylogenetically conserved core of genes of host origin, secondarily reinforced by gene acquisition from common hosts or co-infecting viruses within the host. A single viral genome can be thought of as a mutualistic network, with the core genes acting as an effective host and the satellite genes as effective symbionts. Larger virus genomes show a greater departure from linkage equilibrium between core and satellites functions. PMID:18941535

Dynamic Diglyme-Mediated Self-Assembly of Gold Nanoclusters.

PubMed

Compel, W Scott; Wong, O Andrea; Chen, Xi; Yi, Chongyue; Geiss, Roy; Häkkinen, Hannu; Knappenberger, Kenneth L; Ackerson, Christopher J

2015-12-22

We report the assembly of gold nanoclusters by the nonthiolate ligand diglyme into discrete and dynamic assemblies. To understand this surprising phenomenon, the assembly of Au20(SC2H4Ph)15-diglyme into Au20(SC2H4Ph)15-diglyme-Au20(SC2H4Ph)15 is explored in detail. The assembly is examined by high-angle annular dark field scanning transmission electron microscopy, size exclusion chromatography, mass spectrometry, IR spectroscopy, and calorimetry. We establish a dissociation constant for dimer to monomer conversion of 20.4 μM. Theoretical models validated by transient absorption spectroscopy predict a low-spin monomer and a high-spin dimer, with assembly enabled through weak diglyme oxygen-gold interactions. Close spatial coupling allows electron delocalization between the nanoparticle cores. The resulting assemblies thus possess optical and electronic properties that emerge as a result of assembly.

Archaeal Haloarcula californiae Icosahedral Virus 1 Highlights Conserved Elements in Icosahedral Membrane-Containing DNA Viruses from Extreme Environments.

PubMed

Demina, Tatiana A; Pietilä, Maija K; Svirskaitė, Julija; Ravantti, Janne J; Atanasova, Nina S; Bamford, Dennis H; Oksanen, Hanna M

2016-07-19

Despite their high genomic diversity, all known viruses are structurally constrained to a limited number of virion morphotypes. One morphotype of viruses infecting bacteria, archaea, and eukaryotes is the tailless icosahedral morphotype with an internal membrane. Although it is considered an abundant morphotype in extreme environments, only seven such archaeal viruses are known. Here, we introduce Haloarcula californiae icosahedral virus 1 (HCIV-1), a halophilic euryarchaeal virus originating from salt crystals. HCIV-1 also retains its infectivity under low-salinity conditions, showing that it is able to adapt to environmental changes. The release of progeny virions resulting from cell lysis was evidenced by reduced cellular oxygen consumption, leakage of intracellular ATP, and binding of an indicator ion to ruptured cell membranes. The virion contains at least 12 different protein species, lipids selectively acquired from the host cell membrane, and a 31,314-bp-long linear double-stranded DNA (dsDNA). The overall genome organization and sequence show high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. Phylogenetic analysis based on the major conserved components needed for virion assembly-the major capsid proteins and the packaging ATPase-placed HCIV-1 along with the alphasphaerolipoviruses in a distinct, well-supported clade. On the basis of its virion morphology and sequence similarities, most notably, those of its core virion components, we propose that HCIV-1 is a member of the PRD1-adenovirus structure-based lineage together with other sphaerolipoviruses. This addition to the lineage reinforces the notion of the ancient evolutionary links observed between the viruses and further highlights the limits of the choices found in nature for formation of a virion. Under conditions of extreme salinity, the majority of the organisms present are archaea, which encounter substantial selective pressure, being constantly attacked by viruses. Regardless of the enormous viral sequence diversity, all known viruses can be clustered into a few structure-based viral lineages based on their core virion components. Our description of a new halophilic virus-host system adds significant insights into the largely unstudied field of archaeal viruses and, in general, of life under extreme conditions. Comprehensive molecular characterization of HCIV-1 shows that this icosahedral internal membrane-containing virus exhibits conserved elements responsible for virion organization. This places the virus neatly in the PRD1-adenovirus structure-based lineage. HCIV-1 further highlights the limited diversity of virus morphotypes despite the astronomical number of viruses in the biosphere. The observed high conservation in the core virion elements should be considered in addressing such fundamental issues as the origin and evolution of viruses and their interplay with their hosts. Copyright © 2016 Demina et al.

Grouper iridovirus GIV66 is a Bcl-2 protein that inhibits apoptosis by exclusively sequestering Bim.

PubMed

Banjara, Suresh; Mao, Jiahao; Ryan, Timothy M; Caria, Sofia; Kvansakul, Marc

2018-04-13

Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode functional and structural homologs of Bcl-2 to counter premature host-cell apoptosis and ensure viral proliferation or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the Iridoviridae family and harbors GIV66, a putative Bcl-2-like protein and mitochondrially localized apoptosis inhibitor. However, the molecular and structural basis of GIV66-mediated apoptosis inhibition is currently not understood. To gain insight into GIV66's mechanism of action, we systematically evaluated its ability to bind peptides spanning the BH3 domain of pro-apoptotic Bcl-2 family members. Our results revealed that GIV66 harbors an unusually high level of specificity for pro-apoptotic Bcl-2 and displays affinity only for Bcl-2-like 11 (Bcl2L11 or Bim). Using crystal structures of both apo-GIV66 and GIV66 bound to the BH3 domain from Bim, we unexpectedly found that GIV66 forms dimers via an interface that results in occluded access to the canonical Bcl-2 ligand-binding groove, which breaks apart upon Bim binding. This observation suggests that GIV66 dimerization may affect GIV66's ability to bind host pro-death Bcl-2 proteins and enables highly targeted virus-directed suppression of host apoptosis signaling. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single-specificity, pro-survival Bcl-2 protein and identifying a pivotal role of Bim in GIV-mediated inhibition of apoptosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Photoluminescence of double core/shell infrared (CdSeTe)/ZnS quantum dots conjugated to Pseudo rabies virus antibodies

NASA Astrophysics Data System (ADS)

Torchynska, T. V.; Casas Espinola, J. L.; Jaramillo Gómez, J. A.; Douda, J.; Gazarian, K.

2013-06-01

Double core CdSeTe/ZnS quantum dots (QDs) with emission at 800 nm (1.60 eV) have been studied by photoluminescence (PL) and Raman scattering methods in the non-conjugated state and after the conjugation to the Pseudo rabies virus (PRV) antibodies. The transformation of PL spectra, stimulated by the electric charge of antibodies, has been detected for the bioconjugated QDs. Raman scattering spectra are investigated with the aim to reveal the CdSeTe core compositions. The double core QD energy diagrams were designed that help to analyze the PL spectra and their transformation at the bioconjugation. It is revealed that the interface in double core QDs has the type II quantum well character that permits to explain the near IR optical transition (1.60 eV) in the double core QDs. It is shown that the essential transformation of PL spectra is useful for the study of QD bioconjugation with specific antibodies and can be a powerful technique in early medical diagnostics.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, H.-P.; Hsieh, S.-C.; King, C.-C.

In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibitedmore » by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.« less

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

PubMed

Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

2003-10-01

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Indirect Detection Of Bacillus Anthracis (Anthrax) Using Amplified Gamma Phage-Based Assays

DTIC Science & Technology

2007-11-01

bacteria and viruses . Biologist Kary Mullis invented PCR, described as being to genes as the Gutenberg press was to the written word, in 1983 and... Viruses are particles with a genome that exist only to reproduce. Since viruses are pseudo-living particles that lack ribosomes and have no machinery...for producing energy, they require living host cells in which to replicate. All viruses have an inner core of nucleic acid, either RNA or DNA

Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Zhongliang; Liu, Yanfeng; Luo, Mengjun

Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt −85 to −11 in eBHBV1 Cp was critical formore » the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt −64 to −50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt −58 to −54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt −21 to −17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses. - Highlights: • Endogenous budgerigar hepadnavirus (eBHBV) core promoters (Cps) are active in cells. • NF-Y binding site exists in the Cps of eBHBVs and all the extant avihepadnaviruses. • NF-Y binding and mediated upregulation is critical for eBHBV Cp activity.« less

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

PubMed Central

Chen, Augustine; Kao, Y. F.; Brown, Chris M.

2005-01-01

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins. PMID:15731337

Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

PubMed

Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

2011-12-01

Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

The H3-H4 N-Terminal Tail Domains Are the Primary Mediators of Transcription Factor IIIA Access to 5S DNA within a Nucleosome

PubMed Central

Vitolo, Joseph M.; Thiriet, Christophe; Hayes, Jeffrey J.

2000-01-01

Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B–DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508–2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693–20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA. PMID:10688663

Nucleic acid chaperone activity of retroviral Gag proteins.

PubMed

Rein, Alan

2010-01-01

Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive binding of inositol hexakisphosphate to the matrix domain.

Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.

PubMed

Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L

1999-12-17

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Full-spectrum volumetric solar thermal conversion via photonic nanofluids.

PubMed

Liu, Xianglei; Xuan, Yimin

2017-10-12

Volumetric solar thermal conversion is an emerging technique for a plethora of applications such as solar thermal power generation, desalination, and solar water splitting. However, achieving broadband solar thermal absorption via dilute nanofluids is still a daunting challenge. In this work, full-spectrum volumetric solar thermal conversion is demonstrated over a thin layer of the proposed 'photonic nanofluids'. The underlying mechanism is found to be the photonic superposition of core resonances, shell plasmons, and core-shell resonances at different wavelengths, whose coexistence is enabled by the broken symmetry of specially designed composite nanoparticles, i.e., Janus nanoparticles. The solar thermal conversion efficiency can be improved by 10.8% compared with core-shell nanofluids. The extinction coefficient of Janus dimers with various configurations is also investigated to unveil the effects of particle couplings. This work provides the possibility to achieve full-spectrum volumetric solar thermal conversion, and may have potential applications in efficient solar energy harvesting and utilization.

Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

PubMed

Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

2008-05-01

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

Occult hepatitis B virus infection in hematopoietic stem cell donors in a hepatitis B virus endemic area.

PubMed

Hui, Chee-kin; Sun, Jian; Au, Wing-yan; Lie, Albert K W; Yueng, Yui-hung; Zhang, Hai-ying; Lee, Nikki P; Hou, Jin-ling; Liang, Raymond; Lau, George K K

2005-06-01

The acquisition of hepatitis B virus (HBV) infection following organ transplantation from donors with occult HBV infection is an important cause of morbidity and mortality. The aim of this study is to determine the prevalence of occult HBV in allogeneic hematopoietic stem cell (HSC) transplantation donors. We performed a retrospective study on 124 consecutive hepatitis B surface antigen negative HSC donors. Their serum samples were analyzed by PCR for the pre-S/S, pre-core/core and X regions of the virus. Samples reactive by at least two PCR assays were considered HBV-DNA positive. Nineteen of the 124 HSC donors (15.3%) had occult HBV infection. Sixteen of these 19 donors with occult HBV infection (84.2%) tested positive for hepatitis B core antibody while 78 of 105 subjects (74.3%) without occult HBV infection were also positive (P=0.56). Fourteen of the 19 donors (73.7%) with occult HBV infection tested positive for hepatitis B surface antibody while 67 of the 105 subjects without occult HBV infection were also positive (P=0.45). The prevalence of occult HBV infection among HSC donors in Hong Kong is high. Anti-HBc and anti-HBs status had no significant correlation with the presence of occult HBV infection.

A GFP Expressing Influenza A Virus to Report In Vivo Tropism and Protection by a Matrix Protein 2 Ectodomain-Specific Monoclonal Antibody

PubMed Central

Van den Hoecke, Silvie; Smet, Anouk; Schotsaert, Michael; Job, Emma R.; Roose, Kenny; Schepens, Bert; Fiers, Walter; Saelens, Xavier

2015-01-01

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1–73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1–73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1–73)GFP virus, indicate that this virus is genetically and phenotypically stable. PMID:25816132

A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.

PubMed

De Baets, Sarah; Verhelst, Judith; Van den Hoecke, Silvie; Smet, Anouk; Schotsaert, Michael; Job, Emma R; Roose, Kenny; Schepens, Bert; Fiers, Walter; Saelens, Xavier

2015-01-01

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.

Molecular characterization and experimental host range of an isolate of Wissadula golden mosaic St. Thomas virus.

PubMed

Collins, A M; Mujaddad-ur-Rehman, Malik; Brown, J K; Reddy, C; Wang, A; Fondong, V; Roye, M E

2009-12-01

Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field conditions, confirming that this virus is a causal agent of Wissadula yellow mosaic disease in W. amplissima.

Isolation and Spectroscopic Characterization of Reactive Species in Atmospheric and Interstellar Reactions

NASA Astrophysics Data System (ADS)

Relph, Rachael A.

2011-12-01

A critical element to the study of chemical reactions is the characterization of reaction intermediates. Methods have been developed to isolate these transient species in the gas phase and when combined with infrared spectroscopy have proven to be excellent tools for determining the structure and reactivity of key intermediates. The studies presented here exploit these technologies to better understand the chemistry of species involved in atmospheric and interstellar reactions. An excellent example of their utility is in the study of the formation of proton hydrates and HONO in the upper atmosphere by sequential addition of water molecules onto the nitrosonium ion. This reaction only proceeds to products after addition of the fourth water molecule, and isolation and characterization of the intermediate trihydrate, NO+(H 2O)3, shows that this species is formed in three isomeric forms, each with a different water network that controls the degree of bond formation between the nitrosonium ion and an activated water molecule. Many isomeric structures are also seen in the clustering reactions of acetylene which may be a mechanism for the formation of benzene cation in interstellar space. The spectroscopy of the trimer, (C2H2)3 + indicates that this species exists in two major isomer classes; covalent forms, one of which may be benzene, and an ion-molecule complex, comprised of a loosely bound acetylene on a dimer core. Interestingly, this dimer core is different from the cyclobutadiene-like structure observed in dimerized acetylene, and proves to be a robust species on the potential energy surface as it survives further clustering events. Two structural isomers of CO3 -and NO3 - are also investigated, and found to have drastically different infrared spectra which are analyzed in the context of their electronic structure. Isomers in these systems are prepared under different expansion conditions which accounts for their unique spectral signatures.

Synthesis and structural characterization of lithium, sodium and potassium complexes supported by a tridentate amino-bisphenolate ligand

NASA Astrophysics Data System (ADS)

Durango-García, Clara J.; Rufino-Felipe, Ernesto; López-Cardoso, Marcela; Muñoz-Hernández, Miguel-Ángel; Montiel-Palma, Virginia

2018-07-01

Reactions of methylamino-N,N-bis(2-methylene-4,6-di-tert-butylphenol) (1) with one or two equivalents of bulk Li, Na or K metals in THF or DMSO render mono or dialkali metal complexes depending on the stoichiometric ratio of the reactants. The metal-methylamino-N-(2-methylene-4,6-tert-butylphenol)sbnd N-(2-methylene-4,6-tert-butylphenolate) complexes, 2Li, 2Na and 2K, are generated upon the substitution of a single phenol hydrogen of 1. In the solid state, complex 2Na is a dimer due to the establishment of two symmetric hydrogen bonds between two adjacent molecules. The Na center also engages into the formation of a ten-membered metallacycle ring with a butterfly-like structure. Due to dimerization, an intermolecular six-membered core is formed involving two sodium and four oxygen atoms. The weakly coordinated nitrogen atom from the ligand is nearly perpendicular to the hexagonal core. The dimetal-methylamino-N,N‧-bis(2-methylene-4,6-di-tert-butylphenolate) complexes, 3Li, 3Na and 3K result from metal substitution of the two phenol hydrogens from ligand 1. The SC-XRD structures of 3Li and 3Na are discreet, each incorporating two metal atoms in different coordination environments. Ten-membered rings with boat-boat conformations are also observed as are rhombic central M2O2 cores. The molecular structure of 3K in DMSO shows a higher degree of aggregation. It effectively comprises four K atoms, two ligand backbones and seven solvent molecules forming a central four-membered K2O2 ring perpendicular to an eight-membered structure formed also by K and O atoms spanning over the two ligand moieties.

The solely motif-doped Au36-xAgx(SPh-tBu)24 (x = 1-8) nanoclusters: X-ray crystal structure and optical properties.

PubMed

Fan, Jiqiang; Song, Yongbo; Chai, Jinsong; Yang, Sha; Chen, Tao; Rao, Bo; Yu, Haizhu; Zhu, Manzhou

2016-08-18

We report the observation of new doping behavior in Au36-xAgx(SR)24 nanoclusters (NCs) with x = 1 to 8. The atomic arrangements of Au and Ag atoms are determined by X-ray crystallography. The new gold-silver bimetallic NCs share the same framework as that of the homogold counterpart, i.e. possessing an fcc-type Au28 kernel, four dimeric AuAg(SR)3 staple motifs and twelve simple bridging SR ligands. Interestingly, all the Ag dopants in the Au36-xAgx(SR)24 NCs are selectively incorporated into the surface motifs, which is in contrast to the previously reported Au-Ag alloy structures with the Ag dopants preferentially displacing the core gold atoms. This distinct doping behavior implies that the previous assignments of an fcc Au28 core with four dimers and 12 bridging thiolates for Au36(SR)24 are more justified than other assignments of core vs. surface motifs. The UV-Vis adsorption spectrum of Au36-xAgx(SR)24 is almost the same as that of Au36(SR)24, indicating that the Ag dopants in the motifs do not change the optical properties. The similar UV-Vis spectra are further confirmed by TD-DFT calculations. DFT also reveals that the energies of the HOMO and LUMO of the motif-doped AuAg alloy NC are comparable to those of the homogold Au36 NC, indicating that the electronic structure is not disturbed by the motif Ag dopants. Overall, this study reveals a new silver-doping mode in alloy NCs.

Targets of small interfering RNA restriction during human immunodeficiency virus type 1 replication.

PubMed

Gao, Yong; Lobritz, Michael A; Roth, Justin; Abreha, Measho; Nelson, Kenneth N; Nankya, Immaculate; Moore-Dudley, Dawn M; Abraha, Awet; Gerson, Stanton L; Arts, Eric J

2008-03-01

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5alpha did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5' of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3'-to-5' siRNA amplification and spreading. In contrast, HIV-1 RNA 3' of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively "naked" cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5alpha, or other host restriction factors.

Selection of Optimal Polypurine Tract Region Sequences during Moloney Murine Leukemia Virus Replication

PubMed Central

Robson, Nicole D.; Telesnitsky, Alice

2000-01-01

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT. PMID:11044073

Chest pain in a young patient: an unusual complication of Epstein-Barr virus.

PubMed

Raman, Lavanya; Rathod, Krishnaraj Sinhji; Banka, Rajesh

2014-03-31

A 29-year-old man presented with sudden left-sided pleuritic chest pain on a background of sore throat during the preceding week. On examination he had tender cervical lymphadenopathy, he was tachycardic and had a 24 mm Hg blood pressure difference between the left and right arms. Bloods revealed deranged liver function tests and a lymphocytosis. His D-dimer was raised, hence he was treated for presumed pulmonary embolism before imaging was available. Monospot test was positive. He subsequently had both a CT pulmonary angiogram and a CT angiogram of the aorta to exclude pulmonary embolism and aortic dissection. The CT revealed splenomegaly with a large subdiaphragmatic haematoma secondary to splenic rupture. This had likely caused referred pain through diaphragmatic irritation. He was taken to theatre for urgent splenectomy. The unifying diagnosis was infectious mononucleosis complicated by spontaneous splenic rupture secondary to Epstein-Barr virus infection.

Small-angle X-Ray analysis of macromolecular structure: the structure of protein NS2 (NEP) in solution

NASA Astrophysics Data System (ADS)

Shtykova, E. V.; Bogacheva, E. N.; Dadinova, L. A.; Jeffries, C. M.; Fedorova, N. V.; Golovko, A. O.; Baratova, L. A.; Batishchev, O. V.

2017-11-01

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological pH value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

PubMed

Meier, Markus; Moya-Torres, Aniel; Krahn, Natalie J; McDougall, Matthew D; Orriss, George L; McRae, Ewan K S; Booy, Evan P; McEleney, Kevin; Patel, Trushar R; McKenna, Sean A; Stetefeld, Jörg

2018-06-01

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8[W

PubMed Central

O’Hara, Andrew; Jenkins, Gareth I.

2012-01-01

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the β-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function. PMID:23012433

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA.

PubMed

Mustafa, Farah; Vivet-Boudou, Valérie; Jabeen, Ayesha; Ali, Lizna M; Kalloush, Rawan M; Marquet, Roland; Rizvi, Tahir A

2018-06-21

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Core labeling of adenovirus with EGFP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le, Long P.; Le, Helen N.; Nelson, Amy R.

2006-08-01

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expressionmore » vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.« less

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System

PubMed Central

Taniguchi, Miki; Tasaka-Fujita, Megumi; Nakagawa, Mina; Watanabe, Takako; Kawai-Kitahata, Fukiko; Otani, Satoshi; Goto, Fumio; Nagata, Hiroko; Kaneko, Shun; Nitta, Sayuri; Murakawa, Miyako; Nishimura-Sakurai, Yuki; Azuma, Seishin; Itsui, Yasuhiro; Mori, Kenichi; Yagi, Shintaro; Kakinuma, Sei; Asahina, Yasuhiro; Watanabe, Mamoru

2016-01-01

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment. PMID:27047766

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus.

PubMed

Lai, Qingying; Wu, Wenbi; Li, Ao; Wang, Wei; Yuan, Meijin; Yang, Kai

2018-05-01

Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K ( ac98 ) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene ( ac100 ) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. Copyright © 2018 Lai et al.

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus

PubMed Central

Lai, Qingying; Li, Ao; Wang, Wei; Yuan, Meijin

2018-01-01

ABSTRACT Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. PMID:29444944

Coexistence of Epstein-Barr virus and Parvovirus B19 in tonsillar tissue samples: quantitative measurement by real-time PCR.

PubMed

Sahiner, Fatih; Gümral, Ramazan; Yildizoğlu, Üzeyir; Babayiğit, Mustafa Alparslan; Durmaz, Abdullah; Yiğit, Nuri; Saraçli, Mehmet Ali; Kubar, Ayhan

2014-08-01

In this study, we aimed to investigate the presence and copy number of six different viruses in tonsillar tissue samples removed surgically because of chronic recurrent tonsillitis or chronic obstructive tonsillar hypertrophy. In total, 56 tissue samples (tonsillar core) collected from 44 children and 12 adults were included in this study. The presence of viruses was investigated using a new TaqMan-based quantitative real-time PCR assay. Of the 56 tissue samples, 67.9% (38/56) were positive for at least one of the six viruses. Epstein-Barr virus was the most frequently detected virus, being found in 53.6% (30/56), followed by human Parvovirus B19 21.4% (12/56), human adenovirus 12.5% (7/56), human Cytomegalovirus 5.4% (3/56), BK polyomavirus 1.8% (1/56), and Herpes simplex virus 1.8% (1/56). Precancerous or cancerous changes were not detected in the tonsillar tissue samples by pathologic examination, whereas lymphoid hyperplasia was observed in 24 patients. In contrast to other viruses, B19 virus was present in high copy number in tonsillar tissues. The rates of EBV and B19 virus with high copy number (>500.000 copies/ml) were higher in children than in adults, and a positive relationship was also found between the presence of EBV and the presence of B19 virus with high copy number (P=0.037). It is previously reported that some viral agents are associated with different chronic tonsillar pathologies. In the present study, the presence of B19 virus in tonsillar core samples was investigated quantitatively for the first time, and our data suggests that EBV infections could be associated with B19 virus infections or could facilitate B19 virus replication. However, further detailed studies are needed to clarify this observation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage

PubMed Central

Luque, Daniel; Gómez-Blanco, Josué; Garriga, Damiá; Brilot, Axel F.; González, José M.; Havens, Wendy M.; Carrascosa, José L.; Trus, Benes L.; Verdaguer, Nuria; Ghabrial, Said A.; Castón, José R.

2014-01-01

Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single “hotspot” at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage. PMID:24821769

Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

PubMed Central

Schmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.

2013-01-01

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. PMID:23870263

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

PubMed

Chang, Chung-ke; Chen, Chia-Min Michael; Chiang, Ming-hui; Hsu, Yen-lan; Huang, Tai-huang

2013-01-01

The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

PubMed Central

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-01-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

PubMed

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-11-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyn, Rodney K.; Department of Chemistry, University of Ottawa, Ottawa; Kennedy, David C.

Research highlights: {yields} Hepatitis C virus uses lipid droplets (LD) onto which HCV core proteins bind. {yields} HCV core proteins on LDs facilitate viral particle assembly. {yields} We used a novel combination of CARS, two-photon fluorescence, and DIC microscopies. {yields} Particle tracking experiments show that core slowly affects LD localization. {yields} Particle tracking measured the change in speed and directionality of LD movement. -- Abstract: The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV coremore » proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.« less

ICTV Virus taxonomy profile: Asfarviridae

USDA-ARS?s Scientific Manuscript database

The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virons have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope. Infection of domestic pigs and wild boar results i...

Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen.

PubMed

Mueller, Niklaus H; Pattabiraman, Nagarajan; Ansarah-Sobrinho, Camilo; Viswanathan, Prasanth; Pierson, Theodore C; Padmanabhan, R

2008-09-01

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen approximately 32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K(i) values of 3.2 +/- 0.3 microM and 3.4 +/- 0.6 microM, respectively. These compounds inhibited the dengue virus type 2 protease with K(i) values of 28.6 +/- 5.1 microM and 30.2 +/- 8.6 microM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing.

HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

PubMed Central

Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

2015-01-01

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

The influence of ligand charge and length on the assembly of Brome mosaic virus derived virus-like particles with magnetic core

NASA Astrophysics Data System (ADS)

Mieloch, Adam A.; Krecisz, Monika; Rybka, Jakub D.; Strugała, Aleksander; Krupiński, Michał; Urbanowicz, Anna; Kozak, Maciej; Skalski, Bohdan; Figlerowicz, Marek; Giersig, Michael

2018-03-01

Virus-like particles (VLPs) have sparked a great interest in the field of nanobiotechnology and nanomedicine. The introduction of superparamagnetic nanoparticles (SPIONs) as a core, provides potential use of VLPs in the hyperthermia therapy, MRI contrast agents and magnetically-powered delivery agents. Magnetite NPs also provide a significant improvement in terms of VLPs stability. Moreover employing viral structural proteins as self-assembling units has opened a new paths for targeted therapy, drug delivery systems, vaccines design, and many more. In many cases, the self-assembly of a virus strongly depends on electrostatic interactions between positively charged groups of the capsid proteins and negatively charged nucleic acid. This phenomenon imposes the negative net charge as a key requirement for the core nanoparticle. In our experiments, Brome mosaic virus (BMV) capsid proteins isolated from infected plants Hordeum vulgare were used. Superparamagnetic iron oxide nanoparticles (Fe3O4) with 15 nm in diameter were synthesized by thermal decomposition and functionalized with COOH-PEG-PL polymer or dihexadecylphosphate (DHP) in order to provide water solubility and negative charge required for the assembly. Nanoparticles were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transformed Infrared Spectroscopy (FTIR) and Superconducting Quantum Interference Device (SQUID) magnetometry. TEM and DLS study were conducted to verify VLPs creation. This study demonstrates that the increase of negative surface charge is not a sufficient factor determining successful assembly. Additional steric interactions provided by longer ligands are crucial for the assembly of BMV SPION VLPs and may enhance the colloidal stability.

Calculation of binary magnetic properties and potential energy curve in xenon dimer: second virial coefficient of (129)Xe nuclear shielding.

PubMed

Hanni, Matti; Lantto, Perttu; Runeberg, Nino; Jokisaari, Jukka; Vaara, Juha

2004-09-22

Quantum chemical calculations of the nuclear shielding tensor, the nuclear quadrupole coupling tensor, and the spin-rotation tensor are reported for the Xe dimer using ab initio quantum chemical methods. The binary chemical shift delta, the anisotropy of the shielding tensor Delta sigma, the nuclear quadrupole coupling tensor component along the internuclear axis chi( parallel ), and the spin-rotation constant C( perpendicular ) are presented as a function of internuclear distance. The basis set superposition error is approximately corrected for by using the counterpoise correction (CP) method. Electron correlation effects are systematically studied via the Hartree-Fock, complete active space self-consistent field, second-order Møller-Plesset many-body perturbation, and coupled-cluster singles and doubles (CCSD) theories, the last one without and with noniterative triples, at the nonrelativistic all-electron level. We also report a high-quality theoretical interatomic potential for the Xe dimer, gained using the relativistic effective potential/core polarization potential scheme. These calculations used valence basis set of cc-pVQZ quality supplemented with a set of midbond functions. The second virial coefficient of Xe nuclear shielding, which is probably the experimentally best-characterized intermolecular interaction effect in nuclear magnetic resonance spectroscopy, is computed as a function of temperature, and compared to experiment and earlier theoretical results. The best results for the second virial coefficient, obtained using the CCSD(CP) binary chemical shift curve and either our best theoretical potential or the empirical potentials from the literature, are in good agreement with experiment. Zero-point vibrational corrections of delta, Delta sigma, chi (parallel), and C (perpendicular) in the nu=0, J=0 rovibrational ground state of the xenon dimer are also reported.

Structural and Mechanistic Basis of Zinc Regulation Across the E. coli Zur Regulon

PubMed Central

Gilston, Benjamin A.; Wang, Suning; Marcus, Mason D.; Canalizo-Hernández, Mónica A.; Swindell, Elden P.; Xue, Yi; Mondragón, Alfonso; O'Halloran, Thomas V.

2014-01-01

Commensal microbes, whether they are beneficial or pathogenic, are sensitive to host processes that starve or swamp the prokaryote with large fluctuations in local zinc concentration. To understand how microorganisms coordinate a dynamic response to changes in zinc availability at the molecular level, we evaluated the molecular mechanism of the zinc-sensing zinc uptake regulator (Zur) protein at each of the known Zur-regulated genes in Escherichia coli. We solved the structure of zinc-loaded Zur bound to the PznuABC promoter and show that this metalloregulatory protein represses gene expression by a highly cooperative binding of two adjacent dimers to essentially encircle the core element of each of the Zur-regulated promoters. Cooperativity in these protein-DNA interactions requires a pair of asymmetric salt bridges between Arg52 and Asp49′ that connect otherwise independent dimers. Analysis of the protein-DNA interface led to the discovery of a new member of the Zur-regulon: pliG. We demonstrate this gene is directly regulated by Zur in a zinc responsive manner. The pliG promoter forms stable complexes with either one or two Zur dimers with significantly less protein-DNA cooperativity than observed at other Zur regulon promoters. Comparison of the in vitro Zur-DNA binding affinity at each of four Zur-regulon promoters reveals ca. 10,000-fold variation Zur-DNA binding constants. The degree of Zur repression observed in vivo by comparison of transcript copy number in wild-type and Δzur strains parallels this trend spanning a 100-fold difference. We conclude that the number of ferric uptake regulator (Fur)-family dimers that bind within any given promoter varies significantly and that the thermodynamic profile of the Zur-DNA interactions directly correlates with the physiological response at different promoters. PMID:25369000

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

PubMed Central

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

2006-01-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

2006-04-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

Fowlpox Virus Encodes a Novel DNA Repair Enzyme, CPD-Photolyase, That Restores Infectivity of UV Light-Damaged Virus

PubMed Central

Srinivasan, Viswanathan; Schnitzlein, William M.; Tripathy, Deoki N.

2001-01-01

Fowlpox virus (FPV), a pathogen of poultry, can persist in desiccated scabs shed from infected hosts. Although the mechanisms which ensure virus survival are unknown, it is likely that some type of remedial action against environmentally induced damage is required. In this regard, we have identified an open reading frame (ORF) coding for a putative class II cyclobutane pyrimidine dimer (CPD)-photolyase in the genome of FPV. This enzyme repairs the UV light-induced formation of CPDs in DNA by using blue light as an energy source and thus could enhance the viability of FPV during its exposure to sunlight. Based on transcriptional analyses, the photolyase gene was found to be expressed late during the FPV replicative cycle. That the resultant protein retained DNA repair activity was demonstrated by the ability of the corresponding FPV ORF to complement functionally a photolyase-deficient Escherichia coli strain. Interestingly, insertional inactivation of the FPV photolyase gene did not impair the replication of such a genetically altered virus in cultured cells. However, greater sensitivity of this mutant than of the parental virus to UV light irradiation was evident when both were subsequently photoreactivated in the absence of host participation. Therefore, FPV appears to incorporate its photolyase into mature virions where the enzyme can promote their survival in the environment. Although expression of a homologous protein has been predicted for some chordopoxviruses, this report is the first to demonstrate that a poxvirus can utilize light to repair damage to its genome. PMID:11160666

Sweepoviruses Cause Disease in Sweet Potato and Related Ipomoea spp.: Fulfilling Koch's Postulates for a Divergent Group in the Genus Begomovirus

PubMed Central

Márquez-Martín, Belén; Moriones, Enrique; Navas-Castillo, Jesús

2011-01-01

Sweet potato (Ipomoea batatas) and related Ipomoea species are frequently infected by monopartite begomoviruses (genus Begomovirus, family Geminiviridae), known as sweepoviruses. Unlike other geminiviruses, the genomes of sweepoviruses have been recalcitrant to rendering infectious clones to date. Thus, Koch's postulates have not been fullfilled for any of the viruses in this group. Three novel species of sweepoviruses have recently been described in Spain: Sweet potato leaf curl Lanzarote virus (SPLCLaV), Sweet potato leaf curl Spain virus (SPLCSV) and Sweet potato leaf curl Canary virus (SPLCCaV). Here we describe the generation of the first infectious clone of an isolate (ES:MAL:BG30:06) of SPLCLaV. The clone consisted of a complete tandem dimeric viral genome in a binary vector. Successful infection by agroinoculation of several species of Ipomoea (including sweet potato) and Nicotiana benthamiana was confirmed by PCR, dot blot and Southern blot hybridization. Symptoms observed in infected plants consisted of leaf curl, yellowing, growth reduction and vein yellowing. Two varieties of sweet potato, ‘Beauregard’ and ‘Promesa’, were infected by agroinoculation, and symptoms of leaf curl and interveinal loss of purple colouration were observed, respectively. The virus present in agroinfected plants was readily transmitted by the whitefly Bemisia tabaci to I. setosa plants. The progeny virus population present in agroinfected I. setosa and sweet potato plants was isolated and identity to the original isolate was confirmed by sequencing. Therefore, Koch's postulates were fulfilled for the first time for a sweepovirus. PMID:22073314

Sweepoviruses cause disease in sweet potato and related Ipomoea spp.: fulfilling Koch's postulates for a divergent group in the genus begomovirus.

PubMed

Trenado, Helena P; Orílio, Anelise F; Márquez-Martín, Belén; Moriones, Enrique; Navas-Castillo, Jesús

2011-01-01

Sweet potato (Ipomoea batatas) and related Ipomoea species are frequently infected by monopartite begomoviruses (genus Begomovirus, family Geminiviridae), known as sweepoviruses. Unlike other geminiviruses, the genomes of sweepoviruses have been recalcitrant to rendering infectious clones to date. Thus, Koch's postulates have not been fullfilled for any of the viruses in this group. Three novel species of sweepoviruses have recently been described in Spain: Sweet potato leaf curl Lanzarote virus (SPLCLaV), Sweet potato leaf curl Spain virus (SPLCSV) and Sweet potato leaf curl Canary virus (SPLCCaV). Here we describe the generation of the first infectious clone of an isolate (ES:MAL:BG30:06) of SPLCLaV. The clone consisted of a complete tandem dimeric viral genome in a binary vector. Successful infection by agroinoculation of several species of Ipomoea (including sweet potato) and Nicotiana benthamiana was confirmed by PCR, dot blot and Southern blot hybridization. Symptoms observed in infected plants consisted of leaf curl, yellowing, growth reduction and vein yellowing. Two varieties of sweet potato, 'Beauregard' and 'Promesa', were infected by agroinoculation, and symptoms of leaf curl and interveinal loss of purple colouration were observed, respectively. The virus present in agroinfected plants was readily transmitted by the whitefly Bemisia tabaci to I. setosa plants. The progeny virus population present in agroinfected I. setosa and sweet potato plants was isolated and identity to the original isolate was confirmed by sequencing. Therefore, Koch's postulates were fulfilled for the first time for a sweepovirus.

HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme

PubMed Central

2015-01-01

Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein–protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy. PMID:24654558

Non-structural protein 1 from avian influenza virus H9N2 is an efficient RNA silencing suppressor with characteristics that differ from those of Tomato bushy stunt virus p19.

PubMed

Yu, Ru; Jing, Xiuli; Li, Wenjing; Xu, Jie; Xu, Yang; Geng, Liwei; Zhu, Changxiang; Liu, Hongmei

2018-06-01

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional dimeric protein that contains a conserved N-terminal RNA binding domain. Studies have shown that NS1 suppresses RNA silencing and the NS1 proteins encoded by different influenza A virus strains exhibit differential RNA silencing suppression activities. In this study, we showed that the NS1 protein from avian influenza virus (AIV) H9N2 suppressed systemic RNA silencing induced by sense RNA or dsRNA. It resulted in more severe Potato virus X symptom, but could not reverse established systemic green fluorescent protein silencing in Nicotiana benthamiana. In addition, its systemic silencing suppression activity was much weaker than that of p19. The local silencing suppression activity of AIV H9N2 NS1 was most powerful at 7 dpi and was even stronger than that of p19. And the inhibition ability to RNA silencing of NS1 is stronger than that of p19 in human cells. Collectively, these results indicate that AIV H9N2 NS1 is an effective RNA silencing suppressor that likely targets downstream step(s) of dsRNA formation at an early stage in RNA silencing. Although NS1 and p19 both bind siRNA, their suppression mechanisms seem to differ because of differences in their suppression activities at various times post-infiltration and because p19 can reverse established systemic RNA silencing, but NS1 cannot.

Plant rhabdoviruses.

PubMed

Redinbaugh, M G; Hogenhout, S A

2005-01-01

This chapter provides an overview of plant rhabdovirus structure and taxonomy, genome structure, protein function, and insect and plant infection. It is focused on recent research and unique aspects of rhabdovirus biology. Plant rhabdoviruses are transmitted by aphid, leafhopper or planthopper vectors, and the viruses replicate in both their insect and plant hosts. The two plant rhabdovirus genera, Nucleorhabdovirus and Cytorhabdovirus, can be distinguished on the basis of their intracellular site of morphogenesis in plant cells. All plant rhabdoviruses carry analogs of the five core genes: the nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large or polymerase (L). However, compared to vesiculoviruses that are composed of the five core genes, all plant rhabdoviruses encode more than these five genes, at least one of which is inserted between the P and M genes in the rhabdoviral genome. Interestingly, while these extra genes are not similar among plant rhabdoviruses, two encode proteins with similarity to the 30K superfamily of plant virus movement proteins. Analysis of nucleorhabdoviral protein sequences revealed nuclear localization signals for the N, P, M and L proteins, consistent with virus replication and morphogenesis of these viruses in the nucleus. Plant and insect factors that limit virus infection and transmission are discussed.

Dimeric and trimeric hydrolyzable tannins from Quercus coccifera and Quercus suber.

PubMed

Ito, Hideyuki; Yamaguchi, Koji; Kim, Tae-Hoon; Khennouf, Seddik; Gharzouli, Kamel; Yoshida, Takashi

2002-03-01

Three new hydrolyzable tannins, cocciferins D(1) (1), D(2) (2), and T(1) (4), were isolated from the leaves of Quercus coccifera. Cocciferin D(2) (2) and two additional new tannins, cocciferins D(3) (3) and T(2) (5), were also obtained from the leaves of Quercus suber. Their oligomeric structures were elucidated on the basis of spectroscopic methods and chemical evidence. Compounds 2, 3, and 5 were rare oligomers possessing glucose cores with both open-chain and pyranose forms.

Prevalence of mutations in hepatitis C virus core protein associated with alteration of NF-kappaB activation.

PubMed

Mann, Elizabeth A; Stanford, Sandra; Sherman, Kenneth E

2006-10-01

The hepatitis C virus (HCV) core protein is a key structural element of the virion but also affects a number of cellular pathways, including nuclear factor kappaB (NF-kappaB) signaling. NF-kappaB is a transcription factor that regulates both anti-apoptotic and pro-inflammatory genes and its activation may contribute to HCV-mediated pathogenesis. Amino acid sequence divergence in core is seen at the genotype level as well as within patient isolates. Recent work has implicated amino acids 9-11 of core in the modulation of NF-kappaB activation. We report that the sequence RKT is highly conserved (93%) at this position across all HCV genotypes, based on sequences collected in the Los Alamos HCV database. Of the 13 types of variants present in the database, the two most prevalent substitutions are RQT and RKP. We further show that core encoding RKP fails to activate NF-kappaB signaling in vitro while NF-kappaB activation by core encoding RQT does not differ from control RKT core. The effect of RKP core is specific to NF-kappaB signaling as activator protein 1 (AP-1) activity is not altered. Further studies are needed to assess potential associations between specific amino acid substitutions at positions 9-11 and liver disease progression and/or response to treatment in individual patients.

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

PubMed Central

Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M

2017-01-01

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence. PMID:28915104

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

PubMed

Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

2017-09-15

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV).

PubMed

Hick, Paul M; Subramaniam, Kuttichantran; Thompson, Patrick M; Waltzek, Thomas B; Becker, Joy A; Whittington, Richard J

2017-11-01

Low genetic diversity of Epizootic haematopoietic necrosis virus (EHNV) was determined for the complete genome of 16 isolates spanning the natural range of hosts, geography and time since the first outbreaks of disease. Genomes ranged from 125,591-127,487 nucleotides with 97.47% pairwise identity and 106-109 genes. All isolates shared 101 core genes with 121 potential genes predicted within the pan-genome of this collection. There was high conservation within 90,181 nucleotides of the core genes with isolates separated by average genetic distance of 3.43 × 10 -4 substitutions per site. Evolutionary analysis of the core genome strongly supported historical epidemiological evidence of iatrogenic spread of EHNV to naïve hosts and establishment of endemic status in discrete ecological niches. There was no evidence of structural genome reorganization, however, the complement of non-core genes and variation in repeat elements enabled fine scale molecular epidemiological investigation of this unpredictable pathogen of fish. Copyright © 2017 Elsevier Inc. All rights reserved.

An intramolecular disulfide bridge between Cys-7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus.

PubMed Central

Nassal, M; Rieger, A

1993-01-01

Hepatitis B virus, the prototypic member of the Hepadnaviridae, is a small enveloped DNA virus that replicates via reverse transcription. Efficient usage of its compact 3.2-kb genome is exemplified by the pre-C/C gene from which two proteins with largely overlapping primary sequences but distinctly different properties are synthesized: the self-assembling core protein p21c (hepatitis B core antigen [HbcAg]) and the secretory, nonparticulate protein p17e (hepatitis B e antigen [HbeAg]). Mature p17e carries a 10-amino-acid N-terminal extension with a Cys residue (Cys-7). Using transient transfection of a human liver cell line with constructs expressing wild-type p17 or a series of Cys mutants of p17, we show that Cys-7 forms an intramolecular S-S bond to Cys61, which in assembly-competent core proteins is available for intermolecular disulfide bonds between two neighboring subunits. Removal of the Cys-7/Cys61 bond by mutating either residue has differential effects: in the absence of Cys-7, secretion is relatively efficient and independent of Cys61; however, the molecules are exported as homodimers exhibiting both HBe and HBc antigenicity. In the absence of Cys61, the nonpaired Cys-7 interferes with secretion efficiency. The amino acid sequence flanking Cys-7 also contributes to the formation of the proper intramolecular S-S bond. These results suggest that the Cys-7/Cys61 bond imposes on p17e a conformation that is critical for its secretion and distinct biophysical and antigenic properties. This mechanism adds selective disulfide formation to the repertoire of hepatitis B virus for efficient use of its tiny genome. Images PMID:8510224

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Cong; Sawaya, Michael R.; Eisenberg, David

{beta}{sub 2}-microglobulin ({beta}{sub 2}-m) is the light chain of the type I major histocompatibility complex. It deposits as amyloid fibrils within joints during long-term hemodialysis treatment. Despite the devastating effects of dialysis-related amyloidosis, full understanding of how fibrils form from soluble {beta}{sub 2}-m remains elusive. Here we show that {beta}{sub 2}-m can oligomerize and fibrillize via three-dimensional domain swapping. Isolating a covalently bound, domain-swapped dimer from {beta}{sub 2}-m oligomers on the pathway to fibrils, we were able to determine its crystal structure. The hinge loop that connects the swapped domain to the core domain includes the fibrillizing segment LSFSKD, whosemore » atomic structure we also determined. The LSFSKD structure reveals a class 5 steric zipper, akin to other amyloid spines. The structures of the dimer and the zipper spine fit well into an atomic model for this fibrillar form of {beta}{sub 2}-m, which assembles slowly under physiological conditions.« less

Heptacopper(II) and dicopper(II)-adenine complexes: synthesis, structural characterization, and magnetic properties

DOE PAGES

Leite Ferreira, B. J. M.; Brandão, Paula; Dos Santos, A. M.; ...

2015-07-13

The syntheses, crystal structures, and magnetic properties of two new copper(II) complexes with molecular formulas [Cu 7(μ 2-OH 2) 6(μ 3-O) 6(adenine) 6(NO 3) 26H 2O (1) and [Cu 2(μ 2-H 2O) 2(adenine) 2(H 2O) 4](NO 3) 42H 2O (2) are reported. We composed the heptanuclear compound of a central octahedral CuO 6 core sharing edges with six adjacent copper octahedra. In 2, the copper octahedra shares one equatorial edge. In both compounds, these basic copper cluster units are further linked by water bridges and bridging adenine ligands through N3 and N9 donors. All copper(II) centers exhibit Jahn-Teller distorted octahedralmore » coordination characteristic of a d 9 center. Our study of the magnetic properties of the heptacopper complex revealed a dominant ferromagnetic intra-cluster interaction, while the dicopper complex exhibits antiferromagnetic intra-dimer interactions with weakly ferromagnetic inter-dimer interaction.« less

Static Holes in Geometrically Frustrated Bow Tie Ladder

NASA Astrophysics Data System (ADS)

Martins, George; Brenig, Wolfram

2007-03-01

Doping of the geometrically frustrated bow-tie spin ladder with static holes is investigated by a complementary approach using exact diagonalization and hard-core quantum dimers. Results for the thermodynamics in the undoped case, the singlet density of states, the hole-binding energy, and the spin correlations will be presented. We find that the static holes polarize their vicinity by a localization of singlets in order to reduce the frustration. As a consequence the singlet polarization cloud induces short range repulsive forces between the holes with oscillatory longer range behavior. For those systems we have studied, most results for the quantum dimer approach are found to be qualitatively if not quantitatively in agreement with exact diagonalization. The ground state of the undoped system is non-degenerate with translationally invariant nearest-neighbor spin correlations up to a few unit cells, which is consistent with a spin liquid state or a valence bond crystal with very large unit cell. C. Waldtmann, A. Kreutzmann, U. Schollwock, K. Maisinger, and H.-U. Everts, Phys. Rev. B 62, 9472 (2000).

Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis.

PubMed

Moreno-Morcillo, María; Grande-García, Araceli; Ruiz-Ramos, Alba; Del Caño-Ochoa, Francisco; Boskovic, Jasminka; Ramón-Maiques, Santiago

2017-06-06

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Torque modulates nucleosome stability and facilitates H2A/H2B dimer loss

PubMed Central

Sheinin, Maxim Y.; Li, Ming; Soltani, Mohammad; Luger, Karolin; Wang, Michelle D.

2013-01-01

The nucleosome, the fundamental packing unit of chromatin, has a distinct chirality: 147 bp of DNA are wrapped around the core histones in a left-handed, negative superhelix. It has been suggested that this chirality has functional significance, particularly in the context of the cellular processes that generate DNA supercoiling, such as transcription and replication. However, the impact of torsion on nucleosome structure and stability is largely unknown. Here we perform a detailed investigation of single nucleosome behavior on the high affinity 601 positioning sequence under tension and torque using the angular optical trapping technique. We find that torque has only a moderate effect on nucleosome unwrapping. In contrast, we observe a dramatic loss of H2A/H2B dimers upon nucleosome disruption under positive torque, while (H3/H4)2 tetramers are efficiently retained irrespective of torsion. These data indicate that torque could regulate histone exchange during transcription and replication. PMID:24113677

Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus.

PubMed

Wanas, E; Efler, S; Ghosh, K; Ghosh, H P

1999-12-01

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family HERPESVIRIDAE: Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.

The vaccinia virus E6 protein influences virion protein localization during virus assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Condit, Richard C., E-mail: condit@mgm.ufl.edu; Moussatche, Nissin

2015-08-15

Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulatemore » in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.« less

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: relevance to drug resistance.

PubMed

Sayer, Jane M; Agniswamy, Johnson; Weber, Irene T; Louis, John M

2010-11-01

The mature protease from Group N human immunodeficiency virus Type 1 (HIV-1) (PR1(N)) differs in 20 amino acids from the extensively studied Group M protease (PR1(M)) at positions corresponding to minor drug-resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1(N) with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1(M), but with a slightly larger inhibitor-binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor-binding residues 29-32. However, kinetic parameters of PR1(N) closely resemble those of PR1(M), and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug-specific major DRMs. A miniprecursor (TFR 1-61-PR1(N)) comprising the transframe region (TFR) fused to the N-terminus of PR1(N) undergoes autocatalytic cleavage at the TFR/PR1(N) site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (∼6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1-61/PR1(N) cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1(N) relative to PR1(M) include its suitability for column fractionation by size under native conditions and >10-fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.

PubMed

Wang, Qiang; Ma, Xiaonan; Qian, ShaSha; Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O; Li, Zhenghe

2015-10-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

PubMed Central

Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O.; Li, Zhenghe

2015-01-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses. PMID:26484673

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

Single-virus fusion experiments reveal proton influx into vaccinia virions and hemifusion lag times.

PubMed

Schmidt, Florian I; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S

2013-07-16

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

[The maturation steps of human immunodeficiency virus and the role of proteolysis].

PubMed

Bukrinskaia, A G; Grigor'ev, V B; Korablina, E V; Gur'ev, E L; Vorkunova, G K

2010-01-01

HIV-1 virions are as immature noninfectious particles lacking a central core. Shortly after budding, virions temporally mature and acquire cores and infectious activity. The cause of maturation remains poorly studied. We have revealed that the virions produced early after infection following 24-36 hours, never mature and remain noninfectious, and only virions produced 48-72 hours after infection mature. The mature virions contain 3 times more genomic viral RNA than "early" virus. The "early" virions contain the same proteolytically cleaved Gag proteins as mature virions in contrast to the accepted version. The virus protease inhibitor Indinavir sulfate (IS) fully blocks infectivity when added early after infection. The early proteolysis of Gag precursor in the infected cells and inclusion into the virions of cellularly cleaved matrix protein (cMA) are shown in the IS-treated cells. cMA is associated with genomic viral RNA.

Diagnostic Value of Anti-Hepatitis C Virus (HCV) Core Immunoglobulin M in Recurrence of HCV Infection after Orthotopic Liver Transplantation†

PubMed Central

Casino, Carmela; Lilli, Daniela; Rivanera, Daniela; Comanducci, Antonella; Rossi, Massimo; Casciaro, Giovanni; Pecorella, Irene; Alfani, Dario; Mancini, Carlo

1999-01-01

The significance of anti-hepatitis C virus (HCV) core immunoglobulin M (IgM) and its relationship with genotypes, alanine aminotransferase abnormality, and histological data were studied for 18 patients who had undergone orthotopic liver transplantation due to HCV-related end-stage disease. During follow-up, IgM response seemed to be associated with the recurrence of HCV infection but did not correlate with abnormal alanine aminotransferase levels and histological data. In addition, the results of this study indicated that the detection of HCV RNA is critical for diagnosis of reinfection in liver transplantation. PMID:10405433

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations.

PubMed

Elizalde, María Mercedes; Pérez, Paula Soledad; Sevic, Ina; Grasso, Daniel; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations

PubMed Central

Pérez, Paula Soledad; Sevic, Ina; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy. PMID:29738548

The X40×10 Halogen Bonding Benchmark Revisited: Surprising Importance of (n-1)d Subvalence Correlation.

PubMed

Kesharwani, Manoj K; Manna, Debashree; Sylvetsky, Nitai; Martin, Jan M L

2018-03-01

We have re-evaluated the X40×10 benchmark for halogen bonding using conventional and explicitly correlated coupled cluster methods. For the aromatic dimers at small separation, improved CCSD(T)-MP2 "high-level corrections" (HLCs) cause substantial reductions in the dissociation energy. For the bromine and iodine species, (n-1)d subvalence correlation increases dissociation energies and turns out to be more important for noncovalent interactions than is generally realized; (n-1)sp subvalence correlation is much less important. The (n-1)d subvalence term is dominated by core-valence correlation; with the smaller cc-pVDZ-F12-PP and cc-pVTZ-F12-PP basis sets, basis set convergence for the core-core contribution becomes sufficiently erratic that it may compromise results overall. The two factors conspire to generate discrepancies of up to 0.9 kcal/mol (0.16 kcal/mol RMS) between the original X40×10 data and the present revision.

Pathophysiology of ocular surface squamous neoplasia

PubMed Central

Gichuhi, Stephen; Ohnuma, Shin-ichi; Sagoo, Mandeep S.; Burton, Matthew J.

2014-01-01

The incidence of ocular surface squamous neoplasia (OSSN) is strongly associated with solar ultraviolet (UV) radiation, HIV and human papilloma virus (HPV). Africa has the highest incidence rates in the world. Most lesions occur at the limbus within the interpalpebral fissure particularly the nasal sector. The nasal limbus receives the highest intensity of sunlight. Limbal epithelial crypts are concentrated nasally and contain niches of limbal epithelial stem cells in the basal layer. It is possible that these are the progenitor cells in OSSN. OSSN arises in the basal epithelial cells spreading towards the surface which resembles the movement of corneo-limbal stem cell progeny before it later invades through the basement membrane below. UV radiation damages DNA producing pyrimidine dimers in the DNA chain. Specific CC → TT base pair dimer transformations of the p53 tumour-suppressor gene occur in OSSN allowing cells with damaged DNA past the G1-S cell cycle checkpoint. UV radiation also causes local and systemic photoimmunosuppression and reactivates latent viruses such as HPV. The E7 proteins of HPV promote proliferation of infected epithelial cells via the retinoblastoma gene while E6 proteins prevent the p53 tumour suppressor gene from effecting cell-cycle arrest of DNA-damaged and infected cells. Immunosuppression from UV radiation, HIV and vitamin A deficiency impairs tumour immune surveillance allowing survival of aberrant cells. Tumour growth and metastases are enhanced by; telomerase reactivation which increases the number of cell divisions a cell can undergo; vascular endothelial growth factor for angiogenesis and matrix metalloproteinases (MMPs) that destroy the intercellular matrix between cells. Despite these potential triggers, the disease is usually unilateral. It is unclear how HPV reaches the conjunctiva. PMID:25447808

The bipolar filaments formed by Herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing

PubMed Central

Makhov, Alexander M.; Sen, Anindito; Yu, Xiong; Simon, Martha N.; Griffith, Jack D.; Egelman, Edward H.

2009-01-01

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single strand binding protein and recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic (EM) studies showed that ICP8 will form long left-handed helical filaments. Here EM image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using Scanning Transmission Electron Microscopy. The pitch of the filaments is ~ 250 Å, with ~ 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing ~ 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA, based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary single stranded DNA into double-stranded DNA, where each strand runs in opposite directions. PMID:19138689

The bipolar filaments formed by herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing.

PubMed

Makhov, Alexander M; Sen, Anindito; Yu, Xiong; Simon, Martha N; Griffith, Jack D; Egelman, Edward H

2009-02-20

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is approximately 250 A, with approximately 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing approximately 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makhov, A.M.; Simon, M.; Sen, A.

2009-02-20

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments ismore » {approx} 250 {angstrom}, with {approx} 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing {approx} 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.« less

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

PubMed

Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

2013-03-01

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural Impact of Three Parkinsonism-Associated Missense Mutations on Human DJ-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lakshminarasimhan, M.; Maldonado, M.T.; Zhou, W.

2009-05-20

A number of missense mutations in the oxidative stress response protein DJ-1 are implicated in rare forms of familial Parkinsonism. The best-characterized Parkinsonian DJ-1 missense mutation, L166P, disrupts homodimerization and results in a poorly folded protein. The molecular basis by which the other Parkinsonism-associated mutations disrupt the function of DJ-1, however, is incompletely understood. In this study we show that three different Parkinsonism-associated DJ-1 missense mutations (A104T, E163K, and M26I) reduce the thermal stability of DJ-1 in solution by subtly perturbing the structure of DJ-1 without causing major folding defects or loss of dimerization. Atomic resolution X-ray crystallography shows thatmore » the A104T substitution introduces water and a discretely disordered residue into the core of the protein, E163K disrupts a key salt bridge with R145, and M26I causes packing defects in the core of the dimer. The deleterious effect of each Parkinsonism-associated mutation on DJ-1 is dissected by analysis of engineered substitutions (M26L, A104V, and E163K/R145E) that partially alleviate each of the defects introduced by the A104T, E163K and M26I mutations. In total, our results suggest that the protective function of DJ-1 can be compromised by diverse perturbations in its structural integrity, particularly near the junctions of secondary structural elements.« less

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule

PubMed Central

Nigro, Ersilia; Colavita, Irene; Sarnataro, Daniela; Scudiero, Olga; Zambrano, Gerardo; Granata, Vincenzo; Daniele, Aurora; Carotenuto, Alfonso; Galdiero, Stefania; Folliero, Veronica; Galdiero, Massimiliano; Urbanowicz, Richard A.; Ball, Jonathan K.; Salvatore, Francesco; Pessi, Antonello

2015-01-01

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated “γ-core motif”. We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents. PMID:26688341

The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

NASA Astrophysics Data System (ADS)

Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

2015-10-01

Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

DNA origami based Au-Ag-core-shell nanoparticle dimers with single-molecule SERS sensitivity

NASA Astrophysics Data System (ADS)

Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.; Bald, I.

2016-03-01

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA bands, SEM images, additional AFM images, FDTD simulations, additional reference spectra for Cy3 and detailed description of EF estimation, simulated absorption and scattering spectra. See DOI: 10.1039/c5nr08674d

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication. Copyright © 2017 American Society for Microbiology.

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

PubMed

Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Synonymous Mutations in the Core Gene Are Linked to Unusual Serological Profile in Hepatitis C Virus Infection

PubMed Central

Budkowska, Agata; Kakkanas, Athanassios; Nerrienet, Eric; Kalinina, Olga; Maillard, Patrick; Horm, Srey Viseth; Dalagiorgou, Geena; Vassilaki, Niki; Georgopoulou, Urania; Martinot, Michelle; Sall, Amadou Alpha; Mavromara, Penelope

2011-01-01

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99–124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection. PMID:21283512

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

PubMed

Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

2017-08-22

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

[Analysis of core virion polypeptides from the pathogen causing chicken egg-drop syndrome].

PubMed

Iurov, G K; Dadykov, V A; Neugodova, G L; Naroditskiĭ, B S

1998-01-01

The cores of egg-drop syndrome virus (EDS-76) were isolated by the pyridine technique. EDS-76 proved to be much more resistant to pyridine disruption than other adenoviruses and treatment with 10% pyridine did not lead to complete dissociation of capsid and cores; only increase of pyridine concentration to 20% produced satisfactory results. At least three polypeptides (24, 10.5, and 6.5 kDa) were found in the core by SDS-PAGE, whereas the 40 kDa reacting with the core is most probably not a core component. Much more intensive reactions of the core with EDS-76 virion capsid suggest that its virion structure differs from that of other adenoviruses.

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

PubMed Central

Kalloush, Rawan M.; Vivet-Boudou, Valérie; Ali, Lizna M.; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

2016-01-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. PMID:27095024

Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability

USGS Publications Warehouse

Hall, Jeffrey S.; TeSlaa, Joshua L.; Nashold, Sean W.; Halpin, Rebecca A.; Stockwell, Timothy; Wentworth, David E.; Dugan, Vivien; Ip, Hon S.

2013-01-01

Background: The role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes. Methods: We isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds. Results: We analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs. Conclusions: This process appears similar to genetic shifts seen with swine influenza where a stable "triple reassortant internal gene" core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the need for continued surveillance in gull and waterfowl populations, both on the Pacific and especially Atlantic coasts of North America, to document virus intercontinental movement and the role of gull species in the evolution and epidemiology of AI.

Characterization of the immune response to canine parvovirus induced by vaccination with chimaeric plant viruses.

PubMed

Nicholas, Benjamin L; Brennan, F R; Martinez-Torrecuadrada, J L; Casal, J I; Hamilton, W D; Wakelin, D

2002-06-21

NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV) constructs expressing a 17-mer peptide sequence from canine parvovirus (CPV) as monomers or dimers on the small or large protein surface subunits. Responses to the chimaeric virus particles (CVPs) were compared with those of mice immunized with the native virus or with parvovirus peptide conjugated to keyhole limpet haemocyanin (KLH). The characteristics of the immune response to vaccination were examined by measuring serum and mucosal antibody responses in ELISA, in vitro antigen-induced spleen cell proliferation and cytokine responses. Mice made strong antibody responses to the native plant virus and peptide-specific responses to two of the four CVP constructs tested which were approximately 10-fold lower than responses to native plant virus. The immune response generated by the CVP constructs showed a marked TH1 bias, as determined by a predominantly IgG(2a) isotype peptide-specific antibody response and the release of IFN-gamma but not IL-4 or IL-5 from lymphocytes exposed to antigen in vitro. In comparison, parvovirus peptide conjugated to KLH generated an IgG(1)-biased (TH2) response. These data indicate that the presentation of peptides on viral particles could be used to bias the immune response in favor of a TH1 response.Anti-viral and anti-peptide IgA were detected in intestinal and bronchial lavage fluid of immunized mice, demonstrating that a mucosal immune response to CPV can be generated by systemic and mucosal immunization with CVP vaccines. Serum antibody from both subcutaneously-vaccinated and intranasally-vaccinated mice showed neutralizing activity against CPV in vitro.

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gromowski, Gregory D.; Barrett, Alan D.T.

2007-09-30

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity ofmore » each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.« less

Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

PubMed Central

Keller, Jenny; Leulliot, Nicolas; Cambillau, Christian; Campanacci, Valérie; Porciero, Stéphanie; Prangishvili, David; Forterre, Patrick; Cortez, Diego; Quevillon-Cheruel, Sophie; van Tilbeurgh, Herman

2007-01-01

The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes. PMID:17241456

Hepatitis C virus inhibitor synergism suggests multistep interactions between heat-shock protein 90 and hepatitis C virus replication

PubMed Central

Kubota, Naoko; Nomoto, Masataka; Hwang, Gi-Wook; Watanabe, Toshihiko; Kohara, Michinori; Wakita, Takaji; Naganuma, Akira; Kuge, Shusuke

2016-01-01

AIM: To address the effect of heat-shock protein 90 (HSP90) inhibitors on the release of the hepatitis C virus (HCV), a cell culture-derived HCV (JFH1/HCVcc) from Huh-7 cells was examined. METHODS: We quantified both the intracellular and extracellular (culture medium) levels of the components (RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A (CsA) or interferon. Finally, we statistically examined the combined effect of radicicol and CsA using the combination index (CI) and graphical representation proposed by Chou and Talalay. RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor (CsA or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy (CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release. PMID:26925202

Crispoic acid, a new compound from Laelia marginata (Orchidaceae), and biological evaluations against parasites, human cancer cell lines and Zika virus.

PubMed

Belloto, Andrezza C; Souza, Gredson K; Perin, Paula C; Schuquel, Ivania T A; Santin, Silvana M O; Chiavelli, Lucas U R; Garcia, Francielle P; Kaplum, Vanessa; Rodrigues, Jean H S; Scariot, Débora B; Delvecchio, Rodrigo; Machado-Ferreira, Erik; Santana Aguiar, Renato; Soares, Carlos A G; Nakamura, Celso V; Pomini, Armando M

2017-11-08

The phytochemical study of Laelia marginata (Lindl.) L. O. Williams (Orchidaceae) led to the isolation of a new natural product named crispoic acid (1), together with six other known compounds (2-7). The new natural product was identified as a dimer of eucomic acid and was structurally characterised based upon 1D and 2D NMR and HRMS data. Biological assays with plant crude extract, fractions and isolated compounds were performed against two human cancer cell lines (Hela and Siha), and the tropical parasites Trypanosoma cruzi and Leishmania (Leishmania) amazonensis. The phenantrenoid 9,10-dihydro-4-methoxyphenanthren-2,7-diol 2 was active against Hela and Siha cells (CC 50 5.86 ± 0.19 and 20.78 ± 2.72 μg/mL, respectively). Sub-lethal concentrations of the flavone rhamnazin 4 were not able to rescue the viability of the Vero cells infected by Zika virus.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ting; Ooi, Amy; Lee, Hooi Chen

An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pHmore » 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.« less

Synthetic peptides for the immunodiagnosis of hepatitis A virus infection.

PubMed

Gauna, A; Losada, S; Lorenzo, M; Bermúdez, H; Toledo, M; Pérez, H; Chacón, E; Noya, O

2015-12-01

VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution.

PubMed

Jia, X; Grove, A; Ivancic, M; Hsu, V L; Geiduscheck, E P; Kearns, D R

1996-10-25

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.