Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements

PubMed Central

Wang, Jianglin; Wang, Lin; Li, Xin; Mao, Chuanbin

2013-01-01

Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant. PMID:23393624

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

PubMed

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael; Zlotnick, Adam

2018-01-29

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. © 2017, Schlicksup et al.

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

PubMed Central

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael

2018-01-01

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. PMID:29377794

Unlocking Internal Prestress from Protein Nanoshells

NASA Astrophysics Data System (ADS)

Klug, W. S.; Roos, W. H.; Wuite, G. J. L.

2012-10-01

The capsids of icosahedral viruses are closed shells assembled from a hexagonal lattice of proteins with fivefold angular defects located at the icosahedral vertices. Elasticity theory predicts that these disclinations are subject to an internal compressive prestress, which provides an explanation for the link between size and shape of capsids. Using a combination of experiment and elasticity theory we investigate the question of whether macromolecular assemblies are subject to residual prestress, due to basic geometric incompatibility of the subunits. Here we report the first direct experimental test of the theory: by controlled removal of protein pentamers from the icosahedral vertices, we measure the mechanical response of so-called “whiffle ball” capsids of herpes simplex virus, and demonstrate the signature of internal prestress locked into wild-type capsids during assembly.

RNA encapsidation by SV40-derived nanoparticles follows a rapid two-state mechanism

PubMed Central

Kler, Stanislav; Asor, Roi; Li, Chenglei; Ginsburg, Avi; Harries, Daniel; Oppenheim, Ariella; Zlotnick, Adam; Raviv, Uri

2012-01-01

Remarkably, uniform virus-like particles self-assemble in a process that appears to follow a rapid kinetic mechanism. The mechanisms by which spherical viruses assemble from hundreds of capsid proteins around nucleic acid, however, are yet unresolved. Using Time-Resolved Small-Angle X-ray Scattering (TR-SAXS) we have been able to directly visualize SV40 VP1 pentamers encapsidating short RNA molecules (500 mers). This assembly process yields T = 1 icosahedral particles comprised of 12 pentamers and one RNA molecule. The reaction is nearly 1/3 complete within 35 milliseconds, following a two–state kinetic process with no detectable intermediates. Theoretical analysis of kinetics, using a master equation, shows that the assembly process nucleates at the RNA and continues by a cascade of elongation reactions in which one VP1 pentamer is added at a time, with a rate of approximately 109 M−1 s−1. The reaction is highly robust and faster than the predicted diffusion limit. The emerging molecular mechanism, which appears to be general to viruses that assemble around nucleic acids, implicates long-ranged electrostatic interactions. The model proposes that the growing nucleo-protein complex acts as an electrostatic antenna that attracts other capsid subunits for the encapsidation process. PMID:22329660

A parameter estimation technique for stochastic self-assembly systems and its application to human papillomavirus self-assembly.

PubMed

Kumar, M Senthil; Schwartz, Russell

2010-12-09

Virus capsid assembly has been a key model system for studies of complex self-assembly but it does pose some significant challenges for modeling studies. One important limitation is the difficulty of determining accurate rate parameters. The large size and rapid assembly of typical viruses make it infeasible to directly measure coat protein binding rates or deduce them from the relatively indirect experimental measures available. In this work, we develop a computational strategy to deduce coat-coat binding rate parameters for viral capsid assembly systems by fitting stochastic simulation trajectories to experimental measures of assembly progress. Our method combines quadratic response surface and quasi-gradient descent approximations to deal with the high computational cost of simulations, stochastic noise in simulation trajectories and limitations of the available experimental data. The approach is demonstrated on a light scattering trajectory for a human papillomavirus (HPV) in vitro assembly system, showing that the method can provide rate parameters that produce accurate curve fits and are in good concordance with prior analysis of the data. These fits provide an insight into potential assembly mechanisms of the in vitro system and give a basis for exploring how these mechanisms might vary between in vitro and in vivo assembly conditions.

A parameter estimation technique for stochastic self-assembly systems and its application to human papillomavirus self-assembly

NASA Astrophysics Data System (ADS)

Senthil Kumar, M.; Schwartz, Russell

2010-12-01

Virus capsid assembly has been a key model system for studies of complex self-assembly but it does pose some significant challenges for modeling studies. One important limitation is the difficulty of determining accurate rate parameters. The large size and rapid assembly of typical viruses make it infeasible to directly measure coat protein binding rates or deduce them from the relatively indirect experimental measures available. In this work, we develop a computational strategy to deduce coat-coat binding rate parameters for viral capsid assembly systems by fitting stochastic simulation trajectories to experimental measures of assembly progress. Our method combines quadratic response surface and quasi-gradient descent approximations to deal with the high computational cost of simulations, stochastic noise in simulation trajectories and limitations of the available experimental data. The approach is demonstrated on a light scattering trajectory for a human papillomavirus (HPV) in vitro assembly system, showing that the method can provide rate parameters that produce accurate curve fits and are in good concordance with prior analysis of the data. These fits provide an insight into potential assembly mechanisms of the in vitro system and give a basis for exploring how these mechanisms might vary between in vitro and in vivo assembly conditions.

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

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

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

Modeling Viral Capsid Assembly

PubMed Central

2014-01-01

I present a review of the theoretical and computational methodologies that have been used to model the assembly of viral capsids. I discuss the capabilities and limitations of approaches ranging from equilibrium continuum theories to molecular dynamics simulations, and I give an overview of some of the important conclusions about virus assembly that have resulted from these modeling efforts. Topics include the assembly of empty viral shells, assembly around single-stranded nucleic acids to form viral particles, and assembly around synthetic polymers or charged nanoparticles for nanotechnology or biomedical applications. I present some examples in which modeling efforts have promoted experimental breakthroughs, as well as directions in which the connection between modeling and experiment can be strengthened. PMID:25663722

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

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

Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.

2010-01-12

In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of somemore » antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.« less

Classification of cryo electron microscopy images, noisy tomographic images recorded with unknown projection directions, by simultaneously estimating reconstructions and application to an assembly mutant of Cowpea Chlorotic Mottle Virus and portals of the bacteriophage P22

NASA Astrophysics Data System (ADS)

Lee, Junghoon; Zheng, Yili; Yin, Zhye; Doerschuk, Peter C.; Johnson, John E.

2010-08-01

Cryo electron microscopy is frequently used on biological specimens that show a mixture of different types of object. Because the electron beam rapidly destroys the specimen, the beam current is minimized which leads to noisy images (SNR substantially less than 1) and only one projection image per object (with an unknown projection direction) is collected. For situations where the objects can reasonably be described as coming from a finite set of classes, an approach based on joint maximum likelihood estimation of the reconstruction of each class and then use of the reconstructions to label the class of each image is described and demonstrated on two challenging problems: an assembly mutant of Cowpea Chlorotic Mottle Virus and portals of the bacteriophage P22.

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

PubMed Central

Sherer, Nathan M.; Jin, Jing; Mothes, Walther

2010-01-01

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

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

Lamb, Kristen; Lokesh, G.L.; Sherman, Michael

2010-10-25

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to themore » mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.« less

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

PubMed

Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

2016-06-01

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Rationally engineering natural protein assemblies in nanobiotechnology.

PubMed

Howorka, Stefan

2011-08-01

Multimeric protein assemblies are essential components in viruses, bacteria, eukaryotic cells, and organisms where they act as cytoskeletal scaffold, storage containers, or for directional transport. The bottom-up structures can be exploited in nanobiotechnology by harnessing their built-in properties and combining them with new functional modules. This review summarizes the design principles of natural protein assemblies, highlights recent progress in their structural elucidation, and shows how rational engineering can create new biomaterials for applications in vaccine development, biocatalysis, materials science, and synthetic biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding ▿

PubMed Central

Chen, Benjamin J.; Leser, George P.; Jackson, David; Lamb, Robert A.

2008-01-01

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur. PMID:18701586

Antiviral RNA Recognition and Assembly by RLR Family Innate Immune Sensors

PubMed Central

Bruns, Annie M.; Horvath, Curt M.

2014-01-01

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1–4]. One important group of cytosolic RNA sensors known as the RIG-I like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5, 6]. PMID:25081315

Highly efficient strategy for the heterologous expression and purification of soluble Cowpea chlorotic mottle virus capsid protein and in vitro pH-dependent assembly of virus-like particles.

PubMed

Díaz-Valle, Armando; García-Salcedo, Yardena M; Chávez-Calvillo, Gabriela; Silva-Rosales, Laura; Carrillo-Tripp, Mauricio

2015-12-01

Obtaining pure and soluble viral capsid proteins (CPs) has been a major challenge in the fields of science and technology in recent decades. In many cases, the CPs can self-assemble in the absence of a viral genome, resulting in non-infectious, empty virus-like particles (VLPs) which can be safely handled. The use of VLPs has found great potential in biotechnology and health purposes. In addition, VLPs are a good model system to study protein-protein interactions at the molecular level. In this work, an optimized strategy for the heterologous expression of the Cowpea chlorotic mottle virus (CCMV) CP based in Escherichia coli is described. The method is efficient, inexpensive and it consistently produces higher yields and greater purity levels than those reported so far. Additionally, one of the main advantages of this method is the prevention of the formation of inclusion bodies, thus allowing to directly obtain high amounts of the CP in a soluble and functionally active state with the capacity to readily form VLPs in vitro. The CCMV CP self-assembly pH dependence was also investigated, providing guidelines to easily modulate the process. Copyright © 2015 Elsevier B.V. All rights reserved.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

PubMed

Maassen, Stan J; de Ruiter, Mark V; Lindhoud, Saskia; Cornelissen, Jeroen J L M

2018-05-23

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.

PubMed

Yang, Chunyan; Pan, Huirong; Wei, Minxi; Zhang, Xiao; Wang, Nan; Gu, Ying; Du, Hailian; Zhang, Jun; Li, Shaowei; Xia, Ningshao

2013-03-01

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions. Copyright © 2012 The Protein Society.

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.

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

Gupta, Arunava; Prevelige, Peter E

The primary goal of the project was to develop protein-templated approaches for the synthesis and directed assembly of semiconductor nanomaterials that are efficient for visible light absorption and hydrogen production. In general, visible-light-driven photocatalysis reactions exhibit low quantum efficiency for solar energy conversion primarily because of materials-related issues and limitations, such as the control of the band gap, band structure, photochemical stability, and available reactive surface area of the photocatalyst. Synthesis of multicomponent hierarchical nano-architectures, consisting of semiconductor nanoparticles (NPs) with desired optical properties fabricated to maximize spatial proximity for optimum electron and energy transfer represents an attractive route formore » addressing the problem. Virus capsids are highly symmetrical, self-assembling protein cage nanoparticles that exist in a range of sizes and symmetries. Selective deposition of inorganic, by design, at specific locations on virus capsids affords precise control over the size, spacing, and assembly of nanomaterials, resulting in uniform and reproducible nano-architectures. We utilized the self-assembling capabilities of the 420 subunit, 60 nm icosahedral, P22 virus capsid to direct the nucleation, growth, and proximity of a range of component materials. Controlled fabrication on the exterior of the temperature stable shell was achieved by genetically encoding specific binding peptides into an externally exposed loop which is displayed on each of the 420 coat protein subunits. Localization of complimentary materials to the interior of the particle was achieved through the use “scaffolding-fusion proteins. The scaffolding domain drives coat protein polymerization resulting in a coat protein shell surrounding a core of approximately 300 scaffolding/fusion molecules. The fusion domain comprises a peptide which specifically binds the semiconductor material of interest.« less

The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

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

Li, Long; Lok, Shee-Mei; Yu, I-Mei

2008-09-17

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventingmore » fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.« less

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis

DOE PAGES

Uchida, Masaki; McCoy, Kimberly; Fukuto, Masafumi; ...

2017-11-13

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from anmore » amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. As a result, this study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.« less

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis

PubMed Central

Uchida, Masaki; McCoy, Kimberly; Fukuto, Masafumi; Yang, Lin; Yoshimura, Hideyuki; Miettinen, Heini M.; LaFrance, Ben; Patterson, Dustin P.; Schwarz, Benjamin; Karty, Jonathan A.; Prevelige, Peter E.; Lee, Byeongdu; Douglas, Trevor

2018-01-01

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy; the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles, and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales, and exhibits properties and function that arise from the interaction between individual building blocks. PMID:29131580

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis

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

Uchida, Masaki; McCoy, Kimberly; Fukuto, Masafumi

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from anmore » amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. As a result, this study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.« less

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

PubMed

Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption

PubMed Central

Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae

2013-01-01

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications, but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions. PMID:23899192

Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle▿

PubMed Central

Bourne, Christina; Lee, Sejin; Venkataiah, Bollu; Lee, Angela; Korba, Brent; Finn, M. G.; Zlotnick, Adam

2008-01-01

The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors. PMID:18684823

RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane.

PubMed

Vanover, Daryll; Smith, Daisy V; Blanchard, Emmeline L; Alonas, Eric; Kirschman, Jonathan L; Lifland, Aaron W; Zurla, Chiara; Santangelo, Philip J

2017-09-22

The human respiratory syncytial virus G protein plays an important role in the entry and assembly of filamentous virions. Here, we report the use of fluorescently labeled soybean agglutinin to selectively label the respiratory syncytial virus G protein in living cells without disrupting respiratory syncytial virus infectivity or filament formation and allowing for interrogations of respiratory syncytial virus virion assembly. Using this approach, we discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis. This event is then followed by the dynamic formation of filamentous and branched respiratory syncytial virus particles, and assembly with genomic ribonucleoproteins and caveolae-associated vesicles prior to re-insertion into the plasma membrane. We demonstrate that these processes are halted by the disruption of microtubules and inhibition of molecular motors. Collectively, our results show that for respiratory syncytial virus assembly, viral filaments are produced and loaded with genomic RNA prior to insertion into the plasma membrane.Assembly of filamentous RSV particles is incompletely understood due to a lack of techniques suitable for live-cell imaging. Here Vanover et al. use labeled soybean agglutinin to selectively label RSV G protein and show how filamentous RSV assembly, initiated in the cytoplasm, uses G protein recycled from the plasma membrane.

Virus assembly occurs following a pH- or Ca2+-triggered switch in the thermodynamic attraction between structural protein capsomeres.

PubMed

Chuan, Yap P; Fan, Yuan Y; Lua, Linda H L; Middelberg, Anton P J

2010-03-06

Viral self-assembly is of tremendous virological and biomedical importance. Although theoretical and crystallographic considerations suggest that controlled conformational change is a fundamental regulatory mechanism in viral assembly, direct proof that switching alters the thermodynamic attraction of self-assembling components has not been provided. Using the VP1 protein of polyomavirus, we report a new method to quantitatively measure molecular interactions under conditions of rapid protein self-assembly. We show, for the first time, that triggering virus capsid assembly through biologically relevant changes in Ca(2+) concentration, or pH, is associated with a dramatic increase in the strength of protein molecular attraction as quantified by the second virial coefficient (B(22)). B(22) decreases from -2.3 x 10(-4) mol ml g(-2) (weak protein-protein attraction) to -2.4 x 10(-3) mol ml g(-2) (strong protein attraction) for metastable and Ca(2+)-triggered self-assembling capsomeres, respectively. An assembly-deficient mutant (VP1CDelta63) is conversely characterized by weak protein-protein repulsion independently of chemical change sufficient to cause VP1 assembly. Concomitant switching of both VP1 assembly and thermodynamic attraction was also achieved by in vitro changes in ammonium sulphate concentration, consistent with protein salting-out behaviour. The methods and findings reported here provide new insight into viral assembly, potentially facilitating the development of new antivirals and vaccines, and will open the way to a more fundamental physico-chemical description of complex protein self-assembly systems.

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

Diversity in virus assembly: biology makes things complicated

NASA Astrophysics Data System (ADS)

Zlotnick, Adam

2008-03-01

Icosahedral viruses have an elegance of geometry that implies a general path of assembly. However, structure alone provides insufficient information. Cowpea Chlorotic Mottle Virus (CCMV), an important system for studying virus assembly, consists of 90 coat protein (CP) homodimers condensed around an RNA genome. The crystal structure (Speir et al, 1995) reveals that assembly causes burial of hydrophobic surface and formation of β hexamers, the intertwining of N-termini of the CPs surrounding a quasi-sixfold. This structural view leads to reasonable and erroneous predictions: (i) CCMV capsids are extremely stable, and (ii) β hexamer formation is critical to assembly. Experimentally, we have found that capsids are based on a network of extremely weak (4-5 kT) pairwise interactions and that pentamer formation is the critical step in assembly kinetics. Because of the fragility of CP-Cp interaction, we can redirect assembly to generate and dissociate tubular nanostructures. The dynamic behavior of CCMV reflects the requirements and peculiarities of an evolved biological system; it does not necessarily reflect the behavior predicted from a more static picture of the virus.

Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

PubMed

Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

2016-10-01

Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

PubMed

Hong, Lewis Z; Hong, Shuzhen; Wong, Han Teng; Aw, Pauline P K; Cheng, Yan; Wilm, Andreas; de Sessions, Paola F; Lim, Seng Gee; Nagarajan, Niranjan; Hibberd, Martin L; Quake, Stephen R; Burkholder, William F

2014-01-01

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

Dynamics of HIV-1 Assembly and Release

PubMed Central

Ivanchenko, Sergey; Godinez, William J.; Lampe, Marko; Kräusslich, Hans-Georg; Eils, Roland; Rohr, Karl; Bräuchle, Christoph; Müller, Barbara; Lamb, Don C.

2009-01-01

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics. PMID:19893629

Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles.

PubMed

Liu, Zhi; Qiao, Jing; Niu, Zhongwei; Wang, Qian

2012-09-21

Viruses belong to a fascinating class of natural supramolecular structures, composed of multiple copies of coat proteins (CPs) that assemble into different shapes with a variety of sizes from tens to hundreds of nanometres. Because of their advantages including simple/economic production, well-defined structural features, unique shapes and sizes, genetic programmability and robust chemistries, recently viruses and virus-like nanoparticles (VLPs) have been used widely in biomedical applications and materials synthesis. In this critical review, we highlight recent advances in the use of virus coat proteins (VCPs) and viral nanoparticles (VNPs) as building blocks in self-assembly studies and materials development. We first discuss the self-assembly of VCPs into VLPs, which can efficiently incorporate a variety of different materials as cores inside the viral protein shells. Then, the self-assembly of VNPs at surfaces or interfaces is summarized. Finally, we discuss the co-assembly of VNPs with different functional materials (178 references).

Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

PubMed

Weller, Romy; Hueging, Kathrin; Brown, Richard J P; Todt, Daniel; Joecks, Sebastian; Vondran, Florian W R; Pietschmann, Thomas

2017-09-15

Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry. IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry. Copyright © 2017 Weller et al.

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.

NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

PubMed Central

Brown, Lola A.; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G.; Kuo, Lillian; Freed, Eric O.; Summers, Michael F.

2015-01-01

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly. PMID:25941825

NMR structure of the myristylated feline immunodeficiency virus matrix protein.

PubMed

Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

2015-04-30

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

PubMed

Huang, Hsiu-Chen; Lee, Chung-Pei; Liu, Hui-Kang; Chang, Ming-Fu; Lai, Yu-Heng; Lee, Yu-Ching; Huang, Cheng

2016-12-09

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro 205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

PubMed

Medrano, María; Fuertes, Miguel Ángel; Valbuena, Alejandro; Carrillo, Pablo J P; Rodríguez-Huete, Alicia; Mateu, Mauricio G

2016-11-30

Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

Mechanisms of Virus Assembly

PubMed Central

Perlmutter, Jason D.; Hagan, Michael F.

2015-01-01

Viruses are nanoscale entities containing a nucleic acid genome encased in a protein shell called a capsid, and in some cases surrounded by a lipid bilayer membrane. This review summarizes the physics that govern the processes by which capsids assembles within their host cells and in vitro. We describe the thermodynamics and kinetics for assembly of protein subunits into icosahedral capsid shells, and how these are modified in cases where the capsid assembles around a nucleic acid or on a lipid bilayer. We present experimental and theoretical techniques that have been used to characterize capsid assembly, and we highlight aspects of virus assembly which are likely to receive significant attention in the near future. PMID:25532951

Identification of Factors Promoting HBV Capsid Self-Assembly by Assembly-Promoting Antivirals.

PubMed

Rath, Soumya Lipsa; Liu, Huihui; Okazaki, Susumu; Shinoda, Wataru

2018-02-26

Around 270 million individuals currently live with hepatitis B virus (HBV) infection. Heteroaryldihydropyrimidines (HAPs) are a family of antivirals that target the HBV capsid protein and induce aberrant self-assembly. The capsids formed resemble the native capsid structure but are unable to propagate the virus progeny because of a lack of RNA/DNA. Under normal conditions, self-assembly is initiated by the viral genome. The mode of action of HAPs, however, remains largely unknown. In this work, using molecular dynamics simulations, we attempted to understand the action of HAP by comparing the dynamics of capsid proteins with and without HAPs. We found that the inhibitor is more stable in higher oligomers. It retains its stability in the hexamer throughout 1 μs of simulation. Our results also show that the inhibitor might help in stabilizing the C-terminus, the HBc 149-183 arginine-rich domain of the capsid protein. The C-termini of dimers interact with each other, assisted by the HAP inhibitor. During capsid assembly, the termini are supposed to directly interact with the viral genome, thereby suggesting that the viral genome might work in a similar way to stabilize the capsid protein. Our results may help in understanding the underlying molecular mechanism of HBV capsid self-assembly, which should be crucial for exploring new drug targets and structure-based drug design.

Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production.

PubMed

El Najjar, Farah; Schmitt, Anthony P; Dutch, Rebecca Ellis

2014-08-07

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.

Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

PubMed Central

El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

2014-01-01

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

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

Huang Jialing, E-mail: hjialing@mail.med.upenn.edu; Lazear, Helen M., E-mail: Hlazear@DOM.wustl.edu; Friedman, Harvey M., E-mail: hfriedma@mail.med.upenn.ed

2011-01-05

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infectedmore » with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.« less

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure.

PubMed

Santos, Jose L R; Aparicio, Ricardo; Joekes, Inés; Silva, Jerson L; Bispo, Jose A C; Bonafe, Carlos F S

2008-05-01

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems.

Measuring phenotypic variability and plasticity in influenza A virus using multispectral viral strains

NASA Astrophysics Data System (ADS)

Vahey, Michael

Despite relevance to human health, the mechanisms of enveloped virus assembly remain largely mysterious. This is particularly true of influenza A virus (IAV), which (unlike viral capsids with stereotyped shape and composition) forms heterogeneous particles whose assembly cannot be described in terms of equilibrium thermodynamics. Although the ability to assemble into particles with diverse size and composition could have important implications for infectivity, understanding how virion-to-virion differences arise and how they ultimately influence virus replication has proven challenging due to the lack of available tools for studying the assembly process. To address this challenge and establish a dynamic picture of how IAV assembles, we have developed virus strains that harbor small, non-disruptive fluorescent tags on each of the virus's five major structural proteins. Using these multispectral strains, we are able to quantify the protein composition and dynamics of virions as they assemble in live infected cells - measurements that have been previously inaccessible, and which reveal subpopulations of virus that favor either the binding or destruction of host receptors. The occupancy of these different subpopulations is malleable, shifting in response to environmental stimuli, including antiviral drugs that block receptor-destruction. In complex environments like the human respiratory tract, this phenotypic diversity could act as an evolutionary hedge. We acknowledge the Burroughs Wellcome Fund and NIH NIGMS for supporting this work.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

PubMed

Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

2010-02-01

Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

Deep Sequencing Analysis of Apple Infecting Viruses in Korea

PubMed Central

Cho, In-Sook; Igori, Davaajargal; Lim, Seungmo; Choi, Gug-Seoun; Hammond, John; Lim, Hyoun-Sub; Moon, Jae Sun

2016-01-01

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time. PMID:27721694

FASEB Science Research Conference on Virus Structure and Assembly

DTIC Science & Technology

2017-10-24

Report: FASEB Science Research Conference on Virus Structure and Assembly The views, opinions and/or findings contained in this report are those of the...SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS (ES) U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 REPORT...Title: FASEB Science Research Conference on Virus Structure and Assembly Report Term: 0-Other Email: srcgrants@faseb.org Distribution Statement

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

PubMed

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

PubMed Central

Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

2014-01-01

ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. PMID:25210190

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

Rewriting nature's assembly manual for a ssRNA virus.

PubMed

Patel, Nikesh; Wroblewski, Emma; Leonov, German; Phillips, Simon E V; Tuma, Roman; Twarock, Reidun; Stockley, Peter G

2017-11-14

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes. Copyright © 2017 the Author(s). Published by PNAS.

Biochemical Requirements of Virus Wrapping by the Endoplasmic Reticulum: Involvement of ATP and Endoplasmic Reticulum Calcium Store during Envelopment of African Swine Fever Virus

PubMed Central

Cobbold, Christian; Brookes, Sharon M.; Wileman, Thomas

2000-01-01

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae. PMID:10666244

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

PubMed

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

PubMed Central

Grzesik, Peter; Voorhies, Alexander A.; Alperovich, Nina; MacMath, Derek; Najera, Claudia D.; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N.; Montague, Michael G.; Friedman, Robert M.; Desai, Prashant J.

2017-01-01

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats. PMID:28928148

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.

PubMed

Bharat, Tanmay A M; Noda, Takeshi; Riches, James D; Kraehling, Verena; Kolesnikova, Larissa; Becker, Stephan; Kawaoka, Yoshihiro; Briggs, John A G

2012-03-13

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

Time-resolved spectroscopy of self-assembly of CCMV protein capsids

NASA Astrophysics Data System (ADS)

Moore, Jelyn; Aronzon, Dina; Manoharan, V. N.

2008-10-01

In order to gain a deeper understanding of the process a virus undergoes to assemble; the purpose of this study to time resolve the self-assembly of a virus. Cowpea Chlorotic Mottle virus (CCMV), an icosahedral type virus, can assemble without its genetic code (RNA) depending on its chemical and physical surroundings. The surface plasmon resonance (SPR) of colloidal gold particles is known to display a shift when the gold interacts with the proteins of a virus. Surface plasmon resonance is the free electron oscillation occurring at the surface of the gold particle resulting in a characteristic peak location at maximal absorbance and peak width. The shift results from the change in the refractive index of the particles as induced by the presence of the proteins. We hope to detect this shift through total internal reflection microscopy (TIRM). The accomplishments of this research are the completion of the TIR setup and the purification of the virus and its proteins.

Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus

NASA Astrophysics Data System (ADS)

Zhou, Zhuo; Cao, Mengmeng; Guo, Yang; Zhao, Lili; Wang, Jingfeng; Jia, Xue; Li, Jianguo; Wang, Conghui; Gabriel, Gülsah; Xue, Qinghua; Yi, Yonghong; Cui, Sheng; Jin, Qi; Wang, Jianwei; Deng, Tao

2014-02-01

The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.

Representation of viruses in the remediated PDB archive

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

Lawson, Catherine L., E-mail: cathy.lawson@rutgers.edu; Dutta, Shuchismita; Westbrook, John D.

2008-08-01

A new data model for PDB entries of viruses and other biological assemblies with regular noncrystallographic symmetry is described. A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies,more » subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive.« less

The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites.

PubMed

Harvey, Stephen C; Zeng, Yingying; Heitsch, Christine E

2013-03-01

There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure--often one or more stem-loops--either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these "fingers" of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

PubMed

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

2016-11-18

Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus

PubMed Central

Mangala Prasad, Vidya

2017-01-01

Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway. PMID:28575072

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus.

PubMed

Mangala Prasad, Vidya; Klose, Thomas; Rossmann, Michael G

2017-06-01

Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

PubMed Central

Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

2014-01-01

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

Integration of plant viruses in electron beam lithography nanostructures.

PubMed

Alonso, Jose M; Ondarçuhu, Thierry; Bittner, Alexander M

2013-03-15

Tobacco mosaic virus (TMV) is the textbook example of a virus, and also of a self-assembling nanoscale structure. This tubular RNA/protein architecture has also found applications as biotemplate for the synthesis of nanomaterials such as wires, as tubes, or as nanoparticle assemblies. Although TMV is, being a biological structure, quite resilient to environmental conditions (temperature, chemicals), it cannot be processed in electron beam lithography (eBL) fabrication, which is the most important and most versatile method of nanoscale structuring. Here we present adjusted eBL-compatible processes that allow the incorporation of TMV in nanostructures made of positive and negative tone eBL resists. The key steps are covering TMV by polymer resists, which are only heated to 50 °C, and development (selective dissolution) in carefully selected organic solvents. We demonstrate the post-lithography biochemical functionality of TMV by selective immunocoating of the viral particles, and the use of immobilized TMV as direct immunosensor. Our modified eBL process should be applicable to incorporate a wide range of sensitive materials in nanofabrication schemes.

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

PubMed

Valbuena, Alejandro; Mateu, Mauricio G

2017-02-28

Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

PubMed

Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui; Lee, Il-Hyung; Hurley, James H

2015-10-23

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. Copyright © 2015, American Association for the Advancement of Science.

Production of Japanese encephalitis virus-like particles in insect cells.

PubMed

Yamaji, Hideki; Konishi, Eiji

2013-01-01

Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as vaccines and diagnostic antigens.

Rab1A is required for assembly of classical swine fever virus particle.

PubMed

Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

2018-01-15

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

PubMed

Nikolaitchik, Olga A; Hu, Wei-Shau

2014-04-01

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts.

Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

PubMed Central

Nikolaitchik, Olga A.

2014-01-01

ABSTRACT A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5′ untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts. PMID:24453371

Designed Proteins Induce the Formation of Nanocage-containing Extracellular Vesicles

PubMed Central

Votteler, Jörg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.

2017-01-01

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids, and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3–5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here, we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as Enveloped Protein Nanocages (EPNs). Robust EPN biogenesis required protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induced EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical and electron cryomicroscopic (cryo-EM) analyses revealed that one design, EPN-01, comprised small (~100 nm) vesicles containing multiple protein nanocages that closely matched the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV-G) could fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These studies show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a novel class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells. PMID:27919066

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

PubMed Central

Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

1996-01-01

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

PubMed Central

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C.; Miller, W. Allen

2016-01-01

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits. PMID:27869713

Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

NASA Astrophysics Data System (ADS)

de Ruiter, Mark V.; Overeem, Nico J.; Singhai, Gaurav; Cornelissen, Jeroen J. L. M.

2018-05-01

Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus.

PubMed

Jo, Yeonhwa; Choi, Hoseong; Kim, Sang-Min; Kim, Sun-Lim; Lee, Bong Choon; Cho, Won Kyong

2016-08-09

Next-generation sequencing (NGS) provides many possibilities for plant virology research. In this study, we performed integrated analyses using plant transcriptome data for plant virus identification using Apple stem grooving virus (ASGV) as an exemplar virus. We used 15 publicly available transcriptome libraries from three different studies, two mRNA-Seq studies and a small RNA-Seq study. We de novo assembled nearly complete genomes of ASGV isolates Fuji and Cuiguan from apple and pear transcriptomes, respectively, and identified single nucleotide variations (SNVs) of ASGV within the transcriptomes. We demonstrated the application of NGS raw data to confirm viral infections in the plant transcriptomes. In addition, we compared the usability of two de novo assemblers, Trinity and Velvet, for virus identification and genome assembly. A phylogenetic tree revealed that ASGV and Citrus tatter leaf virus (CTLV) are the same virus, which was divided into two clades. Recombination analyses identified six recombination events from 21 viral genomes. Taken together, our in silico analyses using NGS data provide a successful application of plant transcriptomes to reveal extensive information associated with viral genome assembly, SNVs, phylogenetic relationships, and genetic recombination.

Problems in understanding the structure and assembly of viruses

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

King, J.

1997-12-01

Though viruses infect the cells of all groups of animals, plants, and microorganisms, their structures follow a limited number of general themes; spherical or cylindrical shells built of hundreds of repeated protein subunits enclosing a nucleic acid - DNA or RNA - genome. Since the 1960s it has been known that the protein shells of spherical viruses in fact conform to icosahedral symmetry or to subtle deviations from icosahedral symmetry. The construction of the shell lattices and the transformations they go through in the different stages of the viral life cycle are not fully understood. The shells contain the nucleicmore » in a highly condensed state, of unknown coiling/organization. Features of the well studied bacterial viruses will be reviewed, with examples from adenoviruses, herpesviruses, poliovirus, and HIV. The emergence of new viral disease has led to increased interest in the development of agents which interfere with virus reproduction at the level of the assembly or function of the organized particle. Recently computational approaches to the problem of virus assembly have made important contributions to clarifying shell assembly processes. 1 ref.« less

HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

PubMed

Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

2016-01-01

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

HIV-1 Viral RNA Dynamics at the Plasma Membrane May Provide Insight into Viral Assembly | Poster

Cancer.gov

Many aspects of how infectious viruses assemble in cells have yet to be completely deciphered. However, as reported in a recent Journal of Virology paper, researchers may be one step closer to understanding how HIV-1, the virus that causes AIDS, assembles and replicates.

Ebola virus VP35 blocks stress granule assembly.

PubMed

Le Sage, Valerie; Cinti, Alessandro; McCarthy, Stephen; Amorim, Raquel; Rao, Shringar; Daino, Gian Luca; Tramontano, Enzo; Branch, Donald R; Mouland, Andrew J

2017-02-01

Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptomic response of the insect vector, Peregrinus maidis, to Maize mosaic rhabdovirus and identification of conserved responses to propagative viruses in hopper vectors.

PubMed

Martin, Kathleen M; Barandoc-Alviar, Karen; Schneweis, Derek J; Stewart, Catherine L; Rotenberg, Dorith; Whitfield, Anna E

2017-09-01

Maize mosaic virus (MMV) is a plant-pathogenic rhabdovirus that is transmitted by the corn planthopper, Peregrinus maidis, in a propagative manner. P. maidis supports long-term MMV infections with no negative effects on insect performance. To elucidate whole-body transcriptome responses to virus infection, RNA-Seq was used to examine differential gene expression of virus-infected adult insects, and libraries were prepared from replicated groups of virus-exposed insects and non-exposed insects. From the 68,003 de novo-assembled transcripts, 144 were differentially-expressed (DE) during viral infection with comparable numbers up- and down-regulated. DE transcripts with similarity to genes associated with transposable elements (i.e., RNA-directed DNA polymerases) were enriched and may represent a mechanisim for modulating virus infection. Comparison of the P. maidis DE transcripts to published propagative virus-responsive transcript databases for two other hopper vectors revealed that 16% of the DE transcripts were shared across the three systems and may represent conserved responses to propagative viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanical and Assembly Units of Viral Capsids Identified via Quasi-Rigid Domain Decomposition

PubMed Central

Polles, Guido; Indelicato, Giuliana; Potestio, Raffaello; Cermelli, Paolo; Twarock, Reidun; Micheletti, Cristian

2013-01-01

Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV) for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available. PMID:24244139

Electrostatic assembly of binary nanoparticle superlattices using protein cages

NASA Astrophysics Data System (ADS)

Kostiainen, Mauri A.; Hiekkataipale, Panu; Laiho, Ari; Lemieux, Vincent; Seitsonen, Jani; Ruokolainen, Janne; Ceci, Pierpaolo

2013-01-01

Binary nanoparticle superlattices are periodic nanostructures with lattice constants much shorter than the wavelength of light and could be used to prepare multifunctional metamaterials. Such superlattices are typically made from synthetic nanoparticles, and although biohybrid structures have been developed, incorporating biological building blocks into binary nanoparticle superlattices remains challenging. Protein-based nanocages provide a complex yet monodisperse and geometrically well-defined hollow cage that can be used to encapsulate different materials. Such protein cages have been used to program the self-assembly of encapsulated materials to form free-standing crystals and superlattices at interfaces or in solution. Here, we show that electrostatically patchy protein cages--cowpea chlorotic mottle virus and ferritin cages--can be used to direct the self-assembly of three-dimensional binary superlattices. The negatively charged cages can encapsulate RNA or superparamagnetic iron oxide nanoparticles, and the superlattices are formed through tunable electrostatic interactions with positively charged gold nanoparticles. Gold nanoparticles and viruses form an AB8fcc crystal structure that is not isostructural with any known atomic or molecular crystal structure and has previously been observed only with large colloidal polymer particles. Gold nanoparticles and empty or nanoparticle-loaded ferritin cages form an interpenetrating simple cubic AB structure (isostructural with CsCl). We also show that these magnetic assemblies provide contrast enhancement in magnetic resonance imaging.

Challenging Students' Intuitions--The Influence of a Tangible Model of Virus Assembly on Students' Conceptual Reasoning about the Process of Self-Assembly

ERIC Educational Resources Information Center

Larsson, Caroline; Tibell, Lena A.

2015-01-01

A well-ordered biological complex can be formed by the random motion of its components, i.e. self-assemble. This is a concept that incorporates issues that may contradict students' everyday experiences and intuitions. In previous studies, we have shown that a tangible model of virus self-assembly, used in a group exercise, helps students to grasp…

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

PubMed

Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

2017-07-15

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly. Copyright © 2017 American Society for Microbiology.

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2015-01-01

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2015-12-03

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

PubMed

Smithson, Chad; Imbery, Jacob; Upton, Chris

2017-09-08

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

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

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

The mathematics of virus shell assembly. Progress report 1995--1996

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

Berger, B.

1996-08-01

This research focuses on applying computational and mathematical techniques to problems in biology, and more specifically to problems in protein folding. Significant progress has been made in the following areas relating to virus shell assembly: the local rules theory has been further developed; development has begun on a second-generation simulator which provides a more physically realistic model of assembly, collaborative efforts have continued with an experimental biologist to verify and inspire the local rules theory; an investigation has been initiated into the mechanics of virus shell assembly; laboratory experiments have been conducted on bacteriophage T4 which verify that the previouslymore » believed structure for the core may be incorrect.« less

PREFACE The physics of virus assembly The physics of virus assembly

NASA Astrophysics Data System (ADS)

Stockley, Peter G.; Twarock, Reidun

2010-12-01

Viruses are pathogens in every kingdom of life and are major causes of human disease and suffering. They are known to encompass a size range that overlaps with that of the smallest bacterial cells, and the largest viruses now seem to be hosts of their own viral pathogens. Recent genomic sequencing efforts show that many organisms have genes that are likely to be descended in evolution from viral progenitors. Even more astonishingly, analysis of the world's oceans has shown that some of the simplest viruses, the tailed dsDNA phages, are the most common biological entities on the planet, with estimates of their numbers ranging up to 1031, with ~ 1021 infection events every second, leading to a turnover of around 20% of the biomass in the sea every few days. These cycles of infection and lysis of oceanic bacteria and algae provide the nutrients for the smallest organisms lying at the bottom of the food chain. Without viruses, therefore, life on Earth would probably not be sustainable. These are remarkable facts for systems that are non-living in the strict sense, and are composed of simple materials—nucleic acids, proteins and lipids. Many viruses consist of little more than a protective protein coat surrounding their genomic nucleic acids, which can be either DNA or RNA. Their simplicity leads to highly symmetrical structures with protein containers based on helical or icosahedral lattices. Many simple viruses self-assemble rapidly and with great fidelity, and many groups are busy trying to exploit these properties to make virus-like particles for a wide range of applications, including targeted drug-delivery, medical imaging and even novel materials. This issue of Physical Biology contains a series of papers describing some of the latest experimental and theoretical research on viruses, their structures and assembly, as well as their regulated disassembly during infection. These range from a dissection of the in vivo assembly mechanism of a filamentous virus, through structure determination using cryo-electron microscopy, to entirely in silico modelling of assembly pathways. This selection illustrates the interdisciplinarity of modern virus research which interests an ever widening community including physicists, mathematicians and engineers, and is reflected by the recent appearance of international conferences and workshops in physical and mathematical virology. We hope this selection of papers gives the readers of Physical Biology an impression of the exciting developments in this field.

Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

PubMed

Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A; Teschke, Carolyn M

2014-05-01

Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.

PubMed Central

Dolja, V V; Haldeman, R; Robertson, N L; Dougherty, W G; Carrington, J C

1994-01-01

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport. Images PMID:7511101

Nanoscale device architectures derived from biological assemblies: The case of tobacco mosaic virus and (apo)ferritin

NASA Astrophysics Data System (ADS)

Calò, Annalisa; Eiben, Sabine; Okuda, Mitsuhiro; Bittner, Alexander M.

2016-03-01

Virus particles and proteins are excellent examples of naturally occurring structures with well-defined nanoscale architectures, for example, cages and tubes. These structures can be employed in a bottom-up assembly strategy to fabricate repetitive patterns of hybrid organic-inorganic materials. In this paper, we review methods of assembly that make use of protein and virus scaffolds to fabricate patterned nanostructures with very high spatial control. We chose (apo)ferritin and tobacco mosaic virus (TMV) as model examples that have already been applied successfully in nanobiotechnology. Their interior space and their exterior surfaces can be mineralized with inorganic layers or nanoparticles. Furthermore, their native assembly abilities can be exploited to generate periodic architectures for integration in electrical and magnetic devices. We introduce the state of the art and describe recent advances in biomineralization techniques, patterning and device production with (apo)ferritin and TMV.

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

Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

NASA Astrophysics Data System (ADS)

Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

Targeting Nucleotide Biosynthesis: A Strategy for Improving the Oncolytic Potential of DNA Viruses

PubMed Central

Irwin, Chad R.; Hitt, Mary M.; Evans, David H.

2017-01-01

The rapid growth of tumors depends upon elevated levels of dNTPs, and while dNTP concentrations are tightly regulated in normal cells, this control is often lost in transformed cells. This feature of cancer cells has been used to advantage to develop oncolytic DNA viruses. DNA viruses employ many different mechanisms to increase dNTP levels in infected cells, because the low concentration of dNTPs found in non-cycling cells can inhibit virus replication. By disrupting the virus-encoded gene(s) that normally promote dNTP biosynthesis, one can assemble oncolytic versions of these agents that replicate selectively in cancer cells. This review covers the pathways involved in dNTP production, how they are dysregulated in cancer cells, and the various approaches that have been used to exploit this biology to improve the tumor specificity of oncolytic viruses. In particular, we compare and contrast the ways that the different types of oncolytic virus candidates can directly modulate these processes. We limit our review to the large DNA viruses that naturally encode homologs of the cellular enzymes that catalyze dNTP biogenesis. Lastly, we consider how this knowledge might guide future development of oncolytic viruses. PMID:29018771

Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.

PubMed

Jolly, Clare; Mitar, Ivonne; Sattentau, Quentin J

2007-06-01

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

PubMed

Daum, Bertram; Quax, Tessa E F; Sachse, Martin; Mills, Deryck J; Reimann, Julia; Yildiz, Özkan; Häder, Sabine; Saveanu, Cosmin; Forterre, Patrick; Albers, Sonja-Verena; Kühlbrandt, Werner; Prangishvili, David

2014-03-11

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Competing Hydrophobic and Screened-Coulomb Interactions in Hepatitis B Virus Capsid Assembly

PubMed Central

Kegel, Willem K.; Schoot, Paul van der

2004-01-01

Recent experiments show that, in the range from ∼15 to 45°C, an increase in the temperature promotes the spontaneous assembly into capsids of the Escherichia coli-expressed coat proteins of hepatitis B virus. Within that temperature interval, an increase in ionic strength up to five times that of standard physiological conditions also acts to promote capsid assembly. To explain both observations we propose an interaction of mean force between the protein subunits that is the sum of an attractive hydrophobic interaction, driving the self-assembly, and a repulsive electrostatic interaction, opposing the self-assembly. We find that the binding strength of the capsid subunits increases with temperature virtually independently of the ionic strength, and that, at fixed temperature, the binding strength increases with the square root of ionic strength. Both predictions are in quantitative agreement with experiment. We point out the similarities of capsid assembly in general and the micellization of surfactants. Finally we make plausible that electrostatic repulsion between the native core subunits of a large class of virus suppresses the formation in vivo of empty virus capsids, that is, without the presence of the charge-neutralizing nucleic acid. PMID:15189887

DNA Origami Inside-Out Viruses.

PubMed

Burns, Jonathan R; Lamarre, Baptiste; Pyne, Alice L B; Noble, James E; Ryadnov, Maxim G

2018-03-16

A synthetic topology for everted viruses is reported. The topology is a single-stranded virion DNA assembled into a hollow cube with exterior decorated with HIV-Tat transduction domains. The cube incorporates a pH-responsive lid allowing for the controlled encapsulation of functional proteins and their transfer and release into live cells. Unlike viruses, which are protein shells with a [3,5]-fold rotational symmetry that encase nucleic acids, these cubes are [3, 4]-fold DNA boxes encapsulating proteins. Like viruses, such everted DNA-built viruses are monodisperse nanoscale assemblies that infect human cells with a specialist cargo. The design offers a bespoke bottom-up platform for engineering nonpolyhedral, nonprotein synthetic viruses.

Structure and assembly of the Ebola virus nucleocapsid

PubMed Central

Wan, William; Kolesnikova, Larissa; Clarke, Mairi; Koehler, Alexander; Noda, Takeshi; Becker, Stephan; Briggs, John A. G.

2017-01-01

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause hemorrhagic fever1. Filoviruses are within the order Mononegavirales2 which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein (NP) and other viral proteins to form a helical nucleocapsid (NC). NC acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into NP-NP interactions have been derived from structural studies of oligomerized, RNA-encapsidating NP3–6 and cryo-electron microscopy (cryo-EM) of NC7–12 or NC-like structures11–13. There have been no high-resolution reconstructions of complete mononegavirus NCs. Here, we have applied cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus NC within intact viruses and recombinant NC-like assemblies. These structures reveal the identity and arrangement of the NC components, and suggest that the formation of an extended alpha-helix from the disordered C-terminal region of NP-core links NP oligomerization, NC condensation, RNA encapsidation, and accessory protein recruitment. PMID:29144446

Molecular Evidence of Sexual Transmission of Ebola Virus

PubMed Central

Mate, S.E.; Kugelman, J.R.; Nyenswah, T.G.; Ladner, J.T.; Wiley, M.R.; Cordier-Lassalle, T.; Christie, A; Schroth, G.P.; Gross, S.M.; Davies-Wayne, G.J.; Shinde, S.A.; Murugan, R.; Sieh, S.B.; Badio, M.; Fakoli, L.; Taweh, F.; de Wit, E.; van Doremalen, N.; Munster, V.J.; Pettitt, J.; Prieto, K.; Humrighouse, B.W.; Ströher, U.; DiClaro, J.W.; Hensley, L.E.; Schoepp, R.J.; Safronetz, D.; Fair, J.; Kuhn, J.H.; Blackley, D.J.; Laney, A.S.; Williams, D.E.; Lo, T.; Gasasira, A.; Nichol, S.T.; Formenty, P.; Kateh, F.N.; De Cock, K.M.; Bolay, F.; Sanchez-Lockhart, M.; Palacios, G.

2016-01-01

Summary A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.) PMID:26465384

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

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

Wang, Robert Y.L., E-mail: yuwang@mail.cgu.edu.tw; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan; Kuo, Rei-Lin

2013-09-01

Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted tomore » new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone. - Highlights: • Hsp90β is associated with EV71 virion and is secreted with the release virus. • Hsp90β effects on the correct assembly of viral particles. • Viral titer of cultured medium was reduced in the presence of geldanamycin. • Viral titer was also reduced when Hsp90β was suppressed by siRNA treatment. • The extracellular Hsp90β was also observed in other RNA viruses-infected cells.« less

Representation of viruses in the remediated PDB archive

PubMed Central

Lawson, Catherine L.; Dutta, Shuchismita; Westbrook, John D.; Henrick, Kim; Berman, Helen M.

2008-01-01

A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive. PMID:18645236

Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

NASA Astrophysics Data System (ADS)

Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

2017-12-01

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis -acting elements in the viral genome. Copyright © 2017 American Society for Microbiology.

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

A virus-based biocatalyst

NASA Astrophysics Data System (ADS)

Carette, Noëlle; Engelkamp, Hans; Akpa, Eric; Pierre, Sebastien J.; Cameron, Neil R.; Christianen, Peter C. M.; Maan, Jan C.; Thies, Jens C.; Weberskirch, Ralf; Rowan, Alan E.; Nolte, Roeland J. M.; Michon, Thierry; van Hest, Jan C. M.

2007-04-01

Virus particles are probably the most precisely defined nanometre-sized objects that can be formed by protein self-assembly. Although their natural function is the storage and transport of genetic material, they have more recently been applied as scaffolds for mineralization and as containers for the encapsulation of inorganic compounds. The reproductive power of viruses has been used to develop versatile analytical methods, such as phage display, for the selection and identification of (bio)active compounds. To date, the combined use of self-assembly and reproduction has not been used for the construction of catalytic systems. Here we describe a self-assembled system based on a plant virus that has its coat protein genetically modified to provide it with a lipase enzyme. Using single-object and bulk catalytic studies, we prove that the virus-anchored lipase molecules are catalytically active. This anchored biocatalyst, unlike man-made supported catalysts, has the capability to reproduce itself in vivo, generating many independent catalytically active copies.

Membrane dynamics associated with viral infection.

PubMed

de Armas-Rillo, Laura; Valera, María-Soledad; Marrero-Hernández, Sara; Valenzuela-Fernández, Agustín

2016-05-01

Viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. These processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the ER, mitochondria, Golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. In fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. In this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. By defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © 2015 The Authors Reviews in Medical Virology Published by John Wiley & Sons, Ltd.

Nanotechnology-based approaches for the development of diagnostics, therapeutics, and vaccines.

PubMed

Srinivasan, Alagarsamy; Rastogi, Anshu; Ayyavoo, Velpandi; Srivastava, Shiv

2014-06-01

The architecture of nanoparticles of biological origin, generally also known as bionanoparticles, presents several features that are ideal for their use in developing diagnostics, therapeutics, and vaccines. In this regard, particles formed by viral proteins using recombinant DNA technology resemble authentic virus particles. However, they lack infectivity due to the absence of genetic components such as DNA or RNA. Hence, they are designated as virus-like particles (VLP). VLPs possess the following characteristics: (1) they can be generated by either a single or a few viral proteins; (2) their size, formed by viral proteins, is in the range of 20 to100 nm; (3) the number of protein molecules required for particle assembly is from hundreds to thousands, depending on the VLP; (4) the protein(s) responsible for their assembly are amenable for manipulation; and (5) multiple proteins/peptides can be incorporated into a VLP. The potential advantages of VLPs directed by retroviral proteins are discussed in this review.

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

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. PMID:26792745

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

PubMed

Barrero, Roberto A; Napier, Kathryn R; Cunnington, James; Liefting, Lia; Keenan, Sandi; Frampton, Rebekah A; Szabo, Tamas; Bulman, Simon; Hunter, Adam; Ward, Lisa; Whattam, Mark; Bellgard, Matthew I

2017-01-11

Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.

The nucleolar helicase DDX56 redistributes to West Nile virus assembly sites.

PubMed

Reid, Colleen R; Hobman, Tom C

2017-01-01

Flaviviruses, including the human pathogen, West Nile virus (WNV), are known to co-opt many host factors for their replication and propagation. To this end, we previously reported that the nucleolar DEAD-box RNA helicase, DDX56, is important for production of infectious WNV virions. In this study, we show that WNV infection results in relocalization of DDX56 from nucleoli to virus assembly sites on the endoplasmic reticululm (ER), an observation that is consistent with a role for DDX56 in WNV virion assembly. Super-resolution microscopy revealed that capsid and DDX56 localized to the same subcompartment of the ER, however, unexpectedly, stable interaction between these two proteins was only detected in the nucleus. Together, these data suggest that DDX56 relocalizes to the site of virus assembly during WNV infection and that its interaction with WNV capsid in the cytoplasm may occur transiently during virion morphogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Final Scientific Report - Electromagnetic Interactions in Self-Assembled Metallo-Dielectric Biomaterials

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

Dragnea, Bogdan G.

Achievements which resulted from previous DOE funding include: templated virus-like particle assembly thermodynamics, development of single particle photothermal absorption spectroscopy and dark- field spectroscopy instrumentation for the measurement of optical properties of virus-like nanoparticles, electromagnetic simulations of coupled nanoparticle cluster systems, virus contact mechanics, energy transfer and fluorescence quenching in multichromophore systems supported on biomolecular templates, and photo physical work on virus-aptamer systems. A current total of eight published research articles and a book chapter are acknowledging DOE support for the period 2013-2016.

Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

PubMed

Johnston, Gunner P; Contreras, Erik M; Dabundo, Jeffrey; Henderson, Bryce A; Matz, Keesha M; Ortega, Victoria; Ramirez, Alfredo; Park, Arnold; Aguilar, Hector C

2017-05-15

Nipah virus (NiV), a paramyxovirus in the genus Henipavirus , has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery. IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the fusion glycoprotein, in the incorporation of other viral proteins into viral particles. By identifying several regions in the fusion glycoprotein that drive viral assembly, we further our understanding of how these viruses assemble and egress from infected cells. The results presented will likely be useful toward designing treatments targeting this aspect of the viral life cycle and for the production of new viral particle-based vaccines. Copyright © 2017 American Society for Microbiology.

De Novo Foliar Transcriptome of Chenopodium amaranticolor and Analysis of Its Gene Expression During Virus-Induced Hypersensitive Response

PubMed Central

Zhang, Yongqiang; Pei, Xinwu; Zhang, Chao; Lu, Zifeng; Wang, Zhixing; Jia, Shirong; Li, Weimin

2012-01-01

Background The hypersensitive response (HR) system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. Methodology and Principal Findings Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10−5. Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. Conclusions To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory pathways of the HR-type of virus resistance in Chenopodium. PMID:23029338

Kinetics of human immunodeficiency virus budding and assembly

NASA Astrophysics Data System (ADS)

Zhang, Rui; Nguyen, Toan

2009-03-01

Human immunodeficiency virus (HIV) belongs to a large family of RNA viruses, retroviruses. Unlike budding of regular enveloped viruses, retroviruses bud concurrently with the assembly of retroviral capsids on the cell membrane. The kinetics of HIV (and other retroviruses) budding and assembly is therefore strongly affected by the elastic energy of the membrane and fundamentally different from regular viruses. The main result of this work shows that the kinetics is tunable from a fast budding process to a slow and effectively trapped partial budding process, by varying the attractive energy of retroviral proteins (call Gags), relative to the membrane elastic energy. When the Gag-Gag attraction is relatively high, the membrane elastic energy provides a kinetic barrier for the two pieces of the partial capsids to merge. This energy barrier determines the slowest step in the kinetics and the budding time. In the opposite limit, the membrane elastic energy provides not only a kinetic energy barrier, but a free energy barrier. The budding and assembly is effectively trapped at local free energy minimum, corresponding to a partially budded state. The time scale to escape from this metastable state is exponentially large. In both cases, our result fit with experimental measurements pretty well.

Packaging signals in single-stranded RNA viruses: nature's alternative to a purely electrostatic assembly mechanism.

PubMed

Stockley, Peter G; Twarock, Reidun; Bakker, Saskia E; Barker, Amy M; Borodavka, Alexander; Dykeman, Eric; Ford, Robert J; Pearson, Arwen R; Phillips, Simon E V; Ranson, Neil A; Tuma, Roman

2013-03-01

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA-coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

Dynamic and Kinetic Assembly Studies of an Icosahedral Virus Capsid

NASA Astrophysics Data System (ADS)

Lee, Kelly

2011-03-01

Hepatitis B virus has an icosahedrally symmetrical core particle (capsid), composed of either 90 or 120 copies of a dimeric protein building block. We are using time-resolved, solution small-angle X-ray scattering and single-molecule fluorescence microscopy to probe the core particle assembly reaction at the ensemble and individual assembly levels. Our experiments to date reveal the assembly process to be highly cooperative with minimal population of stable intermediate species. Solution conditions, particularly salt concentration, appears to influence the partitioning of assembly products into the two sizes of shells. Funding from NIH R00-GM080352 and University of Washington.

Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

PubMed Central

Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

2015-01-01

Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton

PubMed Central

2018-01-01

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that has the ability to infect and replicate within epithelial cells and neurons and establish a life-long latent infection in sensory neurons. HSV-1 depends on the host cellular cytoskeleton for entry, replication, and exit. Therefore, HSV-1 has adapted mechanisms to promote its survival by exploiting the microtubule and actin cytoskeletons to direct its active transport, infection, and spread between neurons and epithelial cells during primary and recurrent infections. This review will focus on the currently known mechanisms utilized by HSV-1 to harness the neuronal cytoskeleton, molecular motors, and the secretory and exocytic pathways for efficient virus entry, axonal transport, replication, assembly, and exit from the distinct functional compartments (cell body and axon) of the highly polarized sensory neurons. PMID:29473915

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion

PubMed Central

Mankouri, Jamel; Walter, Cheryl; Stewart, Hazel; Bentham, Matthew; Park, Wei Sun; Heo, Won Do; Fukuda, Mitsunori

2016-01-01

ABSTRACT The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways. PMID:27226379

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

PubMed Central

Eaton, Heather E.; Kobayashi, Takeshi; Dermody, Terence S.; Johnston, Randal N.

2017-01-01

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure. PMID:28298603

Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

PubMed Central

Lee, Ji-Young; Acosta, Eliana G.; Stoeck, Ina Karen; Long, Gang; Hiet, Marie-Sophie; Mueller, Birthe; Fackler, Oliver T.; Kallis, Stephanie

2014-01-01

ABSTRACT The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles but likely contributes to the quality control of secreted infectious virions. These results shed new light on the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE. PMID:25122793

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.

Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

PubMed

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

High-resolution structure, interactions, and dynamics of self-assembled virus-like partilces

NASA Astrophysics Data System (ADS)

Raviv, Uri; Asor, R.; Ben-Shaul, O.; Oppenheim, A.; Schlicksup, L. C.; Seltzer, L.; Jarrold, M. F.; Zlotnick, A.

Using SAXS, in combination with Monte Carlo simulations, and our unique solution x-ray scattering data analysis program, we resolved at high spatial resolution, the manner by which wtSV40 packages its 5.2kb circular DNA about 20 histone octamers in the virus capsid (Figure 1). This structure, known as a mini-chromosome, is highly dynamic and could not be resolved by microscopy methods. Using time-resolved solution SAXS, stopped-flow, and flow-through setups the assembly process of VP1, the major caspid protein of the SV40 virus, with RNA or DNA to form virus-like particles (VLPs) was studied in msec temporal resolution. By mixing the nucleotides and the capsid protein, virus-like particles formed within 35 msec, in the case of RNA that formed T =1 particles, and within 15 seconds in the case of DNA that formed T =7 particles, similar to wt SV40. The structural changes leading to the particle formation were followed in detail. More recently, we have extended this work to study the assembly of HBV virus-like particles.

WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-kappaB activation.

PubMed

Wang, Yan-Yi; Liu, Li-Juan; Zhong, Bo; Liu, Tian-Tian; Li, Ying; Yang, Yan; Ran, Yong; Li, Shu; Tien, Po; Shu, Hong-Bing

2010-01-12

Viral infection causes activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. The mitochondrial outer membrane protein VISA acts as a critical adapter for assembling a virus-induced complex that signals NF-kappaB and IRF3 activation. Using a biochemical purification approach, we identified the WD repeat protein WDR5 as a VISA-associated protein. WDR5 was recruited to VISA in a viral infection dependent manner. Viral infection also caused translocation of WDR5 from the nucleus to mitochondria. Knockdown of WDR5 impaired the formation of virus-induced VISA-associated complex. Consistently, knockdown of WDR5 inhibited virus-triggered activation of IRF3 and NF-kappaB as well as transcription of the IFNB1 gene. These findings suggest that WDR5 is essential in assembling a virus-induced VISA-associated complex and plays an important role in virus-triggered induction of type I IFNs.

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

PubMed Central

Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

2015-01-01

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues. PMID:26067441

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

PubMed

Zou, Xiao-Hui; Bi, Zhi-Xiang; Guo, Xiao-Juan; Zhang, Zun; Zhao, Yang; Wang, Min; Zhu, Ya-Lu; Jie, Hong-Ying; Yu, Yang; Hung, Tao; Lu, Zhuo-Zhuang

2018-07-01

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics. Copyright © 2018 Elsevier B.V. All rights reserved.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

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

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.

2010-09-30

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granularmore » bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.« less

Packaging of Polyelectrolytes in Viral Capsids: The Interplay Between Polymer Length and Capsid Size

NASA Astrophysics Data System (ADS)

Knobler, Charles

2008-03-01

Each particle of the Cowpea Chlorotic Mottle Virus (CCMV) has a very small ``parts list,'' consisting of two components: a molecule of single-stranded RNA and a 190-residue protein that makes up the 28-nm diameter icosahedral capsid. When purified viral RNA and capsid protein are mixed in solution at an appropriate pH and ionic strength, infectious wild-type viruses form spontaneously. Virus-like particles (VLPs) are formed when the protein self assembles around other anionic polymers such as poly(styrene sulfonate) (PSS). Under different pH and ionic strength conditions the capsid protein can assemble by itself into empty capsids, multishell structures, tubes and sheets. To explore the effect on virion size of the competition between the preferred curvature of the protein and the size of the packaged cargo we have examined the formation of VLPs around PSS polymers with molecular weights ranging from 400 kDa to 3.4 MDa. Two distinct sizes are observed -- 22 nm for the lower molecular weights, jumping to 27 nm at 2 MDa. While under given conditions the size of PSS in solution is directly determined by its molecular weight, the self-complementarity of RNA makes its solution structure dependent on the nucleotide sequence as well. We have therefore employed Small-Angle X-ray Scattering and Fluorescence Correlation Spectroscopy to examine the sizes of viral and non-viral RNAs of identical lengths. A model for the assembly that includes both the self-interactions of the polyelectrolyte and the capsid proteins and the interactions between them provides insight into the experimental results.

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

Ningnanmycin inhibits tobacco mosaic virus virulence by binding directly to its coat protein discs

PubMed Central

Li, Xiangyang; Hao, Gefei; Wang, Qingmin; Chen, Zhuo; Ding, Yan; Yu, Lu; Hu, Deyu; Song, Baoan

2017-01-01

Tobacco mosaic virus (TMV) causes severe plant diseases worldwide; however, effective antiviral agents for controlling TMV infections are not available. This lack of effective antiviral agents is mainly due to the poor understanding of potential targets associated with TMV infections. During infection, the coat protein (CP), which is delivered by viral particles into susceptible host cells, provides protection for viral RNA. Here, we found that Ningnanmycin (NNM), a commercially used plant antibacterial agent, inhibits the assembly of the CP by directly binding several residues. These interactions cause the disassembly of the CP from discs into monomers, leading to an almost complete loss of pathogenicity. Substitutions in the involved binding residues resulted in mutants that were significantly less sensitive to NNM. Thus, targeting the binding of viral CPs through small molecular agents offers an effective strategy to study the mechanism of NNM. PMID:29137277

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

PubMed

Manuel-Cabrera, Carlos Alberto; Vallejo-Cardona, Alba Adriana; Padilla-Camberos, Eduardo; Hernández-Gutiérrez, Rodolfo; Herrera-Rodríguez, Sara Elisa; Gutiérrez-Ortega, Abel

2016-11-29

Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

PubMed

Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

2014-02-01

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

Self-assembled virus-like particles with magnetic cores.

PubMed

Huang, Xinlei; Bronstein, Lyudmila M; Retrum, John; Dufort, Chris; Tsvetkova, Irina; Aniagyei, Stella; Stein, Barry; Stucky, Galen; McKenna, Brandon; Remmes, Nicholas; Baxter, David; Kao, C Cheng; Dragnea, Bogdan

2007-08-01

Efficient encapsulation of functionalized spherical nanoparticles by viral protein cages was found to occur even if the nanoparticle is larger than the inner cavity of the native capsid. This result raises the intriguing possibility of reprogramming the self-assembly of viral structural proteins. The iron oxide nanotemplates used in this work are superparamagnetic, with a blocking temperature of about 250 K, making these virus-like particles interesting for applications such as magnetic resonance imaging and biomagnetic materials. Another novel feature of the virus-like particle assembly described in this work is the use of an anionic lipid micelle coat instead of a molecular layer covalently bound to the inorganic nanotemplate. Differences between the two functionalization strategies are discussed.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Study of rubella candidate vaccine based on a structurally modified plant virus.

PubMed

Trifonova, Ekaterina A; Zenin, Vladimir A; Nikitin, Nikolai A; Yurkova, Maria S; Ryabchevskaya, Ekaterina M; Putlyaev, Egor V; Donchenko, Ekaterina K; Kondakova, Olga A; Fedorov, Alexey N; Atabekov, Joseph G; Karpova, Olga V

2017-08-01

A novel rubella candidate vaccine based on a structurally modified plant virus - spherical particles (SPs) - was developed. SPs generated by the thermal remodelling of the tobacco mosaic virus are promising platforms for the development of vaccines. SPs combine unique properties: biosafety, stability, high immunogenicity and the effective adsorption of antigens. We assembled in vitro and characterised complexes (candidate vaccine) based on SPs and the rubella virus recombinant antigen. The candidate vaccine induced a strong humoral immune response against rubella. The IgG isotypes ratio indicated the predominance of IgG1 which plays a key role in immunity to natural rubella infection. The immune response was generally directed against the rubella antigen within the complexes. We suggest that SPs can act as a platform (depot) for the rubella antigen, enhancing specific immune response. Our results demonstrate that SPs-antigen complexes can be an effective and safe candidate vaccine against rubella. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-step Channel Conformational Changes Common to DNA Packaging Motors of Bacterial Viruses T3, T4, SPP1, and Phi29

PubMed Central

Wang, Shaoying; Ji, Zhouxiang; Yan, Erfu; Haque, Farzin; Guo, Peixuan

2016-01-01

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the “One-way Revolution” mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. It is proposed that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation of DNA movement in the reverse direction during ejection. PMID:27181501

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

PubMed

Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses

PubMed Central

Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W. R.; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry. PMID:26226615

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

PubMed

Tinazzi, Elisa; Merlin, Matilde; Bason, Caterina; Beri, Ruggero; Zampieri, Roberta; Lico, Chiara; Bartoloni, Elena; Puccetti, Antonio; Lunardi, Claudio; Pezzotti, Mario; Avesani, Linda

2015-01-01

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

Study and characterization of tobacco mosaic virus head-to-tail assembly assisted by aniline polymerization.

PubMed

Niu, Zhongwei; Bruckman, Michael; Kotakadi, Venkata S; He, Jinbo; Emrick, Todd; Russell, Thomas P; Yang, Lin; Wang, Qian

2006-07-28

One-dimensional composite nanofibres with narrow dispersity, high aspect ratio and high processibility have been fabricated by head-to-tail self-assembly of rod-like tobacco mosaic virus assisted by aniline polymerization, which can promote many potential applications including electronics, optics, sensing and biomedical engineering.

RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

USDA-ARS?s Scientific Manuscript database

Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

New Drug Self-Assembles and Delivers Itself to Its Target | Center for Cancer Research

Cancer.gov

Viruses self-assemble with high precision and disassemble upon fusion with the cell membrane and deliver this way their cargo to the interior of the cells. Using this talent, viruses are able to inject whole proteins and large nucleic acid molecules into certain types of cells.

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

PubMed Central

Hafrén, Anders; Macia, Jean-Luc; Love, Andrew J.; Milner, Joel J.; Drucker, Martin; Hofius, Daniel

2017-01-01

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana. We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis. PMID:28223514

Virus-Based Nanoparticles of Simian Virus 40 in the Field of Nanobiotechnology.

PubMed

Zhang, Wenjing; Zhang, Xian-En; Li, Feng

2017-12-26

Biomolecular nanostructures derived from living organisms, such as protein cages, fibers, and layers are drawing increasing interests as natural biomaterials. The virus-based nanoparticles (VNPs) of simian virus 40 (SV40), with a cage-like structure assembled from the major capsid protein of SV40, have been developed as a platform for nanobiotechnology in the recent decade. Foreign nanomaterials (e.g., quantum dots (QDs) and gold nanoparticles (AuNPs)) can be positioned in the inner cavity or on the outer surface of SV40 VNPs, through self-assembly by engineering the nanoparticle (NP)-protein interfacial interactions. Construction of these hybrid nanostructures has enabled integration of different functionalities. This review briefly summarizes the applications of SV40 VNPs in this multidisciplinary field, including NP encapsulation, templated assembly of nanoarchitectures, nanophotonics, and fluorescence imaging. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

PubMed Central

Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

2015-01-01

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID:26136573

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

PubMed Central

Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves

2014-01-01

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera. PMID:24335294

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

Badia-Martinez, Daniel; Peralta, Bibiana; Andres, German

Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

PubMed

Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

2017-08-16

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses.

PubMed

Bajimaya, Shringkhala; Frankl, Tünde; Hayashi, Tsuyoshi; Takimoto, Toru

2017-10-01

Cholesterol-rich lipid raft microdomains in the plasma membrane are considered to play a major role in the enveloped virus lifecycle. However, the functional role of cholesterol in assembly, infectivity and stability of respiratory RNA viruses is not fully understood. We previously reported that depletion of cellular cholesterol by cholesterol-reducing agents decreased production of human parainfluenza virus type 1 (hPIV1) particles by inhibiting virus assembly. In this study, we analyzed the role of cholesterol on influenza A virus (IAV) and respiratory syncytial virus (RSV) production. Unlike hPIV1, treatment of human airway cells with the agents did not decrease virus particle production. However, the released virions were less homogeneous in density and unstable. Addition of exogenous cholesterol to the released virions restored virus stability and infectivity. Collectively, these data indicate a critical role of cholesterol in maintaining IAV and RSV membrane structure that is essential for sustaining viral stability and infectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

PubMed

Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

2017-02-14

Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature. Copyright © 2017 Jose et al.

Tunable protease-activatable virus nanonodes.

PubMed

Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

2014-05-27

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Lipid biosensor interactions with wild type and matrix deletion HIV-1 Gag proteins.

PubMed

Barklis, Eric; Staubus, August O; Mack, Andrew; Harper, Logan; Barklis, Robin Lid; Alfadhli, Ayna

2018-05-01

The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

Preparation and Characterization of Monomodal Grapevine Virus A Capsid Protein.

PubMed

Santana, Vinícius S; Mariutti, Ricardo B; Eberle, Raphael J; Ullah, Anwar; Caruso, Icaro P; Arni, Raghuvir K

2015-01-01

Grapevine virus A (GVA), a flexible filament of approximately 800 nm in length is composed of capsid subunits that spontaneously assembles around a positive sense genomic RNA. In addition to encapsidation, plant viruses capsid proteins (CPs) participate in other processes throughout infection and GVA CP is involved in cell-to-cell translocation of the virus. A protocol was developed to obtain low-molecular weight GVA-CP that is not prone to aggregation and spontaneous assembly and this was characterized by circular dichroism and dynamic light scattering. These results indicate the suitably of GVA-CP for X-ray crystallographic and NMR studies that should lead to the elucidation of the first three-dimensional structure of a flexible filamentous virus from the Betaflexiviridae family.

Self-assembly of a double-helical complex of sodium.

PubMed

Bell, T W; Jousselin, H

1994-02-03

Spontaneous self-organization of helical and multiple-helical molecular structures occurs on several levels in living organisms. Key examples are alpha-helical polypeptides, double-helical nucleic acids and helical protein structures, including F-actin, microtubules and the protein sheath of the tobacco mosaic virus. Although the self-assembly of double-helical transition-metal complexes bears some resemblance to the molecular organization of double-stranded DNA, selection between monohelical, double-helical and triple-helical structures is determined largely by the size and geometrical preference of the tightly bound metal. Here we present an example of double-helical assembly induced by the weaker and non-directional interactions of an alkali-metal ion with an organic ligand that is pre-organized into a coil. We have characterized the resulting complex by two-dimensional NMR and fast-atom-bombardment mass spectrometry. These results provide a step toward the creation of molecular tubes or ion channels consisting of intertwined coils.

Assessing the Impact of Assemblers on Virus Detection in a De Novo Metagenomic Analysis Pipeline.

PubMed

White, Daniel J; Wang, Jing; Hall, Richard J

2017-09-01

Applying high-throughput sequencing to pathogen discovery is a relatively new field, the objective of which is to find disease-causing agents when little or no background information on disease is available. Key steps in the process are the generation of millions of sequence reads from an infected tissue sample, followed by assembly of these reads into longer, contiguous stretches of nucleotide sequences, and then identification of the contigs by matching them to known databases, such as those stored at GenBank or Ensembl. This technique, that is, de novo metagenomics, is particularly useful when the pathogen is viral and strong discriminatory power can be achieved. However, recently, we found that striking differences in results can be achieved when different assemblers were used. In this study, we test formally the impact of five popular assemblers (MIRA, VELVET, METAVELVET, SPADES, and OMEGA) on the detection of a novel virus and assembly of its whole genome in a data set for which we have confirmed the presence of the virus by empirical laboratory techniques, and compare the overall performance between assemblers. Our results show that if results from only one assembler are considered, biologically important reads can easily be overlooked. The impacts of these results on the field of pathogen discovery are considered.

Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

PubMed

Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

2015-12-02

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

PubMed

Milles, Sigrid; Jensen, Malene Ringkjøbing; Communie, Guillaume; Maurin, Damien; Schoehn, Guy; Ruigrok, Rob W H; Blackledge, Martin

2016-08-01

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Degenerate RNA packaging signals in the genome of Satellite Tobacco Necrosis Virus: implications for the assembly of a T=1 capsid.

PubMed

Bunka, David H J; Lane, Stephen W; Lane, Claire L; Dykeman, Eric C; Ford, Robert J; Barker, Amy M; Twarock, Reidun; Phillips, Simon E V; Stockley, Peter G

2011-10-14

Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem-loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem-loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem-loops displaying the sequence motif AxxA. The implication is that there are many stem-loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem-loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA. Copyright © 2011 Elsevier Ltd. All rights reserved.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

PubMed

Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

2016-04-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

PubMed Central

Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

2016-01-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

Simulations of curved assemblies in soft matter and biological systems

NASA Astrophysics Data System (ADS)

Qiao, Cong

Viruses are small infectious agents that replicate only inside living cells of other organisms. In the viral life cycle, the self-assembly of the outer protein shell (capsid) is an essential step. We study this process in the hope of shedding light on development of antiviral drugs, gene therapy and other virus-related technologies that can benefit the humankind. More fundamentally, learning about the process of viral capsid assembly can elucidate the assembly mechanisms of a wide range of complex structures. In this work, we use molecular dynamics simulations and coarse-grained computational models to study viral capsid assembly in several situations where geometric constraints play a role in dictating assembly outcomes. We first focus on icosahedral viruses with single-stranded RNA genomes, in which case the capsid usually assembles around the genomic RNA. It is consistently observed in experiments that such viral particles are ''overcharged'', meaning the net negative charge on the viral genome is greater than the net positive charge on the viral capsid. We computationally investigate the mechanisms that lead to ``overcharging'', and more broadly, how the encapsidated genome length is influenced by the capsid. We perform both dynamical simulations of the assembly process and equilibrium calculations to determine the optimal genome length (meaning that which maximizes the assembly yield and/or minimizes the free energy of the assembled virus). We find that the optimal genome length is determined by the interplay between capsid size, net capsid charge, distribution of capsid charge and nucleic acid structures. Our simulations demonstrate that overcharging results from a combination of electrostatic screening and the geometric constraints associated with encapsulating a nucleic acid inside of a spherical virus. We then study the assembly of the immature HIV. In contrast to icosahedral viruses, the immature HIV forms an asymmetric particle, consisting of continuous regularly packed regions with local hexagonal order and vacancies. A similar lattice structure has been observed in experiments in which mutually attractive colloidal particles pack on the surface of a spherical droplet (G. Meng, J. Paulose, D. R. Nelson, and V. N. Manoharan, ''Elastic instability of a crystal growing on a curved surface'', Science 343, 634-637 (2014).), suggesting that the two systems experience a similar form of geometric frustration. We therefore study the adsorption and packing of spherical particles on a spherical template, as a function of the strength and range of interparticle attractions, as well as the radius of the spherical template. We observe that the adsorbed particles form two different classes of packing arrangements, one with icosahedrally ordered topological defects, and the other with highly disordered defects and vacancies. The latter regime is consistent with experiments on colloidal packing on spherical droplets and the immature HIV lattice. Our results suggest that the transition between these regimes is controlled by the range of the interparticle attractions. In the last chapter, we study a model for the assembly and budding of a capsid on a membrane, such as occurs during the exit of the immature HIV virus from a cell. We use a coarse-grained subunit model to represent the capsid proteins, and a fluid membrane model to represent the cell membrane. We find that the size and structure of the assembled capsid depends sensitively on the timescale of budding.

Roles for the Cytoplasmic Tails of the Fusion and Hemagglutinin-Neuraminidase Proteins in Budding of the Paramyxovirus Simian Virus 5

PubMed Central

Waning, David L.; Schmitt, Anthony P.; Leser, George P.; Lamb, Robert A.

2002-01-01

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependant on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding. PMID:12186912

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

PubMed Central

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc

2017-01-01

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly. PMID:28053108

Molecular Recognition Directed Self-Assembly of Supramolecular Architectures

DTIC Science & Technology

1994-06-30

TMV-Like SupraxlecuiarArchiteturc _ TMV is a simple virus consisting only of a single type of protein molecule and of a strand of ribonucleic acid (RNA...experiments have demonstrated that various substituted gallic acid derivatives can be used to construct exo-receptors with a tapered shape. 1.2...with 3,4,5-tris(p - dodecyloxybenzyloxy)benzoic acid {12-ABCr) resulted in the taper shaped structural units. 12- ABG-B1SC5 and 12-ABG-15C5

VirusDetect: An automated pipeline for efficient virus discovery using deep sequencing of small RNAs

USDA-ARS?s Scientific Manuscript database

Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep sequencing and assembly of virus-derived siRNAs has proven to be a highly efficient approach for virus discovery. However, to date no computational tools specifically designed for both k...

Packaging signals in two single-stranded RNA viruses imply a conserved assembly mechanism and geometry of the packaged genome.

PubMed

Dykeman, Eric C; Stockley, Peter G; Twarock, Reidun

2013-09-09

The current paradigm for assembly of single-stranded RNA viruses is based on a mechanism involving non-sequence-specific packaging of genomic RNA driven by electrostatic interactions. Recent experiments, however, provide compelling evidence for sequence specificity in this process both in vitro and in vivo. The existence of multiple RNA packaging signals (PSs) within viral genomes has been proposed, which facilitates assembly by binding coat proteins in such a way that they promote the protein-protein contacts needed to build the capsid. The binding energy from these interactions enables the confinement or compaction of the genomic RNAs. Identifying the nature of such PSs is crucial for a full understanding of assembly, which is an as yet untapped potential drug target for this important class of pathogens. Here, for two related bacterial viruses, we determine the sequences and locations of their PSs using Hamiltonian paths, a concept from graph theory, in combination with bioinformatics and structural studies. Their PSs have a common secondary structure motif but distinct consensus sequences and positions within the respective genomes. Despite these differences, the distributions of PSs in both viruses imply defined conformations for the packaged RNA genomes in contact with the protein shell in the capsid, consistent with a recent asymmetric structure determination of the MS2 virion. The PS distributions identified moreover imply a preferred, evolutionarily conserved assembly pathway with respect to the RNA sequence with potentially profound implications for other single-stranded RNA viruses known to have RNA PSs, including many animal and human pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

3D tissue-like assemblies: A novel approach to investigate virus-cell interactions.

PubMed

Goodwin, Thomas J; McCarthy, Maureen; Cohrs, Randall J; Kaufer, Benedikt B

2015-11-15

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV). Copyright © 2015 Elsevier Inc. All rights reserved.

3D Tissue-Like Assemblies: A Novel Approach to Investigate Virus-Cell Interactions

PubMed Central

Goodwin, Thomas J.; McCarthy, Maureen; Cohrs, Randall J.; Kaufer, Benedikt B.

2017-01-01

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV) PMID:25986169

Membrane Remodeling by the Double-Barrel Scaffolding Protein of Poxvirus

PubMed Central

Hijnen, Marcel; Schult, Philipp; Pettikiriarachchi, Anne; Mitra, Alok K.; Coulibaly, Fasséli

2011-01-01

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors. PMID:21931553

Viral Impact in Autoimmune Diseases: Expanding the “X Chromosome–Nucleolus Nexus” Hypothesis

PubMed Central

Brooks, Wesley H.

2017-01-01

Viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. We get some insight by considering demands a virus places on host cells. Viruses not only require production of their own proteins, RNA and/or DNA, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. Since the nucleolus is a major site of RNA processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral RNA and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. Polyamines are at high levels in nucleoli to assist in RNA folding. The size and activity of nucleoli increase directly with increases in polyamines. Nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive X chromosome, leading to expression of previously sequestered DNA. Sudden expression of a large concentration of Alu elements from the disrupted inactive X can compete with RNA transcripts containing intronic Alu sequences that normally maintain nucleolar structural integrity. Such disruption of nucleolar activity can lead to misfolded RNAs, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. Many autoantigens in lupus are, at least transiently, components of the nucleolus. Considering these effects of viruses, the “X chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive X by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered Alu elements, which can fragment the nucleolus, leading to generation of autoantigens. PMID:29234321

Soft materials design via self assembly of functionalized icosahedral particles

NASA Astrophysics Data System (ADS)

Muthukumar, Vidyalakshmi Chockalingam

In this work we simulate self assembly of icosahedral building blocks using a coarse grained model of the icosahedral capsid of virus 1m1c. With significant advancements in site-directed functionalization of these macromolecules [1], we propose possible application of such self-assembled materials for drug delivery. While there have been some reports on organization of viral particles in solution through functionalization, exploiting this behaviour for obtaining well-ordered stoichiometric structures has not yet been explored. Our work is in well agreement with the earlier simulation studies of icosahedral gold nanocrystals, giving chain like patterns [5] and also broadly in agreement with the wet lab works of Finn, M.G. et al., who have shown small predominantly chain-like aggregates with mannose-decorated Cowpea Mosaic Virus (CPMV) [22] and small two dimensional aggregates with oligonucleotide functionalization on the CPMV capsid [1]. To quantify the results of our Coarse Grained Molecular Dynamics Simulations I developed analysis routines in MATLAB using which we found the most preferable nearest neighbour distances (from the radial distribution function (RDF) calculations) for different lengths of the functional groups and under different implicit solvent conditions, and the most frequent coordination number for a virus particle (histogram plots further using the information from RDF). Visual inspection suggests that our results most likely span the low temperature limits explored in the works of Finn, M.G. et al., and show a good degree of agreement with the experimental results in [1] at an annealing temperature of 4°C. Our work also reveals the possibility of novel stoichiometric N-mer type aggregates which could be synthesized using these capsids with appropriate functionalization and solvent conditions.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons

PubMed Central

Anderson, Fenja; Rother, Franziska; Rudolph, Kathrin; Prank, Ute; Binz, Anne; Hügel, Stefanie; Hartmann, Enno; Bader, Michael; Bauerfeind, Rudolf; Sodeik, Beate

2018-01-01

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons. PMID:29304174

Synthesis and Evaluation of N-phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators inhibiting Hepatitis B Virus (HBV).

PubMed

Vandyck, Koen; Rombouts, Geert; Stoops, Bart; Tahri, Abdellah; Vos, Ann; Verschueren, Wim; Wu, Yiming; Yang, Jingmei; Hou, Fuliang; Huang, Bing; Vergauwen, Karen; Dehertogh, Pascale; Berke, Jan-Martin; Raboisson, Pierre Jean Marie Bernard

2018-06-15

Small molecule induced Hepatitis B virus (HBV) capsid assembly modulation is considered an attractive approach for new antiviral therapies against HBV. Here we describe efforts towards the discovery of a HBV capsid assembly modulator in a hit-to-lead optimization, resulting in JNJ-632, a tool compound used to further profile the mode of action. Administration of JNJ-632 (54) in HBV genotype D infected chimeric mice, resulted in a 2.77 log reduction of the HBV DNA viral load.

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

PubMed Central

Londrigan, Sarah L.; Short, Kirsty R.; Ma, Joel; Gillespie, Leah; Rockman, Steven P.; Brooks, Andrew G.

2015-01-01

ABSTRACT Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. PMID:26423941

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle.

PubMed

Londrigan, Sarah L; Short, Kirsty R; Ma, Joel; Gillespie, Leah; Rockman, Steven P; Brooks, Andrew G; Reading, Patrick C

2015-12-01

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Open membranes are the precursors for assembly of large DNA viruses

PubMed Central

Suárez, Cristina; Welsch, Sonja; Chlanda, Petr; Hagen, Wim; Hoppe, Simone; Kolovou, Androniki; Pagnier, Isabelle; Raoult, Didier; Locker, Jacomine Krijnse

2014-01-01

Summary Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convex side of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open-eight’ conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis. PMID:23751082

Evidence that viral RNAs have evolved for efficient, two-stage packaging.

PubMed

Borodavka, Alexander; Tuma, Roman; Stockley, Peter G

2012-09-25

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

Collective helicity switching of a DNA-coat assembly

NASA Astrophysics Data System (ADS)

Kim, Yongju; Li, Huichang; He, Ying; Chen, Xi; Ma, Xiaoteng; Lee, Myongsoo

2017-07-01

Hierarchical assemblies of biomolecular subunits can carry out versatile tasks at the cellular level with remarkable spatial and temporal precision. As an example, the collective motion and mutual cooperation between complex protein machines mediate essential functions for life, such as replication, synthesis, degradation, repair and transport. Nucleic acid molecules are far less dynamic than proteins and need to bind to specific proteins to form hierarchical structures. The simplest example of these nucleic acid-based structures is provided by a rod-shaped tobacco mosaic virus, which consists of genetic material surrounded by coat proteins. Inspired by the complexity and hierarchical assembly of viruses, a great deal of effort has been devoted to design similarly constructed artificial viruses. However, such a wrapping approach makes nucleic acid dynamics insensitive to environmental changes. This limitation generally restricts, for example, the amplification of the conformational dynamics between the right-handed B form to the left-handed Z form of double-stranded deoxyribonucleic acid (DNA). Here we report a virus-like hierarchical assembly in which the native DNA and a synthetic coat undergo repeated collective helicity switching triggered by pH change under physiological conditions. We also show that this collective helicity inversion occurs during translocation of the DNA-coat assembly into intracellular compartments. Translating DNA conformational dynamics into a higher level of hierarchical dynamics may provide an approach to create DNA-based nanomachines.

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion.

PubMed

Wu, Ren-Huang; Tsai, Ming-Han; Chao, Day-Yu; Yueh, Andrew

2015-04-01

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Synthetic approaches to construct viral capsid-like spherical nanomaterials.

PubMed

Matsuura, Kazunori

2018-06-06

This feature article describes recent progress in synthetic strategies to construct viral capsid-like spherical nanomaterials using the self-assembly of peptides and/or proteins. By mimicking the self-assembly of spherical viral capsids and clathrin, trigonal peptide conjugates bearing β-sheet-forming peptides, glutathiones, or coiled-coil-forming peptides were developed to construct viral capsid-like particles. β-Annulus peptides from tomato bushy stunt virus self-assembled into viral capsid-like nanocapsules with a size of 30-50 nm, which could encapsulate various guest molecules and be decorated with different molecules on their surface. Rationally designed fusion proteins bearing symmetric assembling units afforded precise viral capsid-like polyhedral assemblies. These synthetic approaches to construct artificial viruses could become useful guidelines to develop novel drug carriers, vaccine platforms, nanotemplates and nanoreactors.

Field-induced assembly of colloidal ellipsoids into well-defined microtubules

PubMed Central

Crassous, Jérôme J.; Mihut, Adriana M.; Wernersson, Erik; Pfleiderer, Patrick; Vermant, Jan; Linse, Per; Schurtenberger, Peter

2014-01-01

Current theoretical attempts to understand the reversible formation of stable microtubules and virus shells are generally based on shape-specific building blocks or monomers, where the local curvature of the resulting structure is explicitly built-in via the monomer geometry. Here we demonstrate that even simple ellipsoidal colloids can reversibly self-assemble into regular tubular structures when subjected to an alternating electric field. Supported by model calculations, we discuss the combined effects of anisotropic shape and field-induced dipolar interactions on the reversible formation of self-assembled structures. Our observations show that the formation of tubular structures through self-assembly requires much less geometrical and interaction specificity than previously thought, and advance our current understanding of the minimal requirements for self-assembly into regular virus-like structures. PMID:25409686

Evidence for retrovirus and paramyxovirus infection of multiple bat species in china.

PubMed

Yuan, Lihong; Li, Min; Li, Linmiao; Monagin, Corina; Chmura, Aleksei A; Schneider, Bradley S; Epstein, Jonathan H; Mei, Xiaolin; Shi, Zhengli; Daszak, Peter; Chen, Jinping

2014-05-16

Bats are recognized reservoirs for many emerging zoonotic viruses of public health importance. Identifying and cataloguing the viruses of bats is a logical approach to evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats comprising 20 common species, which were sampled in three locations of Yunnan province, by combining reverse transcription polymerase chain reaction (RT-PCR) assays and next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR using degenerate primers, and these paramyxoviruses were mainly classified into three genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of 7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus), 17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the 103 vertebrate viral contigs, 79 displayed low identity (<70%) to known viruses including human viruses at the amino acid level, suggesting that these belong to novel and genetically divergent viruses. Overall, the most frequently identified viruses, particularly in bats from the family Hipposideridae, were retroviruses. The present study expands our understanding of the bat virome in species commonly found in Yunnan, China, and provides insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into humans.

Evidence for Retrovirus and Paramyxovirus Infection of Multiple Bat Species in China

PubMed Central

Yuan, Lihong; Li, Min; Li, Linmiao; Monagin, Corina; Chmura, Aleksei A.; Schneider, Bradley S.; Epstein, Jonathan H.; Mei, Xiaolin; Shi, Zhengli; Daszak, Peter; Chen, Jinping

2014-01-01

Bats are recognized reservoirs for many emerging zoonotic viruses of public health importance. Identifying and cataloguing the viruses of bats is a logical approach to evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats comprising 20 common species, which were sampled in three locations of Yunnan province, by combining reverse transcription polymerase chain reaction (RT-PCR) assays and next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR using degenerate primers, and these paramyxoviruses were mainly classified into three genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of 7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus), 17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the 103 vertebrate viral contigs, 79 displayed low identity (<70%) to known viruses including human viruses at the amino acid level, suggesting that these belong to novel and genetically divergent viruses. Overall, the most frequently identified viruses, particularly in bats from the family Hipposideridae, were retroviruses. The present study expands our understanding of the bat virome in species commonly found in Yunnan, China, and provides insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into humans. PMID:24841387

Tunable Protease-Activatable Virus Nanonodes

PubMed Central

2015-01-01

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery. PMID:24796495

Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

PubMed Central

Tobaly-Tapiero, Joelle; Bittoun, Patricia; Giron, Marie-Lou; Neves, Manuel; Koken, Marcel; Saïb, Ali; de Thé, Hugues

2001-01-01

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly. PMID:11287585

Host-guest encapsulation of materials by assembled virus protein cages

NASA Astrophysics Data System (ADS)

Douglas, Trevor; Young, Mark

1998-05-01

Self-assembled cage structures of nanometre dimensions can be used as constrained environments for the preparation of nanostructured materials, and the encapsulation of guest molecules, with potential applications in drug delivery and catalysis. In synthetic systems the number of subunits contributing to cage structures is typically rather small,. But the protein coats of viruses (virions) commonly comprise hundreds of subunits that self-assemble into a cage for transporting viral nucleic acids. Many virions, moreover, can undergo reversible structural changes that open or close gated pores to allow switchable access to their interior. Here we show that such a virion - that of the cowpea chlorotic mottle virus - can be used as a host for the synthesis of materials. We report the mineralization of two polyoxometalate species (paratungstate and decavanadate) and the encapsulation of an anionic polymer inside this virion, controlled by pH-dependent gating of the virion's pores. The diversity in size and shape of such virus particles make this a versatile strategy for materials synthesis and molecular entrapment.

Molecular Mechanism of Arenavirus Assembly and Budding

PubMed Central

Urata, Shuzo; Yasuda, Jiro

2012-01-01

Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2. PMID:23202453

The microviridae: Diversity, assembly, and experimental evolution.

PubMed

Doore, Sarah M; Fane, Bentley A

2016-04-01

The Microviridae, comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and Assembly of Intracellular Mature Vaccinia Virus: Thin-Section Analyses

PubMed Central

Griffiths, Gareth; Roos, Norbert; Schleich, Sybille; Locker, Jacomine Krijnse

2001-01-01

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper. PMID:11602745

Packaging and structural phenotype of brome mosaic virus capsid protein with altered N-terminal {beta}-hexamer structure

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

Wispelaere, Melissanne de; Chaturvedi, Sonali; Wilkens, Stephan

2011-10-10

The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and structural domains that are implicated in the assembly of infectious virions. One such important structural domain encompassing amino acids {sup 28}QPVIV{sup 32}, highly conserved between BMV and cowpea chlorotic mottle virus (CCMV), exhibits a {beta}-hexamer structure. In this study we report that alteration of the {beta}-hexamer structure by mutating {sup 28}QPVIV{sup 32} to {sup 28}AAAAA{sup 32} had no effect either on symptom phenotype, local and systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity to RNasemore » and assembly phenotypes distinguished virions assembled with CP subunits having {beta}-hexamer from those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes of symmetry.« less

Metagenomic characterization of viral communities in Goseong Bay, Korea

NASA Astrophysics Data System (ADS)

Hwang, Jinik; Park, So Yun; Park, Mirye; Lee, Sukchan; Jo, Yeonhwa; Cho, Won Kyong; Lee, Taek-Kyun

2016-12-01

In this study, seawater samples were collected from Goseong Bay, Korea in March 2014 and viral populations were examined by metagenomics assembly. Enrichment of marine viral particles using FeCl3 followed by next-generation sequencing produced numerous sequences. De novo assembly and BLAST search showed that most of the obtained contigs were unknown sequences and only 0.74% of sequences were associated with known viruses. As a result, 138 viruses, including bacteriophages (87%), viruses infecting algae and others (13%) were identified. The identified 138 viruses were divided into 11 orders, 14 families, 34 genera, and 133 species. The dominant viruses were Pelagibacter phage HTVC010P and Roseobacter phage SIO1. The viruses infecting algae, including the Ostreococcus species, accounted for 9.4% of total identified viruses. In addition, we identified pathogenic herpes viruses infecting fishes and giant viruses infecting parasitic acanthamoeba species. This is a comprehensive study to reveal the viral populations in the Goseong Bay using metagenomics. The information associated with the marine viral community in Goseong Bay, Korea will be useful for comparative analysis in other marine viral communities.

Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

PubMed Central

Yu, Zheng; Beer, Christiane; Koester, Mario; Wirth, Manfred

2006-01-01

Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly. PMID:16956408

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

PubMed

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

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

Lee, Eun-Gyung; Linial, Maxine L.

2006-03-30

The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similarmore » to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both {delta}CH1 and {delta}CH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.« less

Design, assembly, and validation of a nose-only inhalation exposure system for studies of aerosolized viable influenza H5N1 virus in ferrets

PubMed Central

2010-01-01

Background The routes by which humans acquire influenza H5N1 infections have not been fully elucidated. Based on the known biology of influenza viruses, four modes of transmission are most likely in humans: aerosol transmission, ingestion of undercooked contaminated infected poultry, transmission by large droplets and self-inoculation of the nasal mucosa by contaminated hands. In preparation of a study to resolve whether H5N1 viruses are transmissible by aerosol in an animal model that is a surrogate for humans, an inhalation exposure system for studies of aerosolized H5N1 viruses in ferrets was designed, assembled, and validated. Particular attention was paid towards system safety, efficacy of dissemination, the viability of aerosolized virus, and sampling methodology. Results An aerosol generation and delivery system, referred to as a Nose-Only Bioaerosol Exposure System (NBIES), was assembled and function tested. The NBIES passed all safety tests, met expected engineering parameters, required relatively small quantities of material to obtain the desired aerosol concentrations of influenza virus, and delivered doses with high-efficacy. Ferrets withstood a mock exposure trial without signs of stress. Conclusions The NBIES delivers doses of aerosolized influenza viruses with high efficacy, and uses less starting material than other similar designs. Influenza H5N1 and H3N2 viruses remain stable under the conditions used for aerosol generation and sample collection. The NBIES is qualified for studies of aerosolized H5N1 virus. PMID:20573226

Self-Assembly: How Nature Builds

ERIC Educational Resources Information Center

Jones, M. Gail; Falvo, Michael R.; Broadwell, Bethany; Dotger, Sharon

2006-01-01

Self-assembly or spontaneous assembly is a process in which materials build themselves without assistance. This process plays a central role in the construction of biological structures and materials such as cells, viruses, and bone, and also in abiotic processes like phase transitions and crystal formation. The principles of self-assembly help…

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

PubMed

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

2016-11-01

Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies. Copyright © 2016 Barouch-Bentov et al.

[Ultrastructural observation related to cell-to-cell movement and long-distance systemic transport on the hosts infected with BBWV 2].

PubMed

Hong, Jian; Wang, Wei-Bing; Zhou, Xue-Ping; Hu, Dong-Wei

2006-06-01

The alteration of ultrastructure in Pisum sativum and Vicia faba leaf cells infected with B935 isolate of BBWV 2 were investigated by electron microscopy, immunogold-labeling technique. The results showed that the membranous proliferation, virus-formed crystals and tubular structures were found in leaf cells of two hosts. At early stages of infection, the tubules containing virus-like particles associate with plasmodesmata in mesophyll cell. Immunogold particles anti-BBWV 2 were localized to the plasmodesmata modified by tubules passing through them. The membranous proliferation and virus-formed tubules were also found in the parenchyma cells, companion cells and transfer cells of vascular bundle. Some virus-like particles located within sieve tube can be labeled immunogold particles anti-BBWV 2. These suggest that BBWV 2, similar CPMV, produce tubules extending into the plasmodesmata. Virions assembled in the cytoplasm are escorted to the tubular structures through interactions with their MP and are then transported to the adjacent cell. Many 160 nm in diameter virus-formed tubules in the cytoplasm, as a special aggregate, not directly relate to cell-to-cell movement; Intact virions are long-distance sustemic transported possibly through sieve elements.

Structure and organization of paramyxovirus particles.

PubMed

Cox, Robert M; Plemper, Richard K

2017-06-01

The paramyxovirus family comprises major human and animal pathogens such as measles virus (MeV), mumps virus (MuV), the parainfluenzaviruses, Newcastle disease virus (NDV), and the highly pathogenic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface. A number of crystal structures of different paramyxovirus proteins and protein fragments were solved, but the available information concerning overall virion organization remains limited. However, recent studies have reported cryo-electron tomography-based reconstructions of Sendai virus (SeV), MeV, NDV, and human parainfluenza virus type 3 (HPIV3) particles and a surface assessment of NiV-derived virus-like particles (VLPs), which have yielded innovative hypotheses concerning paramyxovirus particle assembly, budding, and organization. Following a summary of the current insight into paramyxovirus virion morphology, this review will focus on discussing the implications of these particle reconstructions on the present models of paramyxovirus assembly and infection. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Insights into the polerovirus-plant interactome revealed by coimmunoprecipitation and mass spectrometry.

PubMed

DeBlasio, Stacy L; Johnson, Richard; Mahoney, Jaclyn; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

2015-04-01

Identification of host proteins interacting with the aphidborne Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step toward understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to coimmunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells, overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and consisted of host proteins interacting directly or indirectly with virions, as well as the nonincorporated readthrough protein (RTP) and three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGO and STRING showed that plant proteins in the PLRV protein interaction network regulate key biochemical processes, including carbon fixation, amino acid biosynthesis, ion transport, protein folding, and trafficking.

Genetic engineering and chemical conjugation of potato virus X.

PubMed

Lee, Karin L; Uhde-Holzem, Kerstin; Fischer, Rainer; Commandeur, Ulrich; Steinmetz, Nicole F

2014-01-01

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

Role of Gag and lipids during HIV-1 assembly in CD4+ T cells and macrophages

PubMed Central

Mariani, Charlotte; Desdouits, Marion; Favard, Cyril; Benaroch, Philippe; Muriaux, Delphine M.

2014-01-01

HIV-1 is an RNA enveloped virus that preferentially infects CD4+ T lymphocytes and also macrophages. In CD4+ T cells, HIV-1 mainly buds from the host cell plasma membrane. The viral Gag polyprotein targets the plasma membrane and is the orchestrator of the HIV assembly as its expression is sufficient to promote the formation of virus-like particles carrying a lipidic envelope derived from the host cell membrane. Certain lipids are enriched in the viral membrane and are thought to play a key role in the assembly process and the envelop composition. A large body of work performed on infected CD4+ T cells has provided important knowledge about the assembly process and the membrane virus lipid composition. While HIV assembly and budding in macrophages is thought to follow the same general Gag-driven mechanism as in T-lymphocytes, the HIV cycle in macrophage exhibits specific features. In these cells, new virions bud from the limiting membrane of seemingly intracellular compartments, where they accumulate while remaining infectious. These structures are now often referred to as Virus Containing Compartments (VCCs). Recent studies suggest that VCCs represent intracellularly sequestered regions of the plasma membrane, but their precise nature remains elusive. The proteomic and lipidomic characterization of virions produced by T cells or macrophages has highlighted the similarity between their composition and that of the plasma membrane of producer cells, as well as their enrichment in acidic lipids, some components of raft lipids and in tetraspanin-enriched microdomains. It is likely that Gag promotes the coalescence of these components into an assembly platform from which viral budding takes place. How Gag exactly interacts with membrane lipids and what are the mechanisms involved in the interaction between the different membrane nanodomains within the assembly platform remains unclear. Here we review recent literature regarding the role of Gag and lipids on HIV-1 assembly in CD4+ T cells and macrophages. PMID:25009540

Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

PubMed

Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

2013-11-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

PubMed Central

Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

2014-01-01

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

PubMed

Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

2011-04-01

Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles.

Viral assembly of oriented quantum dot nanowires

NASA Astrophysics Data System (ADS)

Mao, Chuanbin; Flynn, Christine E.; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M.

2003-06-01

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Viral assembly of oriented quantum dot nanowires.

PubMed

Mao, Chuanbin; Flynn, Christine E; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M

2003-06-10

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Gigadalton-scale shape-programmable DNA assemblies

NASA Astrophysics Data System (ADS)

Wagenbauer, Klaus F.; Sigl, Christian; Dietz, Hendrik

2017-12-01

Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

Gigadalton-scale shape-programmable DNA assemblies.

PubMed

Wagenbauer, Klaus F; Sigl, Christian; Dietz, Hendrik

2017-12-06

Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

Bacteriophage lambda: The path from biology to theranostic agent.

PubMed

Catalano, Carlos E

2018-03-13

Viral particles provide an attractive platform for the engineering of semisynthetic therapeutic nanoparticles. They can be modified both genetically and chemically in a defined manner to alter their surface characteristics, for targeting specific cell types, to improve their pharmacokinetic features and to attenuate (or enhance) their antigenicity. These advantages derive from a detailed understanding of virus biology, gleaned from decades of fundamental genetic, biochemical, and structural studies that have provided mechanistic insight into virus assembly pathways. In particular, bacteriophages offer significant advantages as nanoparticle platforms and several have been adapted toward the design and engineering of "designer" nanoparticles for therapeutic and diagnostic (theranostic) applications. The present review focuses on one such virus, bacteriophage lambda; I discuss the biology of lambda, the tools developed to faithfully recapitulate the lambda assembly reactions in vitro and the observations that have led to cooptation of the lambda system for nanoparticle design. This discussion illustrates how a fundamental understanding of virus assembly has allowed the rational design and construction of semisynthetic nanoparticles as potential theranostic agents and illustrates the concept of benchtop to bedside translational research. This article is categorized under: Biology-Inspired Nanomaterials> Protein and Virus-Based Structures Biology-Inspired Nanomaterials> Nucleic Acid-Based Structures. © 2018 Wiley Periodicals, Inc.

Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview.

PubMed

Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

2017-07-05

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV-host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus-host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding.

Spontaneous curvature as a regulator of the size of virus capsids

NASA Astrophysics Data System (ADS)

Šiber, Antonio; Majdandžić, Antonio

2009-08-01

We investigate the physical reasons underlying the high monodispersity of empty virus capsids assembled in thermodynamical equilibrium in conditions of favorable pH and ionic strength. We propose that the high fidelity of the assembly results from the effective spontaneous curvature of the viral protein assemblies and the corresponding bending rigidity that penalizes curvatures which are larger and smaller from the spontaneous one. On the example of hepatitis B virus, which has been thoroughly studied experimentally in the context of interest to us, we estimate the magnitude of bending rigidity that is needed to suppress the appearance of aberrant capsid structures (˜60kBT) . Our approach also demonstrates that the aberrant capsids that can be classified within the Caspar-Klug framework are in most circumstances likely to be smaller from the regular ones, in agreement with the experimental findings.

Nanocoating for biomolecule delivery using layer-by-layer self-assembly

PubMed Central

Keeney, M.; Jiang, X. Y.; Yamane, M.; Lee, M.; Goodman, S.

2016-01-01

Since its introduction in the early 1990s, layer-by-layer (LbL) self-assembly of films has been widely used in the fields of nanoelectronics, optics, sensors, surface coatings, and controlled drug delivery. The growth of this industry is propelled by the ease of film manufacture, low cost, mild assembly conditions, precise control of coating thickness, and versatility of coating materials. Despite the wealth of research on LbL for biomolecule delivery, clinical translation has been limited and slow. This review provides an overview of methods and mechanisms of loading biomolecules within LbL films and achieving controlled release. In particular, this review highlights recent advances in the development of LbL coatings for the delivery of different types of biomolecules including proteins, polypeptides, DNA, particles and viruses. To address the need for co-delivery of multiple types of biomolecules at different timing, we also review recent advances in incorporating compartmentalization into LbL assembly. Existing obstacles to clinical translation of LbL technologies and enabling technologies for future directions are also discussed. PMID:27099754

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding.

PubMed

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc; Zhong, Jin

2017-03-15

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly. Copyright © 2017 American Society for Microbiology.

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

PubMed

Lee, S Y; Poloumienko, A; Belfry, S; Qu, X; Chen, W; MacAfee, N; Morin, B; Lucarotti, C; Krause, M

1996-01-01

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

PubMed

Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

2016-08-01

Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Coupling of replication and assembly in flaviviruses.

PubMed

Apte-Sengupta, Swapna; Sirohi, Devika; Kuhn, Richard J

2014-12-01

Flaviviruses affect hundreds of millions of people each year causing tremendous morbidity and mortality worldwide. This genus includes significant human pathogens such as dengue, West Nile, yellow fever, tick-borne encephalitis and Japanese encephalitis virus among many others. The disease caused by these viruses can range from febrile illness to hemorrhagic fever and encephalitis. A deeper understanding of the virus life cycle is required to foster development of antivirals and vaccines, which are an urgent need for many flaviviruses, especially dengue. The focus of this review is to summarize our current knowledge of flaviviral replication and assembly, the proteins and lipids involved therein, and how these processes are coordinated for efficient virus production. Copyright © 2014 Elsevier B.V. All rights reserved.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2016-06-07

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2016-01-01

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

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

Hoenen, Antje; Gillespie, Leah; Department of Microbiology and Immunology, University of Melbourne, Melbourne

2014-01-05

Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNV{sub KUN}) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV{sub KUN} particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNV{sub KUN} under conditions ofmore » virus infection. Our results indicate a co-ordinated and compartmentalized WNV{sub KUN} assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNV{sub KUN} assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition.« less

Role of dynamic capsomere supply for viral capsid self-assembly

NASA Astrophysics Data System (ADS)

Boettcher, Marvin A.; Klein, Heinrich C. R.; Schwarz, Ulrich S.

2015-02-01

Many viruses rely on the self-assembly of their capsids to protect and transport their genomic material. For many viral systems, in particular for human viruses like hepatitis B, adeno or human immunodeficiency virus, that lead to persistent infections, capsomeres are continuously produced in the cytoplasm of the host cell while completed capsids exit the cell for a new round of infection. Here we use coarse-grained Brownian dynamics simulations of a generic patchy particle model to elucidate the role of the dynamic supply of capsomeres for the reversible self-assembly of empty T1 icosahedral virus capsids. We find that for high rates of capsomere influx only a narrow range of bond strengths exists for which a steady state of continuous capsid production is possible. For bond strengths smaller and larger than this optimal value, the reaction volume becomes crowded by small and large intermediates, respectively. For lower rates of capsomere influx a broader range of bond strengths exists for which a steady state of continuous capsid production is established, although now the production rate of capsids is smaller. Thus our simulations suggest that the importance of an optimal bond strength for viral capsid assembly typical for in vitro conditions can be reduced by the dynamic influx of capsomeres in a cellular environment.

Discovering Deeply Divergent RNA Viruses in Existing Metatranscriptome Data with Machine Learning

NASA Astrophysics Data System (ADS)

Rivers, A. R.

2016-02-01

Most sampling of RNA viruses and phages has been directed toward a narrow range of hosts and environments. Several marine metagenomic studies have examined the RNA viral fraction in aquatic samples and found a number of picornaviruses and uncharacterized sequences. The lack of homology to known protein families has limited the discovery of new RNA viruses. We developed a computational method for identifying RNA viruses that relies on information in the codon transition probabilities of viral sequences to train a classifier. This approach does not rely on homology, but it has higher information content than other reference-free methods such as tetranucleotide frequency. Training and validation with RefSeq data gave true positive and true negative rates of 99.6% and 99.5% on the highly imbalanced validation sets (0.2% viruses) that, like the metatranscriptomes themselves, contain mostly non-viral sequences. To further test the method, a validation dataset of putative RNA virus genomes were identified in metatransciptomes by the presence of RNA dependent RNA polymerase, an essential gene for RNA viruses. The classifier successfully identified 99.4% of those contigs as viral. This approach is currently being extended to screen all metatranscriptome data sequenced at the DOE Joint Genome Institute, presently 4.5 Gb of assembled data from 504 public projects representing a wide range of marine, aquatic and terrestrial environments.

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

PubMed Central

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

2016-01-01

ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

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

Viral genome structures, charge, and sequences are optimal for capsid assembly

NASA Astrophysics Data System (ADS)

Hagan, Michael

2014-03-01

For many viruses, the spontaneous assembly of a capsid shell around the nu-cleic acid (NA) genome is an essential step in the viral life cycle. Capsid formation is a multicomponent, out-of-equilibrium assembly process for which kinetic effects and thermodynamic constraints compete to determine the outcome. Understand-ing how viral components drive highly efficient assembly under these constraints could promote biomedical efforts to block viral propagation, and would elucidate the factors controlling assembly in a wide range of systems containing proteins and polyelectrolytes. This talk will describe coarse-grained models of capsid proteins and NAs with which we investigate the dynamics and thermodynamics of virus assembly. In con-trast to recent theoretical models, we find that capsids spontaneously `overcharge' that is, the NA length which is kinetically and thermodynamically optimal possess-es a negative charge greater than the positive charge of the capsid. When applied to specific virus capsids, the calculated optimal NA lengths closely correspond to the natural viral genome lengths. These results suggest that the features included in this model (i.e. electrostatics, excluded volume, and NA tertiary structure) play key roles in determining assembly thermodynamics and consequently exert selec-tive pressure on viral evolution. I will then discuss mechanisms by which se-quence-specific interactions between NAs and capsid proteins promote selective encapsidation of the viral genome. This work was supported by NIH R01GM108021 and the Brandeis MRSEC NSF-MRSEC-0820492.

Full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm.

PubMed

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.

Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation, Deep Sequencing, and a Novel Iterative Sequence Classification Algorithm

PubMed Central

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J.; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. PMID:24695106

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

NASA Astrophysics Data System (ADS)

Lizotte, P. H.; Wen, A. M.; Sheen, M. R.; Fields, J.; Rojanasopondist, P.; Steinmetz, N. F.; Fiering, S.

2016-03-01

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The ‘in situ vaccination’ immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic antitumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from cowpea mosaic virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic antitumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the antitumour immune response. CPMV also exhibited clear treatment efficacy and systemic antitumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

PubMed

Martinez-Torrecuadrada, Jorge L; Saubi, Narciís; Pagès-Manté, Albert; Castón, José R; Espuña, Enric; Casal, J Ignacio

2003-07-04

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 micrograms of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

PubMed

Martinez-Torrecuadrada, Jorge L; Saubi, Narcis; Pagès-Manté, Albert; Castón, José R; Espuña, Enric; Casal, J Ignacio

2003-05-16

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3& mgr;g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

A highly oriented hybrid microarray modified electrode fabricated by a template-free method for ultrasensitive electrochemical DNA recognition

NASA Astrophysics Data System (ADS)

Shi, Lei; Chu, Zhenyu; Dong, Xueliang; Jin, Wanqin; Dempsey, Eithne

2013-10-01

Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors.Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors. Electronic supplementary information (ESI) available: Four-probe method for determining the conductivity of the hybrid crystal (Fig. S1); stability comparisons of the hybrid films (Fig. S2); FESEM images of the hybrid microarray (Fig. S3); electrochemical characterizations of the hybrid films (Fig. S4); DFT simulations (Fig. S5); cross-sectional FESEM image of the hybrid microarray (Fig. S6); regeneration and stability tests of the DNA biosensor (Fig. S7). See DOI: 10.1039/c3nr03097k

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

PubMed Central

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica; Bolduc, Benjamin; de la Cruz Peña, Maria Jose; Martínez, Joaquín Martínez; Anton, Josefa; Gasol, Josep M.; Rosselli, Riccardo; Rodriguez-Valera, Francisco; Sullivan, Matthew B.; Acinas, Silvia G.; Martinez-Garcia, Manuel

2017-01-01

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies. PMID:28643787

Expanding the genetic code for site-specific labelling of tobacco mosaic virus coat protein and building biotin-functionalized virus-like particles.

PubMed

Wu, F C; Zhang, H; Zhou, Q; Wu, M; Ballard, Z; Tian, Y; Wang, J Y; Niu, Z W; Huang, Y

2014-04-18

A method for site-specific and high yield modification of tobacco mosaic virus coat protein (TMVCP) utilizing a genetic code expanding technology and copper free cycloaddition reaction has been established, and biotin-functionalized virus-like particles were built by the self-assembly of the protein monomers.

Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses.

PubMed

El Omari, Kamel; Sutton, Geoff; Ravantti, Janne J; Zhang, Hanwen; Walter, Thomas S; Grimes, Jonathan M; Bamford, Dennis H; Stuart, David I; Mancini, Erika J

2013-08-06

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

PubMed

Reed, Jonathan C; Westergreen, Nick; Barajas, Brook C; Ressler, Dylan T B; Phuong, Daryl J; Swain, John V; Lingappa, Vishwanath R; Lingappa, Jaisri R

2018-05-01

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules. IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies. Copyright © 2018 American Society for Microbiology.

Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

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

Brown, Gaie; Jeffree, Chris E.; McDonald, Terence

2004-10-01

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of themore » virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.« less

Mechanisms of Size Control and Polymorphism in Viral Capsid Assembly

PubMed Central

Elrad, Oren M.; Hagan, Michael F.

2009-01-01

We simulate the assembly dynamics of icosahedral capsids from subunits that interconvert between different conformations (or quasi-equivalent states). The simulations identify mechanisms by which subunits form empty capsids with only one morphology, but adaptively assemble into different icosahedral morphologies around nanoparticle cargoes with varying sizes, as seen in recent experiments with brome mosaic virus (BMV) capsid proteins. Adaptive cargo encapsidation requires moderate cargo-subunit interaction strengths; stronger interactions frustrate assembly by stabilizing intermediates with incommensurate curvature. We compare simulation results to experiments with cowpea chlorotic mottle virus empty capsids and BMV capsids assembled on functionalized nanoparticles, and suggest new cargo encapsidation experiments. Finally, we find that both empty and templated capsids maintain the precise spatial ordering of subunit conformations seen in the crystal structure even if interactions that preserve this arrangement are favored by as little as the thermal energy, consistent with experimental observations that different subunit conformations are highly similar. PMID:18950240

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

PubMed

Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C; Wege, Christina

2016-01-01

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles.

PubMed

Vonderstein, Kirstin; Nilsson, Emma; Hubel, Philipp; Nygård Skalman, Lars; Upadhyay, Arunkumar; Pasto, Jenny; Pichlmair, Andreas; Lundmark, Richard; Överby, Anna K

2017-10-18

Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention. Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target. Copyright © 2017 Vonderstein et al.

Inhibition of U snRNP assembly by a virus-encoded proteinase.

PubMed

Almstead, Laura L; Sarnow, Peter

2007-05-01

It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

PubMed

Yang, Yantao; Qu, Na; Tan, Jie; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

2018-06-11

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag ZiL ) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and Gag ZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while Gag ZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not Gag ZiL , was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses. Copyright © 2018 the Author(s). Published by PNAS.

Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

PubMed

Hsin, Wei-Chen; Chang, Chan-Hua; Chang, Chi-You; Peng, Wei-Hao; Chien, Chung-Liang; Chang, Ming-Fu; Chang, Shin C

2018-05-24

Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969) ME ), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969) ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969) ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

Paramyxovirus Assembly and Budding: Building Particles that Transmit Infections

PubMed Central

Harrison, Megan S.; Sakaguchi, Takemasa; Schmitt, Anthony P.

2010-01-01

The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release. PMID:20398786

Core protein: a pleiotropic keystone in the HBV lifecycle

PubMed Central

Zlotnick, Adam; Venkatakrishnan, Balasubramanian; Tan, Zhenning; Lewellyn, Eric; Turner, William; Francis, Samson

2015-01-01

Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals -- while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on “From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story.” PMID:26129969

Maize rayado fino virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display

USDA-ARS?s Scientific Manuscript database

The ability of plant virus coat proteins to self-assemble into virus-like particles (VLPs), coupled with unique properties including three-dimensional structures, orthogonal reactivities, suitability for genetic manipulation and chemical bio-conjugation, provide potential utility in nanotechnology a...

Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses

USDA-ARS?s Scientific Manuscript database

To analyze transcriptome response to virus infection, we have assembled currently available microarray data on changes in gene expression levels in compatible Arabidopsis-virus interactions. We used the mean r (Pearson’s correlation coefficient) for neighboring pairs to estimate pairwise local simil...

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

PubMed Central

Li, Xiangyang; Song, Baoan; Chen, Xi; Wang, Zhenchao; Zeng, Mengjiao; Yu, Dandan; Hu, Deyu; Chen, Zhuo; Jin, Linhong; Yang, Song; Yang, Caiguang; Chen, Baoen

2013-01-01

Background Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination. Methodology/Principal Findings The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP19). Overall, N-His-TMV CP19 protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP19, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP19 layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle. Conclusion The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA. PMID:24223721

Structure of the recombinant alphavirus Western equine encephalitis virus revealed by cryoelectron microscopy.

PubMed

Sherman, Michael B; Weaver, Scott C

2010-10-01

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV's structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.

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

Cryo-electron tomography of bacterial viruses

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

Guerrero-Ferreira, Ricardo C.; Wright, Elizabeth R., E-mail: erwrigh@emory.edu

2013-01-05

Bacteriophage particles contain both simple and complex macromolecular assemblages and machines that enable them to regulate the infection process under diverse environmental conditions with a broad range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible to observe the interactions of bacteriophages with their host cells under native-state conditions at unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of investigation and future directions in the field are also discussed.

The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells*

PubMed Central

Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra

2012-01-01

The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

Student Learning about Biomolecular Self-Assembly Using Two Different External Representations

ERIC Educational Resources Information Center

Host, Gunnar E.; Larsson, Caroline; Olson, Arthur; Tibell, Lena A. E.

2013-01-01

Self-assembly is the fundamental but counterintuitive principle that explains how ordered biomolecular complexes form spontaneously in the cell. This study investigated the impact of using two external representations of virus self-assembly, an interactive tangible three-dimensional model and a static two-dimensional image, on student learning…

Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses.

PubMed

Tse, Longping V; Moller-Tank, Sven; Meganck, Rita M; Asokan, Aravind

2018-04-25

Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly. Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a distinct role and can be engineered for carrying out new functions. Copyright © 2018 American Society for Microbiology.

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

DOE PAGES

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; ...

2016-02-16

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

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

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Biomimetic Antigenic Nanoparticles Elicit Controlled Protective Immune Response to Influenza

PubMed Central

Patterson, Dustin P.; Rynda-Apple, Agnieszka; Harmsen, Ann L.; Harmsen, Allen G.; Douglas, Trevor

2013-01-01

Here we present a biomimetic strategy towards nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multi-strain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response. PMID:23540530

Induced maturation of human immunodeficiency virus.

PubMed

Mattei, Simone; Anders, Maria; Konvalinka, Jan; Kräusslich, Hans-Georg; Briggs, John A G; Müller, Barbara

2014-12-01

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

PubMed

Xu, Jin; Guo, Hui-Chen; Wei, Yan-Quan; Dong, Hu; Han, Shi-Chong; Ao, Da; Sun, De-Hui; Wang, Hai-Ming; Cao, Sui-Zhong; Sun, Shi-Qi

2014-04-01

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

De novo assembly of honey bee RNA viral genomes by tapping into the innate insect antiviral response pathway.

PubMed

Fung, Elisabeth; Hill, Kelly; Hogendoorn, Katja; Glatz, Richard V; Napier, Kathryn R; Bellgard, Matthew I; Barrero, Roberto A

2018-02-01

Bee pollination is critical for improving productivity of one third of all plants or plant products consumed by humans. The health of honey bees is in decline in many countries worldwide, and RNA viruses together with other biological, environmental and anthropogenic factors have been identified as the main causes. The rapid genetic variation of viruses represents a challenge for diagnosis. Thus, application of deep sequencing methods for detection and analysis of viruses has increased over the last years. In this study, we leverage from the innate Dicer-2 mediated antiviral response against viruses to reconstruct complete viral genomes using virus-derived small interfering RNAs (vsiRNAs). Symptomatic A. mellifera larvae collected from hives free of Colony Collapse Disorder (CCD) and the parasitic Varroa mite (Varroa destructor) were used to generate more than 107 million small RNA reads. We show that de novo assembly of insect viral sequences is less fragmented using only 22 nt long vsiRNAs rather than a combination of 21-22 nt small RNAs. Our results show that A. mellifera larvae activate the RNAi immune response in the presence of Sacbrood virus (SBV). We assembled three SBV genomes from three individual larvae from different hives in a single apiary, with 1-2% nucleotide sequence variability among them. We found 3-4% variability between SBV genomes generated in this study and earlier published Australian variants suggesting the presence of different SBV quasispecies within the country. Copyright © 2018. Published by Elsevier Inc.

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System.

PubMed

Liu, Kuancheng; Hu, Jianming

2018-04-20

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.

Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells.

PubMed

Gomez, Candace Y; Hope, Thomas J

2006-09-01

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

DNA-templated assembly of viral protein hydrogel

NASA Astrophysics Data System (ADS)

Xu, Xin; Tao, Ailin; Xu, Yun

2014-11-01

Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs.Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02414a

Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

PubMed

Earley, Lauriel F; Powers, John M; Adachi, Kei; Baumgart, Joshua T; Meyer, Nancy L; Xie, Qing; Chapman, Michael S; Nakai, Hiroyuki

2017-02-01

Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly. Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family. Copyright © 2017 American Society for Microbiology.

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

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

Liu Chao; Li Zhaofei; Wu Wenbi

2008-12-05

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc{sup ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc{sup ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are presentmore » in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.« less

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

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

Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-10-15

Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particlesmore » had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.« less

Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus.

PubMed Central

Santos, N C; Castanho, M A

1996-01-01

The tobacco mosaic virus is used as a model molecular assembly to illustrate the basic potentialities of light scattering techniques (both static and dynamic) to undergraduates. The work has two objectives: a pedagogic one (introducing light scattering to undergraduate students) and a scientific one (stabilization of the virus molecular assembly structure by the nucleic acid). Students are first challenged to confirm the stabilization of the cylindrical shape of the virus by the nucleic acid, at pH and ionic strength conditions where the coat proteins alone do not self-assemble. The experimental intramolecular scattering factor is compared with the theoretical ones for several model geometries. The data clearly suggest that the geometry is, in fact, a rod. Comparing the experimental values of gyration radius and hydrodynamic radius with the theoretical expectations further confirms this conclusion. Moreover, the rod structure is maintained over a wider range of pH and ionic strength than that valid for the coat proteins alone. The experimental values of the diffusion coefficient and radius of gyration are compared with the theoretical expectations assuming the dimensions detected by electron microscopy techniques. In fact, both values are in agreement (length approximately 300 nm, radius approximately 20 nm). PMID:8874039

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

PubMed Central

Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

2002-01-01

Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

PubMed

Vashee, Sanjay; Stockwell, Timothy B; Alperovich, Nina; Denisova, Evgeniya A; Gibson, Daniel G; Cady, Kyle C; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B; Voorhies, Alexander A; Bruening, Eric; Caposio, Patrizia; Früh, Klaus

2017-01-01

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae . Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae . Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

PubMed Central

Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia

2017-01-01

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. PMID:28989973

Actin-myosin network is required for proper assembly of influenza virus particles

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

Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregatedmore » on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.« less

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

PubMed

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

PubMed

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2015-09-04

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Filamentous phages as building blocks for reconfigurable and hierarchical self-assembly

NASA Astrophysics Data System (ADS)

Gibaud, Thomas

2017-12-01

Filamentous bacteriophages such as fd-like viruses are monodisperse rod-like colloids that have well defined properties of diameter, length, rigidity, charge and chirality. Engineering these viruses leads to a library of colloidal rods, which can be used as building blocks for reconfigurable and hierarchical self-assembly. Their condensation in an aqueous solution with additive polymers, which act as depletants to induce attraction between the rods, leads to a myriad of fluid-like micronic structures ranging from isotropic/nematic droplets, colloid membranes, achiral membrane seeds, twisted ribbons, π-wall, pores, colloidal skyrmions, Möbius anchors, scallop membranes to membrane rafts. These structures, and the way that they shape-shift, not only shed light on the role of entropy, chiral frustration and topology in soft matter, but also mimic many structures encountered in different fields of science. On the one hand, filamentous phages being an experimental realization of colloidal hard rods, their condensation mediated by depletion interactions constitutes a blueprint for the self-assembly of rod-like particles and provides a fundamental foundation for bio- or material-oriented applications. On the other hand, the chiral properties of the viruses restrict the generalities of some results but vastly broaden the self-assembly possibilities.

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

DOE PAGES

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica; ...

2017-06-23

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and thatmore » microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.« less

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

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

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and thatmore » microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.« less

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and Its Implication in Virion Assembly and Budding

PubMed Central

Wang, Jibin; Fang, Shouguo; Xiao, Han; Chen, Bo; Tam, James P.; Liu, Ding Xiang

2009-01-01

Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus. PMID:19287488

Model of human immunodeficiency virus budding and self-assembly: Role of the cell membrane

NASA Astrophysics Data System (ADS)

Zhang, Rui; Nguyen, Toan T.

2008-11-01

Budding from the plasma membrane of the host cell is an indispensable step in the life cycle of the human immunodeficiency virus (HIV), which belongs to a large family of enveloped RNA viruses, retroviruses. Unlike regular enveloped viruses, retrovirus budding happens concurrently with the self-assembly of the main retrovirus protein subunits (called Gag protein after the name of the genetic material that codes for this protein: Group-specific AntiGen) into spherical virus capsids on the cell membrane. Led by this unique budding and assembly mechanism, we study the free energy profile of retrovirus budding, taking into account the Gag-Gag attraction energy and the membrane elastic energy. We find that if the Gag-Gag attraction is strong, budding always proceeds to completion. During early stage of budding, the zenith angle of partial budded capsids, α , increases with time as α∝t1/2 . However, if the Gag-Gag attraction is weak, a metastable state of partial budding appears. The zenith angle of these partially spherical capsids is given by α0≃(τ2/κσ)1/4 in a linear approximation, where κ and σ are the bending modulus and the surface tension of the membrane, and τ is a line tension of the capsid proportional to the strength of Gag-Gag attraction. Numerically, we find α0<0.3π without any approximations. Using experimental parameters, we show that HIV budding and assembly always proceed to completion in normal biological conditions. On the other hand, by changing Gag-Gag interaction strength or membrane rigidity, it is relatively easy to tune it back and forth between complete budding and partial budding. Our model agrees reasonably well with experiments observing partial budding of retroviruses including HIV.

C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly

PubMed Central

Castón, José R.; Martínez-Torrecuadrada, Jorge L.; Maraver, Antonio; Lombardo, Eleuterio; Rodríguez, José F.; Casal, J. Ignacio; Carrascosa, José L.

2001-01-01

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis. PMID:11602723

Architecturing hierarchical function layers on self-assembled viral templates as 3D nano-array electrodes for integrated Li-ion microbatteries.

PubMed

Liu, Yihang; Zhang, Wei; Zhu, Yujie; Luo, Yanting; Xu, Yunhua; Brown, Adam; Culver, James N; Lundgren, Cynthia A; Xu, Kang; Wang, Yuan; Wang, Chunsheng

2013-01-09

This work enables an elegant bottom-up solution to engineer 3D microbattery arrays as integral power sources for microelectronics. Thus, multilayers of functional materials were hierarchically architectured over tobacco mosaic virus (TMV) templates that were genetically modified to self-assemble in a vertical manner on current-collectors, so that optimum power and energy densities accompanied with excellent cycle-life could be achieved on a minimum footprint. The resultant microbattery based on self-aligned LiFePO(4) nanoforests of shell-core-shell structure, with precise arrangement of various auxiliary material layers including a central nanometric metal core as direct electronic pathway to current collector, delivers excellent energy density and stable cycling stability only rivaled by the best Li-ion batteries of conventional configurations, while providing rate performance per foot-print and on-site manufacturability unavailable from the latter. This approach could open a new avenue for microelectromechanical systems (MEMS) applications, which would significantly benefit from the concept that electrochemically active components be directly engineered and fabricated as an integral part of the integrated circuit (IC).

Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

NASA Astrophysics Data System (ADS)

Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

2004-11-01

The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

PubMed

Tan, Wui Siew; Lewis, Christina L; Horelik, Nicholas E; Pregibon, Daniel C; Doyle, Patrick S; Yi, Hyunmin

2008-11-04

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

Breach of the nuclear lamina during assembly of herpes simplex viruses.

PubMed

Morrison, Lynda A; DeLassus, Gregory S

2011-01-01

Beneath the inner nuclear membrane lies the dense meshwork of the nuclear lamina, which provides structural support for the nuclear envelope and serves as an important organizing center for a number of nuclear and cytoplasmic constituents and processes. Herpesviruses have a significant and wide-ranging impact on human health, and their capacity to replicate and cause disease includes events that occur in the host cell nucleus. Herpesviruses begin assembly of progeny virus in the nuclei of infected cells and their capsids must escape the confines of the nucleus by budding through the inner nuclear membrane (INM) to proceed with later stages of virion assembly and egress. Access of viral capsids to the INM thus necessitates disruption of the dense nuclear lamina layer. We review herpesvirus effects on the nuclear lamina and in particular the roles of the herpes simplex virus-encoded nuclear envelope complex and viral kinases on lamin phosphorylation, dissociation, and nucleocapsid envelopment at the INM.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

PubMed

Nielsen, H Bjørn; Almeida, Mathieu; Juncker, Agnieszka Sierakowska; Rasmussen, Simon; Li, Junhua; Sunagawa, Shinichi; Plichta, Damian R; Gautier, Laurent; Pedersen, Anders G; Le Chatelier, Emmanuelle; Pelletier, Eric; Bonde, Ida; Nielsen, Trine; Manichanh, Chaysavanh; Arumugam, Manimozhiyan; Batto, Jean-Michel; Quintanilha Dos Santos, Marcelo B; Blom, Nikolaj; Borruel, Natalia; Burgdorf, Kristoffer S; Boumezbeur, Fouad; Casellas, Francesc; Doré, Joël; Dworzynski, Piotr; Guarner, Francisco; Hansen, Torben; Hildebrand, Falk; Kaas, Rolf S; Kennedy, Sean; Kristiansen, Karsten; Kultima, Jens Roat; Léonard, Pierre; Levenez, Florence; Lund, Ole; Moumen, Bouziane; Le Paslier, Denis; Pons, Nicolas; Pedersen, Oluf; Prifti, Edi; Qin, Junjie; Raes, Jeroen; Sørensen, Søren; Tap, Julien; Tims, Sebastian; Ussery, David W; Yamada, Takuji; Renault, Pierre; Sicheritz-Ponten, Thomas; Bork, Peer; Wang, Jun; Brunak, Søren; Ehrlich, S Dusko

2014-08-01

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

PubMed

Thuenemann, Eva C; Meyers, Ann E; Verwey, Jeanette; Rybicki, Edward P; Lomonossoff, George P

2013-09-01

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

PubMed

Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

2018-02-01

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

PubMed

Doležal, Michal; Hadravová, Romana; Kožíšek, Milan; Bednárová, Lucie; Langerová, Hana; Ruml, Tomáš; Rumlová, Michaela

2016-09-23

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Cryo-Electron Tomography of Rubella Virus

PubMed Central

Battisti, Anthony J.; Yoder, Joshua D.; Plevka, Pavel; Winkler, Dennis C.; Mangala Prasad, Vidya; Kuhn, Richard J.; Frey, Teryl K.; Steven, Alasdair C.

2012-01-01

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses. PMID:22855483

Cryo-electron tomography of rubella virus.

PubMed

Battisti, Anthony J; Yoder, Joshua D; Plevka, Pavel; Winkler, Dennis C; Prasad, Vidya Mangala; Kuhn, Richard J; Frey, Teryl K; Steven, Alasdair C; Rossmann, Michael G

2012-10-01

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis

PubMed Central

Komarova, Anastassia V.; Real, Eléonore; Borman, Andrew M.; Brocard, Michèle; England, Patrick; Tordo, Noël; Hershey, John W.B.; Jacob, Yves

2007-01-01

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5′-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein–protein interaction with the cellular translation machinery. PMID:17287294

The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

PubMed

Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

2012-11-01

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. Copyright © 2012 Elsevier B.V. All rights reserved.

A Method for Molecular Dynamics on Curved Surfaces

PubMed Central

Paquay, Stefan; Kusters, Remy

2016-01-01

Dynamics simulations of constrained particles can greatly aid in understanding the temporal and spatial evolution of biological processes such as lateral transport along membranes and self-assembly of viruses. Most theoretical efforts in the field of diffusive transport have focused on solving the diffusion equation on curved surfaces, for which it is not tractable to incorporate particle interactions even though these play a crucial role in crowded systems. We show here that it is possible to take such interactions into account by combining standard constraint algorithms with the classical velocity Verlet scheme to perform molecular dynamics simulations of particles constrained to an arbitrarily curved surface. Furthermore, unlike Brownian dynamics schemes in local coordinates, our method is based on Cartesian coordinates, allowing for the reuse of many other standard tools without modifications, including parallelization through domain decomposition. We show that by applying the schemes to the Langevin equation for various surfaces, we obtain confined Brownian motion, which has direct applications to many biological and physical problems. Finally we present two practical examples that highlight the applicability of the method: 1) the influence of crowding and shape on the lateral diffusion of proteins in curved membranes; and 2) the self-assembly of a coarse-grained virus capsid protein model. PMID:27028633

A Method for Molecular Dynamics on Curved Surfaces

NASA Astrophysics Data System (ADS)

Paquay, Stefan; Kusters, Remy

2016-03-01

Dynamics simulations of constrained particles can greatly aid in understanding the temporal and spatial evolution of biological processes such as lateral transport along membranes and self-assembly of viruses. Most theoretical efforts in the field of diffusive transport have focussed on solving the diffusion equation on curved surfaces, for which it is not tractable to incorporate particle interactions even though these play a crucial role in crowded systems. We show here that it is possible to combine standard constraint algorithms with the classical velocity Verlet scheme to perform molecular dynamics simulations of particles constrained to an arbitrarily curved surface, in which such interactions can be taken into account. Furthermore, unlike Brownian dynamics schemes in local coordinates, our method is based on Cartesian coordinates allowing for the reuse of many other standard tools without modifications, including parallelisation through domain decomposition. We show that by applying the schemes to the Langevin equation for various surfaces, confined Brownian motion is obtained, which has direct applications to many biological and physical problems. Finally we present two practical examples that highlight the applicability of the method: (i) the influence of crowding and shape on the lateral diffusion of proteins in curved membranes and (ii) the self-assembly of a coarse-grained virus capsid protein model.

Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

PubMed

Martinez-Torrecuadrada, J L; Castón, J R; Castro, M; Carrascosa, J L; Rodriguez, J F; Casal, J I

2000-12-20

Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. Copyright 2000 Academic Press.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

PubMed

Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

2011-06-16

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus.

PubMed

Gallo, Araiz; Valli, Adrian; Calvo, María; García, Juan Antonio

2018-05-01

Accurate assembly of viral particles in the potyvirus Plum pox virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans , was assembled into virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection. IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum pox virus (genus Potyvirus ), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle. Copyright © 2018 American Society for Microbiology.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

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

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to exploremore » the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.« less

Forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of HIV-1 gp41

PubMed Central

2013-01-01

Background The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site. Results The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER. Conclusions The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus. PMID:23618462

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

PubMed

Phelps, Jamie P; Dao, Philip; Jin, Hongfan; Rasochova, Lada

2007-02-01

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

The molecular biology of Bluetongue virus replication.

PubMed

Patel, Avnish; Roy, Polly

2014-03-01

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

Engineering building blocks for self-assembling protein nanoparticles

PubMed Central

2010-01-01

Like natural viruses, manmade protein cages for drug delivery are to be ideally formed by repetitive subunits with self-assembling properties, mimicking viral functions and molecular organization. Naturally formed nanostructures (such as viruses, flagella or simpler protein oligomers) can be engineered to acquire specific traits of interest in biomedicine, for instance through the addition of cell targeting agents for desired biodistribution and specific delivery of associated drugs. However, fully artificial constructs would be highly desirable regarding finest tuning and adaptation to precise therapeutic purposes. Although engineering of protein assembling is still in its infancy, arising principles and promising strategies of protein manipulation point out the rational construction of nanoscale protein cages as a feasible concept, reachable through conventional recombinant DNA technologies and microbial protein production. PMID:21192790

Analysis of differential selective forces acting on the coat protein (P3) of the plant virus family Luteoviridae.

PubMed

Torres, Marina W; Corrêa, Régis L; Schrago, Carlos G

2005-12-30

The coat protein (CP) of the family Luteoviridae is directly associated with the success of infection. It participates in various steps of the virus life cycle, such as virion assembly, stability, systemic infection, and transmission. Despite its importance, extensive studies on the molecular evolution of this protein are lacking. In the present study, we investigate the action of differential selective forces on the CP coding region using maximum likelihood methods. We found that the protein is subjected to heterogeneous selective pressures and some sites may be evolving near neutrality. Based on the proposed 3-D model of the CP S-domain, we showed that nearly neutral sites are predominantly located in the region of the protein that faces the interior of the capsid, in close contact with the viral RNA, while highly conserved sites are mainly part of beta-strands, in the protein's major framework.

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis.

PubMed

Fang, Mingli; Diao, Wenzhen; Dong, Boqi; Wei, Hongfei; Liu, Jialin; Hua, Li; Zhang, Miaomin; Guo, Sheng; Xiao, Yue; Yu, Yongli; Wang, Liying; Wan, Min

2015-01-01

Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines. © 2016 S. Karger AG, Basel.

From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein.

PubMed

Hempel, Franziska; Maurer, Michael; Brockmann, Björn; Mayer, Christian; Biedenkopf, Nadine; Kelterbaum, Anne; Becker, Stephan; Maier, Uwe G

2017-07-27

The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

PubMed

Lennemann, Nicholas J; Coyne, Carolyn B

2017-02-01

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

Enhanced assembly and colloidal stabilization of primate erythroparvovirus 1 virus-like particles for improved surface engineering.

PubMed

Sánchez-Rodríguez, Sandra Paola; Morán-García, Areli del Carmen; Bolonduro, Olurotimi; Dordick, Jonathan S; Bustos-Jaimes, Ismael

2016-04-15

Virus-like particles (VLPs) are the product of the self-assembly, either in vivo or in vitro, of structural components of viral capsids. These particles are excellent scaffolds for surface display of biomolecules that can be used in vaccine development and tissue-specific drug delivery. Surface engineering of VLPs requires structural stability and chemical reactivity. Herein, we report the enhanced assembly, colloidal stabilization and fluorescent labeling of primate erythroparvovirus 1 (PE1V), generally referred to as parvovirus B19. In vitro assembly of the VP2 protein of PE1V produces VLPs, which are prone to flocculate and hence undergo limited chemical modification by thiol-specific reagents like the fluorogenic monobromobimane (mBBr). We determined that the addition of 0.2M l-arginine during the assembly process produced an increased yield of soluble VLPs with good dispersion stability. Fluorescent labeling of VLPs suspended in phosphate buffered saline (PBS) added with 0.2M l-Arg was achieved in significantly shorter times than the flocculated VLPs assembled in only PBS buffer. Finally, to demonstrate the potential application of this approach, mBBr-labeled VLPs were successfully used to tag human hepatoma HepG2 cells. This new method for assembly and labeling PE1V VLPs eases its applications and provides insights on the manipulation of this biomaterial for further developments. Application of virus-derived biomaterials sometimes requires surface modification for diverse purposes, including enhanced cell-specific interaction, the inclusion of luminescent probes for bioimaging, or the incorporation of catalytic properties for the production of enzyme nanocarriers. In this research, we reported for the first time the colloidal stabilization of the primate erythroparvovirus 1 (PE1V) virus-like particles (VLPs). Also, we report the chemical modification of the natural Cys residues located on the surface of these VLPs with a fluorescent probe, as well as its application for tagging hepatoma cells in vitro. Keeping in mind that PE1V is a human pathogen, virus-host interactions already exist in human cells, and they can be exploited for therapeutic and research aims. This study will impact on the speed in which the scientific community will be able to manipulate PE1V VLPs for diverse purposes. Additionally, this study may provide insights on the colloidal properties of these VLPs as well as in the effect of different protein additives used for protein stabilization. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

PubMed

Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

2008-02-29

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

Critical Role of the HTLV-1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

PubMed

Martin, Jessica L; Mendonça, Luiza; Marusinec, Rachel; Zuczek, Jennifer; Angert, Isaac; Blower, Ruth J; Mueller, Joachim D; Perilla, Juan R; Zhang, Wei; Mansky, Louis M

2018-04-25

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD) as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses. Importance Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The capsid (CA) domain of Gag is generally thought to possess the key determinants for Gag-Gag interactions, and the present study has discovered several critical amino acid residues in the CA domain of human T-cell leukemia virus type 1 (HTLV-1) Gag, an important cancer-causing human retrovirus, which are distinct from that of human immunodeficiency virus type 1 (HIV-1) as well as other retroviruses studied to date. Altogether, our results provide important new insights into a poorly understood aspect of HTLV-1 replication, which significantly enhances our understanding of the molecular nature of Gag-Gag interaction determinants crucial for virus particle assembly. Copyright © 2018 American Society for Microbiology.

A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production.

PubMed

Geisler, Christoph

2018-02-07

Adventitious viral contamination in cell substrates used for biologicals production is a major safety concern. A powerful new approach that can be used to identify adventitious viruses is a combination of bioinformatics tools with massively parallel sequencing technology. Typically, this involves mapping or BLASTN searching individual reads against viral nucleotide databases. Although extremely sensitive for known viruses, this approach can easily miss viruses that are too dissimilar to viruses in the database. Moreover, it is computationally intensive and requires reference cell genome databases. To avoid these drawbacks, we set out to develop an alternative approach. We reasoned that searching genome and transcriptome assemblies for adventitious viral contaminants using TBLASTN with a compact viral protein database covering extant viral diversity as the query could be fast and sensitive without a requirement for high performance computing hardware. We tested our approach on Spodoptera frugiperda Sf-RVN, a recently isolated insect cell line, to determine if it was contaminated with one or more adventitious viruses. We used Illumina reads to assemble the Sf-RVN genome and transcriptome and searched them for adventitious viral contaminants using TBLASTN with our viral protein database. We found no evidence of viral contamination, which was substantiated by the fact that our searches otherwise identified diverse sequences encoding virus-like proteins. These sequences included Maverick, R1 LINE, and errantivirus transposons, all of which are common in insect genomes. We also identified previously described as well as novel endogenous viral elements similar to ORFs encoded by diverse insect viruses. Our results demonstrate TBLASTN searching massively parallel sequencing (MPS) assemblies with a compact, manually curated viral protein database is more sensitive for adventitious virus detection than BLASTN, as we identified various sequences that encoded virus-like proteins, but had no similarity to viral sequences at the nucleotide level. Moreover, searches were fast without requiring high performance computing hardware. Our study also documents the enhanced biosafety profile of Sf-RVN as compared to other Sf cell lines, and supports the notion that Sf-RVN is highly suitable for the production of safe biologicals.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

PubMed

Bruhn, Jessica F; Kirchdoerfer, Robert N; Urata, Sarah M; Li, Sheng; Tickle, Ian J; Bricogne, Gérard; Saphire, Erica Ollmann

2017-01-15

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. Copyright © 2017 American Society for Microbiology.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

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

Bruhn, Jessica F.; Kirchdoerfer, Robert N.; Urata, Sarah M.

ABSTRACT Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (generaMarburgvirusandEbolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOVmore » VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCEMarburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.« less

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

PubMed

Pessler, F; Pendergrast, P S; Hernandez, N

1997-07-01

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

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

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE PAGES

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc; ...

2017-06-13

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

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.

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

Relative size selection of a conjugated polyelectrolyte in virus-like protein structures.

PubMed

Brasch, Melanie; Cornelissen, Jeroen J L M

2012-02-01

A conjugated polyelectrolyte poly[(2-methoxy-5-propyloxy sulfonate)-phenyl-ene vinylene] (MPS-PPV) drives the assembly of virus capsid proteins to form single virus-like particles (VLPs) and aggregates with more than two VLPs, with a relative selection of high molecular weight polymer in the latter. This journal is © The Royal Society of Chemistry 2012

Pro-Apoptotic Breast Cancer Nanotherapeutics

DTIC Science & Technology

2013-07-01

Examples of nonspherical cell-penetrating particles existing in nature include the fila- mentous viruses such as the tobaccomosaic virus and the potato... virus X.4 Inspired by these examples from biology, Discher et al. have shown that cylindrical micelles have longer circulation times relative to...intended to create a protective corona around the assembled nanostructure, which would limit nonspecific adsorption of proteins and reduce proteolysis

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

PubMed Central

Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S.

2017-01-01

ABSTRACT The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. PMID:28794042

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion.

PubMed

Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S; Zimmerberg, Joshua

2017-11-01

The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. Copyright © 2017 American Society for Microbiology.

Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

PubMed Central

Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

2015-01-01

Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

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

Ibrahim, Amr; Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619; Hutchens, Heather M.

2012-11-25

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed withmore » P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.« less

Icosahedral plant viral nanoparticles - bioinspired synthesis of nanomaterials/nanostructures.

PubMed

Narayanan, Kannan Badri; Han, Sung Soo

2017-10-01

Viral nanotechnology utilizes virus nanoparticles (VNPs) and virus-like nanoparticles (VLPs) of plant viruses as highly versatile platforms for materials synthesis and molecular entrapment that can be used in the nanotechnological fields, such as in next-generation nanoelectronics, nanocatalysis, biosensing and optics, and biomedical applications, such as for targeting, therapeutic delivery, and non-invasive in vivo imaging with high specificity and selectivity. In particular, plant virus capsids provide biotemplates for the production of novel nanostructured materials with organic/inorganic moieties incorporated in a very precise and controlled manner. Interestingly, capsid proteins of spherical plant viruses can self-assemble into well-organized icosahedral three-dimensional (3D) nanoscale multivalent architectures with high monodispersity and structural symmetry. Using viral genetic and protein engineering of icosahedral viruses with a variety of sizes, the interior, exterior and the interfaces between coat protein (CP) subunits can be manipulated to fabricate materials with a wide range of desirable properties allowing for biomineralization, encapsulation, infusion, controlled self-assembly, and multivalent ligand display of nanoparticles or molecules for varied applications. In this review, we discuss the various functional nanomaterials/nanostructures developed using the VNPs and VLPs of different icosahedral plant viruses and their nano(bio)technological and nanomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.

PubMed

Tseng, Ying-Tzu; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

2014-04-27

Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

PubMed

Joseph, Narcisse Ms; Ho, Kok Lian; Tey, Beng Ti; Tan, Chon Seng; Shafee, Norazizah; Tan, Wen Siang

2016-07-08

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016. © 2016 American Institute of Chemical Engineers.

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

PubMed Central

2014-01-01

Background Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. Results SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. Conclusions The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly. PMID:24766657

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

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

Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, whichmore » is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.« less

Activation of DNA damage repair pathways by murine polyomavirus.

PubMed

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L

2016-10-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Influenza vaccines based on virus-like particles

PubMed Central

Kang, Sang-Moo; Song, Jae-Min; Quan, Fu-Shi; Compans, Richard W.

2009-01-01

The simultaneous expression of structural proteins of virus can produce virus-like particles (VLPs) by a self-assembly process in a viral life cycle even in the absence of genomic material. Taking an advantage of structural and morphological similarities of VLPs to native virions, VLPs have been suggested as a promising platform for new viral vaccines. In the light of a pandemic threat, influenza VLPs have been recently developed as a new generation of non-egg based cell culture-derived vaccine candidates against influenza infection. Animals vaccinated with VLPs containing hemagglutinin (HA) or HA and neuraminidase (NA) were protected from morbidity and mortality resulting from lethal influenza infections. Influenza VLPs serve as an excellent model system of an enveloped virus for understanding the properties of VLPs in inducing protective immunity. In this review, we briefly describe the characteristics of influenza VLPs assembled with a lipid bilayer containing glycoproteins, and summarize the current progress on influenza VLPs as an alternative vaccine candidate against seasonal as well as pandemic influenza viruses. In addition, the protective immune correlates induced by vaccination with influenza VLPs are discussed. PMID:19374929

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

PubMed Central

2011-01-01

Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

Membrane Assembly during the Infection Cycle of the Giant Mimivirus

PubMed Central

Mutsafi, Yael; Shimoni, Eyal; Shimon, Amir; Minsky, Abraham

2013-01-01

Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, ∼70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general. PMID:23737745

The Alphavirus Exit Pathway: What We Know and What We Wish We Knew

PubMed Central

2018-01-01

Alphaviruses are enveloped positive sense RNA viruses and include serious human pathogens, such as the encephalitic alphaviruses and Chikungunya virus. Alphaviruses are transmitted to humans primarily by mosquito vectors and include species that are classified as emerging pathogens. Alphaviruses assemble highly organized, spherical particles that bud from the plasma membrane. In this review, we discuss what is known about the alphavirus exit pathway during a cellular infection. We describe the viral protein interactions that are critical for virus assembly/budding and the host factors that are involved, and we highlight the recent discovery of cell-to-cell transmission of alphavirus particles via intercellular extensions. Lastly, we discuss outstanding questions in the alphavirus exit pathway that may provide important avenues for future research. PMID:29470397

Multiscale Modeling of Virus Structure, Assembly, and Dynamics

NASA Astrophysics Data System (ADS)

May, Eric R.; Arora, Karunesh; Mannige, Ranjan V.; Nguyen, Hung D.; Brooks, Charles L.

Viruses are traditionally considered as infectious agents that attack cells and cause illnesses like AIDS, Influenza, Hepatitis, etc. However, recent advances have illustrated the potential for viruses to play positive roles for human health, instead of causing disease [1, 2]. For example, viruses can be employed for a variety of biomedical and biotechnological applications, including gene therapy[3], drug delivery[4], tumor targeting[5], and medical imaging[6]. Therefore, it is important to understand quantitatively how viruses operate such that they can be engineered in a predictive manner for beneficial roles.

Membrane-mediated interaction between retroviral capsids

NASA Astrophysics Data System (ADS)

Zhang, Rui; Nguyen, Toan

2012-02-01

A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

Viruses, Artificial Viruses and Virus-Based Structures for Biomedical Applications.

PubMed

van Rijn, Patrick; Schirhagl, Romana

2016-06-01

Nanobiomaterials such as virus particles and artificial virus particles offer tremendous opportunities to develop new biomedical applications such as drug- or gene-delivery, imaging and sensing but also improve understanding of biological mechanisms. Recent advances within the field of virus-based systems give insights in how to mimic viral structures and virus assembly processes as well as understanding biodistribution, cell/tissue targeting, controlled and triggered disassembly or release and circulation times. All these factors are of high importance for virus-based functional systems. This review illustrates advances in mimicking and enhancing or controlling these aspects to a high degree toward delivery and imaging applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional Redundancy in HIV-1 Viral Particle Assembly

PubMed Central

O'Carroll, Ina P.; Crist, Rachael M.; Mirro, Jane; Harvin, Demetria; Soheilian, Ferri; Kamata, Anne; Nagashima, Kunio

2012-01-01

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly. PMID:22993163

New Materials Based on Spider Silk

DTIC Science & Technology

2007-11-06

encoding the honey bee secretion signal was (genetically) fused to the spider silk protein. A respective recombinant virus was used to infect high...2007). Role of individual spider silk domains during assembly. Poster at the conference on "Protein Assembly in Materials, Biology and Medicine

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress.

PubMed

Garaigorta, Urtzi; Heim, Markus H; Boyd, Bryan; Wieland, Stefan; Chisari, Francis V

2012-10-01

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

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

Hibiscus Chlorotic Ringspot Virus Coat Protein Is Essential for Cell-to-Cell and Long-Distance Movement but Not for Viral RNA Replication

PubMed Central

Niu, Shengniao; Gil-Salas, Francisco M.; Tewary, Sunil Kumar; Samales, Ashwin Kuppusamy; Johnson, John; Swaminathan, Kunchithapadam; Wong, Sek-Man

2014-01-01

Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro. PMID:25402344

Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

PubMed Central

Kolesnikova, Larissa; Welsch, Sonja; Krähling, Verena; Davey, Norman; Parsy, Marie-Laure; Becker, Stephan; Briggs, John A. G.

2011-01-01

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps. PMID:22110401

De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLAST-based assembly pipeline.

PubMed

Lin, You-Yu; Hsieh, Chia-Hung; Chen, Jiun-Hong; Lu, Xuemei; Kao, Jia-Horng; Chen, Pei-Jer; Chen, Ding-Shinn; Wang, Hurng-Yi

2017-04-26

The accuracy of metagenomic assembly is usually compromised by high levels of polymorphism due to divergent reads from the same genomic region recognized as different loci when sequenced and assembled together. A viral quasispecies is a group of abundant and diversified genetically related viruses found in a single carrier. Current mainstream assembly methods, such as Velvet and SOAPdenovo, were not originally intended for the assembly of such metagenomics data, and therefore demands for new methods to provide accurate and informative assembly results for metagenomic data. In this study, we present a hybrid method for assembling highly polymorphic data combining the partial de novo-reference assembly (PDR) strategy and the BLAST-based assembly pipeline (BBAP). The PDR strategy generates in situ reference sequences through de novo assembly of a randomly extracted partial data set which is subsequently used for the reference assembly for the full data set. BBAP employs a greedy algorithm to assemble polymorphic reads. We used 12 hepatitis B virus quasispecies NGS data sets from a previous study to assess and compare the performance of both PDR and BBAP. Analyses suggest the high polymorphism of a full metagenomic data set leads to fragmentized de novo assembly results, whereas the biased or limited representation of external reference sequences included fewer reads into the assembly with lower assembly accuracy and variation sensitivity. In comparison, the PDR generated in situ reference sequence incorporated more reads into the final PDR assembly of the full metagenomics data set along with greater accuracy and higher variation sensitivity. BBAP assembly results also suggest higher assembly efficiency and accuracy compared to other assembly methods. Additionally, BBAP assembly recovered HBV structural variants that were not observed amongst assembly results of other methods. Together, PDR/BBAP assembly results were significantly better than other compared methods. Both PDR and BBAP independently increased the assembly efficiency and accuracy of highly polymorphic data, and assembly performances were further improved when used together. BBAP also provides nucleotide frequency information. Together, PDR and BBAP provide powerful tools for metagenomic data studies.

75 FR 21232 - Application(s) for Duty-Free Entry of Scientific Instruments

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-23

... Institute, Kent State University, Summit Street, PO Box 5190, Kent, OH 44242. Instrument: Electron... W. State Street, Lilly Hall, B126, West Lafayette, IN 47907-2054. Instrument: Electron Microscope... viruses and other macromolecular assemblies. Using cryo-electron microscopy, numerous virus/macromolecular...

Impact of a nonuniform charge distribution on virus assembly

NASA Astrophysics Data System (ADS)

Li, Siyu; Erdemci-Tandogan, Gonca; Wagner, Jef; van der Schoot, Paul; Zandi, Roya

2017-08-01

Many spherical viruses encapsulate their genomes in protein shells with icosahedral symmetry. This process is spontaneous and driven by electrostatic interactions between positive domains on the virus coat proteins and the negative genomes. We model the effect of the nonuniform icosahedral charge distribution from the protein shell instead using a mean-field theory. We find that this nonuniform charge distribution strongly affects the optimal genome length and that it can explain the experimentally observed phenomenon of overcharging of virus and viruslike particles.

Virus and Host Mechanics Support Membrane Penetration and Cell Entry

PubMed Central

2016-01-01

Viruses are quasi-inert macromolecular assemblies. Their metastable conformation changes during entry into cells, when chemical and mechanical host cues expose viral membrane-interacting proteins. This leads to membrane rupture or fusion and genome uncoating. Importantly, virions tune their physical properties and enhance penetration and uncoating. For example, influenza virus softens at low pH to uncoat. The stiffness and pressure of adenovirus control uncoating and membrane penetration. Virus and host mechanics thus present new opportunities for antiviral therapy. PMID:26842477

Charge detection mass spectrometry: Instrumentation & applications to viruses

NASA Astrophysics Data System (ADS)

Pierson, Elizabeth E.

For over three decades, electrospray ionization (ESI) has been used to ionize non-covalent complexes and subsequently transfer the intact ion into the gas phase for mass spectrometry (MS) analysis. ESI generates a distribution of multiple charged ions, resulting in an m/z spectrum comprised of a series of peaks, known as a charge state envelope. To obtain mass information, the number of charges for each peak must be deduced. For smaller biological analytes like peptides, the charge states are sufficiently resolved and this process is straightforward. For macromolecular complexes exceeding ~100 kDa, this process is complicated by the broadening and shifting of charge states due to incomplete desolvation, salt adduction, and inherent mass heterogeneity. As the analyte mass approaches the MDa regime, the m/z spectrum is often comprised of a broad distribution of unresolved charge states. In such cases, mass determination is precluded. Charge detection mass spectrometry (CDMS) is an emerging MS technique for determining the masses of heterogeneous, macromolecular complexes. In CDMS, the m/z and z of single ions are measured concurrently so that mass is easily calculated. With this approach, deconvolution of an m/z spectrum is unnecessary. This measurement is carried out by passing macroions through a conductive cylinder. The induced image charge on the cylindrical detector provides information about m/z and z: the m/z is related to its time-of-flight through the detector, and the z is related to the intensity of the image charge. We have applied CDMS to study the self-assembly of virus capsids. Late-stage intermediates in the assembly of hepatitis B virus, a devastating human pathogen, have been identified. This is the first time that such intermediates have been detected and represent a significant advancement towards understanding virus capsid assembly. CDMS has also been used to identify oversized, non-icosahedral polymorphs in the assembly of woodchuck hepatitis virus capsids. Finally, CDMS has been used to characterize the purity of adeno-associated viral vectors for potential gene therapy applications.

Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

PubMed Central

Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

2013-01-01

SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

Crystal structure of an HIV assembly and maturation switch

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

Wagner, Jonathan M.; Zadrozny, Kaneil K.; Chrustowicz, Jakub

Virus assembly and maturation proceed through the programmed operation of molecular switches, which trigger both local and global structural rearrangements to produce infectious particles. HIV-1 contains an assembly and maturation switch that spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the precursor structural protein, Gag. The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn, and the stem comprises a 6-helix bundle. The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysismore » during maturation. This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses. Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate.« less

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

PubMed

Huang, Zhong; Elkin, Galina; Maloney, Bryan J; Beuhner, Norene; Arntzen, Charles J; Thanavala, Yasmin; Mason, Hugh S

2005-03-07

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.

Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter▿

PubMed Central

Konesky, Kasey L.; Nyborg, Jennifer K.; Laybourn, Paul J.

2006-01-01

Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter. PMID:16943293

Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

PubMed Central

Ren, Suping; Espiritu, Christine; Kelly, Mollie; Lau, Vincent; Zheng, Lingjie; Hartman, George D.; Flores, Osvaldo A.; Klumpp, Klaus

2017-01-01

ABSTRACT The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5′ and 3′ ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA. PMID:28559265

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

PubMed

Wijesinghe, Kaveesha J; Stahelin, Robert V

2015-12-30

Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in West Nile virus-infected cells

PubMed Central

Basu, Mausumi; Courtney, Sean C.

2017-01-01

Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle. PMID:28241074

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses.

PubMed

Roux, Simon; Brum, Jennifer R; Dutilh, Bas E; Sunagawa, Shinichi; Duhaime, Melissa B; Loy, Alexander; Poulos, Bonnie T; Solonenko, Natalie; Lara, Elena; Poulain, Julie; Pesant, Stéphane; Kandels-Lewis, Stefanie; Dimier, Céline; Picheral, Marc; Searson, Sarah; Cruaud, Corinne; Alberti, Adriana; Duarte, Carlos M; Gasol, Josep M; Vaqué, Dolors; Bork, Peer; Acinas, Silvia G; Wincker, Patrick; Sullivan, Matthew B

2016-09-29

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses

NASA Astrophysics Data System (ADS)

2016-09-01

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting ‘global ocean virome’ dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

PubMed

Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

2015-03-01

Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Interaction between Flavivirus and Cytoskeleton during Virus Replication

PubMed Central

Foo, Kar Yue; Chee, Hui-Yee

2015-01-01

Flaviviruses are potentially human pathogens that cause major epidemics worldwide. Flavivirus interacts with host cell factors to form a favourable virus replication site. Cell cytoskeletons have been observed to have close contact with flaviviruses, which expands the understanding of cytoskeleton functions during virus replication, although many detailed mechanisms are still unclear. The interactions between the virus and host cytoskeletons such as actin filaments, microtubules, and intermediate filaments have provided insight into molecular alterations during the virus infection, such as viral entry, in-cell transport, scaffold assembly, and egress. This review article focuses on the utilization of cytoskeleton by Flavivirus and the respective functions during virus replication. PMID:26347881

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

PubMed

Fleith, Renata C; Mears, Harriet V; Leong, Xin Yun; Sanford, Thomas J; Emmott, Edward; Graham, Stephen C; Mansur, Daniel S; Sweeney, Trevor R

2018-06-01

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.

A molecular tug-of-war: Global plant proteome changes during viral infection

USDA-ARS?s Scientific Manuscript database

Plant pathogenic viruses cause a number of economically important diseases in food, fuel, and fiber crops worldwide. As obligate parasites with highly reduced genomes, viruses rely heavily on their hosts for replication, assembly, intra- and intercellular movement, and attraction of vectors for disp...

Complex Virus–Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview

PubMed Central

Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

2017-01-01

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV–host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus–host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding. PMID:28678154

Using Synchrotron Radiation and Electron Microscopy to Map the Huge Structural Changes that Occur in Viruses During Their Life Cycle

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

Rossman, Michael

2011-09-07

The crystallographic techniques for structure determination of proteins and neucleic acids at near atomic resolution using synchrotron X-radiation has become almost automatic. However the limits of this procedure are determined by the availability of crystals. As the size and complexity of the molecular assemblies being studied increases, the likelihood of growing useful crystals diminishes. Cryo electron microscopy and tomography have extended the range of biological objects that can be determined at near atomic resolution. Furthermore it is now becoming apparent that the function of the molecular assemblies most often requires very large conformational changes that could never be contained withinmore » a crystal, Examples will be presented of the structural changes that occur in viruses as they assembly and prepare to infect new cells.« less

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

PubMed

Broce, Sean; Hensley, Lisa; Sato, Tomoharu; Lehrer-Graiwer, Joshua; Essrich, Christian; Edwards, Katie J; Pajda, Jacqueline; Davis, Christopher J; Bhadresh, Rami; Hurt, Clarence R; Freeman, Beverly; Lingappa, Vishwanath R; Kelleher, Colm A; Karpuj, Marcela V

2016-05-14

Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

Three-Dimensional Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent Varicella-Zoster Virus Infection

PubMed Central

Goodwin, Thomas J.; McCarthy, Maureen; Osterrieder, Nikolaus; Cohrs, Randall J.; Kaufer, Benedikt B.

2013-01-01

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells. PMID:23935496

Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

NASA Astrophysics Data System (ADS)

Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

2016-02-01

Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

Epidemiology of Feline Foamy Virus and Feline Immunodeficiency Virus Infections in Domestic and Feral Cats: a Seroepidemiological Study

PubMed Central

Winkler, I. G.; Löchelt, M.; Flower, R. L. P.

1999-01-01

Although foamy viruses (Spumaviruses) have repeatedly been isolated from both healthy and diseased cats, cattle, and primates, the primary mode of transmission of those common viruses remains undefined. A database of the feline foamy virus (FeFV) and feline immunodeficiency virus (FIV) antibody status, age, and sex of 389 domestic cats presented to veterinarians was assembled. A similar database for 66 feral (wild) cats was also assembled. That FeFV antibody status reflects infection was validated by PCR. Both FeFV and FIV infection rates were found to gradually increase with age, and over 70% of cats older than 9 years were seropositive for FeFV. In domestic cats, the prevalence of FeFV infection was similar in both sexes. In feral cats, FeFV infection was more prevalent in female cats than in male cats. Although both FeFV and FIV have been reported to be transmitted by biting, the patterns of infection observed are more consistent with an interpretation that transmission of these two retroviruses is not the same. The prevalence of FIV infection is highest in nondesexed male cats, the animals most likely to display aggressive behavior. The gradual increase in the proportion of FeFV-infected animals is consistent with transmission of foamy viruses by intimate social contact between animals and less commonly by aggressive behavior. PMID:10449463

Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

PubMed

Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani; Yi, MinKyung

2015-04-01

Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

Metagenomic Survey for Viruses in Western Arctic Caribou, Alaska, through Iterative Assembly of Taxonomic Units

PubMed Central

Schürch, Anita C.; Schipper, Debby; Bijl, Maarten A.; Dau, Jim; Beckmen, Kimberlee B.; Schapendonk, Claudia M. E.; Raj, V. Stalin; Osterhaus, Albert D. M. E.; Haagmans, Bart L.; Tryland, Morten; Smits, Saskia L.

2014-01-01

Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-)emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive inventory of viruses present in wildlife. We report a metagenomic viral survey of the Western Arctic herd of barren ground caribou (Rangifer tarandus granti) in Alaska, USA. The presence of mammalian viruses in eye and nose swabs of 39 free-ranging caribou was investigated by random amplification combined with a metagenomic analysis approach that applied exhaustive iterative assembly of sequencing results to define taxonomic units of each metagenome. Through homology search methods we identified the presence of several mammalian viruses, including different papillomaviruses, a novel parvovirus, polyomavirus, and a virus that potentially represents a member of a novel genus in the family Coronaviridae. PMID:25140520

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

Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; McCarthy, M.; Lin, Y-H

2006-01-01

In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

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

Virus Assembly and Maturation

NASA Astrophysics Data System (ADS)

Johnson, John E.

2004-03-01

We use two techniques to look at three-dimensional virus structure: electron cryomicroscopy (cryoEM) and X-ray crystallography. Figure 1 is a gallery of virus particles whose structures Timothy Baker, one of my former colleagues at Purdue University, used cryoEM to determine. It illustrates the variety of sizes of icosahedral virus particles. The largest virus particle on this slide is the Herpes simplex virus, around 1200Å in diameter; the smallest we examined was around 250Å in diameter. Viruses bear their genomic information either as positive-sense DNA and RNA, double-strand DNA, double-strand RNA, or negative-strand RNA. Viruses utilize the various structure and function "tactics" seen throughout cell biology to replicate at high levels. Many of the biological principles that we consider general were in fact discovered in the context of viruses ...

Modulation of a Pore in the Capsid of JC Polyomavirus Reduces Infectivity and Prevents Exposure of the Minor Capsid Proteins

PubMed Central

Nelson, Christian D. S.; Ströh, Luisa J.; Gee, Gretchen V.; O'Hara, Bethany A.; Stehle, Thilo

2015-01-01

ABSTRACT JC polyomavirus (JCPyV) infection of immunocompromised individuals results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The viral capsid of JCPyV is composed primarily of the major capsid protein virus protein 1 (VP1), and pentameric arrangement of VP1 monomers results in the formation of a pore at the 5-fold axis of symmetry. While the presence of this pore is conserved among polyomaviruses, its functional role in infection or assembly is unknown. Here, we investigate the role of the 5-fold pore in assembly and infection of JCPyV by generating a panel of mutant viruses containing amino acid substitutions of the residues lining this pore. Multicycle growth assays demonstrated that the fitness of all mutants was reduced compared to that of the wild-type virus. Bacterial expression of VP1 pentamers containing substitutions to residues lining the 5-fold pore did not affect pentamer assembly or prevent association with the VP2 minor capsid protein. The X-ray crystal structures of selected pore mutants contained subtle changes to the 5-fold pore, and no other changes to VP1 were observed. Pore mutant pseudoviruses were not deficient in assembly, packaging of the minor capsid proteins, or binding to cells or in transport to the host cell endoplasmic reticulum. Instead, these mutant viruses were unable to expose VP2 upon arrival to the endoplasmic reticulum, a step that is critical for infection. This study demonstrated that the 5-fold pore is an important structural feature of JCPyV and that minor modifications to this structure have significant impacts on infectious entry. IMPORTANCE JCPyV is an important human pathogen that causes a severe neurological disease in immunocompromised individuals. While the high-resolution X-ray structure of the major capsid protein of JCPyV has been solved, the importance of a major structural feature of the capsid, the 5-fold pore, remains poorly understood. This pore is conserved across polyomaviruses and suggests either that these viruses have limited structural plasticity in this region or that this pore is important in infection or assembly. Using a structure-guided mutational approach, we showed that modulation of this pore severely inhibits JCPyV infection. These mutants do not appear deficient in assembly or early steps in infectious entry and are instead reduced in their ability to expose a minor capsid protein in the host cell endoplasmic reticulum. Our work demonstrates that the 5-fold pore is an important structural feature for JCPyV. PMID:25609820

Dengue envelope-based 'four-in-one' virus-like particles produced using Pichia pastoris induce enhancement-lacking, domain III-directed tetravalent neutralising antibodies in mice.

PubMed

Rajpoot, Ravi Kant; Shukla, Rahul; Arora, Upasana; Swaminathan, Sathyamangalam; Khanna, Navin

2018-06-05

Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors. Following a three-dose immunisation schedule, the T-mVLPs elicited EDIII-directed antibodies in mice which could neutralise all four DENV serotypes. Importantly, anti-T-mVLP antibodies did not augment sub-lethal DENV-2 infection of dengue-sensitive AG129 mice, based on multiple parameters. The 'four-in-one' tetravalent T-mVLPs possess multiple desirable features which may potentially contribute to safety (non-viral, prM-lacking and ADE potential-lacking), immunogenicity (induction of virus-neutralising antibodies), and low cost (single tetravalent immunogen produced using P. pastoris, an expression system known for its high productivity using simple inexpensive media). These results strongly warrant further exploration of this vaccine candidate.

EXPRESSION AND SELF-ASSEMBLY OF NORWALK VIRUS CAPSID PROTEIN FROM VENEZUELAN EQUINE ENCEPHALITIS VIRUS REPLICONS. (R826139)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral position is available in the Viral Recombination Section (VRS), HIV Dynamics and Replication Program, CCR.  The VRS studies retroviral replication using human immunodeficiency viruses and other retroviruses, with a particular emphasis on the mechanisms of viral RNA biology, specific RNA packaging, virus assembly, and HIV replication.  Molecular tools and

A Novel Self-Replicating Chimeric Lentivirus-Like Particle

PubMed Central

Young, Kelly R.; Madden, Victoria J.; Johnson, Philip R.; Johnston, Robert E.

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4+ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation. PMID:22013035

A novel self-replicating chimeric lentivirus-like particle.

PubMed

Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

Virus-like nanostructures for tuning immune response

NASA Astrophysics Data System (ADS)

Mammadov, Rashad; Cinar, Goksu; Gunduz, Nuray; Goktas, Melis; Kayhan, Handan; Tohumeken, Sehmus; Topal, Ahmet E.; Orujalipoor, Ilghar; Delibasi, Tuncay; Dana, Aykutlu; Ide, Semra; Tekinay, Ayse B.; Guler, Mustafa O.

2015-11-01

Synthetic vaccines utilize viral signatures to trigger immune responses. Although the immune responses raised against the biochemical signatures of viruses are well characterized, the mechanism of how they affect immune response in the context of physical signatures is not well studied. In this work, we investigated the ability of zero- and one-dimensional self-assembled peptide nanostructures carrying unmethylated CpG motifs (signature of viral DNA) for tuning immune response. These nanostructures represent the two most common viral shapes, spheres and rods. The nanofibrous structures were found to direct immune response towards Th1 phenotype, which is responsible for acting against intracellular pathogens such as viruses, to a greater extent than nanospheres and CpG ODN alone. In addition, nanofibers exhibited enhanced uptake into dendritic cells compared to nanospheres or the ODN itself. The chemical stability of the ODN against nuclease-mediated degradation was also observed to be enhanced when complexed with the peptide nanostructures. In vivo studies showed that nanofibers promoted antigen-specific IgG production over 10-fold better than CpG ODN alone. To the best of our knowledge, this is the first report showing the modulation of the nature of an immune response through the shape of the carrier system.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

PubMed

Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

2014-01-01

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

PubMed

Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

2016-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

PubMed

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

2017-10-15

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production. Copyright © 2017 American Society for Microbiology.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

PubMed Central

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard

2017-01-01

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production. PMID:28768875

Endogenous avian leukosis viral loci in the Red Jungle Fowl genome assembly.

PubMed

Benkel, Bernhard; Rutherford, Katherine

2014-12-01

The current build (galGal4) of the genome of the ancestor of the modern chicken, the Red Jungle Fowl, contains a single endogenous avian leukosis viral element (ALVE) on chromosome 1 (designated RSV-LTR; family ERVK). The assembly shows the ALVE provirus juxtaposed with a member of a second family of avian endogenous retroviruses (designated GGERV20; family ERVL); however, the status of the 3' end of the ALVE element as well as its flanking region remain unclear due to a gap in the reference genome sequence. In this study, we filled the gap in the assembly using a combination of long-range PCR (LR-PCR) and a short contig present in the unassembled portion of the reference genome database. Our results demonstrate that the ALVE element (ALVE-JFevB) is inserted into the putative envelope region of a GGERV20 element, roughly 1 kbp from its 3' end, and that ALVE-JFevB is complete, and depending on its expression status, potentially capable of directing the production of virus. Moreover, the unassembled portion of the genome database contains junction fragments for a second, previously characterized endogenous proviral element, ALVE-6. ©2014 Poultry Science Association Inc.

Self-assembly of tetravalent Goldberg polyhedra from 144 small components

NASA Astrophysics Data System (ADS)

Fujita, Daishi; Ueda, Yoshihiro; Sato, Sota; Mizuno, Nobuhiro; Kumasaka, Takashi; Fujita, Makoto

2016-12-01

Rational control of the self-assembly of large structures is one of the key challenges in chemistry, and is believed to become increasingly difficult and ultimately impossible as the number of components involved increases. So far, it has not been possible to design a self-assembled discrete molecule made up of more than 100 components. Such molecules—for example, spherical virus capsids—are prevalent in nature, which suggests that the difficulty in designing these very large self-assembled molecules is due to a lack of understanding of the underlying design principles. For example, the targeted assembly of a series of large spherical structures containing up to 30 palladium ions coordinated by up to 60 bent organic ligands was achieved by considering their topologies. Here we report the self-assembly of a spherical structure that also contains 30 palladium ions and 60 bent ligands, but belongs to a shape family that has not previously been observed experimentally. The new structure consists of a combination of 8 triangles and 24 squares, and has the symmetry of a tetravalent Goldberg polyhedron. Platonic and Archimedean solids have previously been prepared through self-assembly, as have trivalent Goldberg polyhedra, which occur naturally in the form of virus capsids and fullerenes. But tetravalent Goldberg polyhedra have not previously been reported at the molecular level, although their topologies have been predicted using graph theory. We use graph theory to predict the self-assembly of even larger tetravalent Goldberg polyhedra, which should be more stable, enabling another member of this polyhedron family to be assembled from 144 components: 48 palladium ions and 96 bent ligands.

Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production

PubMed Central

Essaidi-Laziosi, Manel; Shevtsova, Anastasia; Gerlier, Denis; Roux, Laurent

2013-01-01

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface. PMID:24339863

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells

PubMed Central

Qiao, Wenjie; Medina, Vicente; Falk, Bryce W.

2017-01-01

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses. PMID:29021801

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

PubMed

Stephen, Sam L; Beales, Lucy; Peyret, Hadrien; Roe, Amy; Stonehouse, Nicola J; Rowlands, David J

2018-01-01

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

New Strategies and Methods to Study Interactions between Tobacco Mosaic Virus Coat Protein and Its Inhibitors

PubMed Central

Li, Xiangyang; Chen, Zhuo; Jin, Linhong; Hu, Deyu; Yang, Song

2016-01-01

Studies of the targets of anti-viral compounds are hot topics in the field of pesticide research. Various efficient anti-TMV (Tobacco Mosaic Virus) compounds, such as Ningnanmycin (NNM), Antofine (ATF), Dufulin (DFL) and Bingqingxiao (BQX) are available. However, the mechanisms of the action of these compounds on targets remain unclear. To further study the mechanism of the action of the anti-TMV inhibitors, the TMV coat protein (TMV CP) was expressed and self-assembled into four-layer aggregate disks in vitro, which could be reassembled into infectious virus particles with TMV RNA. The interactions between the anti-TMV compounds and the TMV CP disk were analyzed by size exclusion chromatography, isothermal titration calorimetry and native-polyacrylamide gel electrophoresis methods. The results revealed that assembly of the four-layer aggregate disk was inhibited by NNM; it changed the four-layer aggregate disk into trimers, and affected the regular assembly of TMV CP and TMV RNA. The four-layer aggregate disk of TMV CP was little inhibited by ATF, DFL and BQX. Our results provide original data, as well as new strategies and methods, for research on the mechanism of action of anti-viral drugs. PMID:26927077

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome.

PubMed

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Pan, Zhao-Yi; Wang, Su-Yun; Wang, Yan-Yi

2017-04-01

Upon viral infection, retinoic acid-inducible gene I-like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses.

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

PubMed Central

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Wang, Su-Yun

2017-01-01

Upon viral infection, retinoic acid–inducible gene I–like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses. PMID:28414768

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

PubMed Central

Pessler, F; Pendergrast, P S; Hernandez, N

1997-01-01

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes. PMID:9199312

A CRISPR toolbox to study virus–host interactions

PubMed Central

Puschnik, Andreas S.; Majzoub, Karim; Ooi, Yaw Shin; Carette, Jan E.

2018-01-01

Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR–Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR–Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR–Cas make it a powerful tool for probing virus–host interactions and for identifying new antiviral targets. PMID:28420884

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

PubMed Central

El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Zhu, Haining; Buchholz, Ursula J.; Moncman, Carole L.; Dutch, Rebecca Ellis

2016-01-01

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. PMID:27683250

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

Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

PubMed

Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

2017-08-04

Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Building polyhedra by self-assembly: theory and experiment.

PubMed

Kaplan, Ryan; Klobušický, Joseph; Pandey, Shivendra; Gracias, David H; Menon, Govind

2014-01-01

We investigate the utility of a mathematical framework based on discrete geometry to model biological and synthetic self-assembly. Our primary biological example is the self-assembly of icosahedral viruses; our synthetic example is surface-tension-driven self-folding polyhedra. In both instances, the process of self-assembly is modeled by decomposing the polyhedron into a set of partially formed intermediate states. The set of all intermediates is called the configuration space, pathways of assembly are modeled as paths in the configuration space, and the kinetics and yield of assembly are modeled by rate equations, Markov chains, or cost functions on the configuration space. We review an interesting interplay between biological function and mathematical structure in viruses in light of this framework. We discuss in particular: (i) tiling theory as a coarse-grained description of all-atom models; (ii) the building game-a growth model for the formation of polyhedra; and (iii) the application of these models to the self-assembly of the bacteriophage MS2. We then use a similar framework to model self-folding polyhedra. We use a discrete folding algorithm to compute a configuration space that idealizes surface-tension-driven self-folding and analyze pathways of assembly and dominant intermediates. These computations are then compared with experimental observations of a self-folding dodecahedron with side 300 μm. In both models, despite a combinatorial explosion in the size of the configuration space, a few pathways and intermediates dominate self-assembly. For self-folding polyhedra, the dominant intermediates have fewer degrees of freedom than comparable intermediates, and are thus more rigid. The concentration of assembly pathways on a few intermediates with distinguished geometric properties is biologically and physically important, and suggests deeper mathematical structure.

The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

PubMed Central

Lu, X; Welsh, T M; Peterlin, B M

1993-01-01

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes. Images PMID:8445708

ATP Depletion Blocks Herpes Simplex Virus DNA Packaging and Capsid Maturation

PubMed Central

Dasgupta, Anindya; Wilson, Duncan W.

1999-01-01

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed. PMID:9971781

Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

PubMed Central

Camunas-Soler, Joan; Kertesz, Michael; De Vlaminck, Iwijn; Koh, Winston; Pan, Wenying; Martin, Lance; Neff, Norma F.; Okamoto, Jennifer; Wong, Ronald J.; Kharbanda, Sandhya; El-Sayed, Yasser; Blumenfeld, Yair; Stevenson, David K.; Shaw, Gary M.; Wolfe, Nathan D.; Quake, Stephen R.

2017-01-01

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated. PMID:28830999

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

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

Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin

2009-10-10

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibersmore » is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.« less

Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

PubMed

Bell, Peter; Vandenberghe, Luk H; Wilson, James M

2014-06-01

Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

NASA Astrophysics Data System (ADS)

Mousnier, Aurélie; Bell, Andrew S.; Swieboda, Dawid P.; Morales-Sanfrutos, Julia; Pérez-Dorado, Inmaculada; Brannigan, James A.; Newman, Joseph; Ritzefeld, Markus; Hutton, Jennie A.; Guedán, Anabel; Asfor, Amin S.; Robinson, Sean W.; Hopkins-Navratilova, Iva; Wilkinson, Anthony J.; Johnston, Sebastian L.; Leatherbarrow, Robin J.; Tuthill, Tobias J.; Solari, Roberto; Tate, Edward W.

2018-06-01

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

Morphology and ultrastructure of retrovirus particles

PubMed Central

Zhang, Wei; Cao, Sheng; Martin, Jessica L.; Mueller, Joachim D.; Mansky, Louis M.

2015-01-01

Retrovirus morphogenesis entails assembly of Gag proteins and the viral genome on the host plasma membrane, acquisition of the viral membrane and envelope proteins through budding, and formation of the core through the maturation process. Although in both immature and mature retroviruses, Gag and capsid proteins are organized as paracrystalline structures, the curvatures of these protein arrays are evidently not uniform within one or among all virus particles. The heterogeneity of retroviruses poses significant challenges to studying the protein contacts within the Gag and capsid lattices. This review focuses on current understanding of the molecular organization of retroviruses derived from the sub-nanometer structures of immature virus particles, helical capsid protein assemblies and soluble envelope protein complexes. These studies provide insight into the molecular elements that maintain the stability, flexibility and infectivity of virus particles. Also reviewed are morphological studies of retrovirus budding, maturation, infection and cell-cell transmission, which inform the structural transformation of the viruses and the cells during infection and viral transmission, and lead to better understanding of the interplay between the functioning viral proteins and the host cell. PMID:26448965

Role of L-Particles during Herpes Simplex Virus Infection.

PubMed

Heilingloh, Christiane S; Krawczyk, Adalbert

2017-01-01

Infection of eukaryotic cells with α-herpesviruses results in the formation and secretion of infectious heavy particles (virions; H-particles) and non-infectious light particles (L-particles). Herpes simplex virus type 1 (HSV-1) H-particles consist of a genome-containing capsid surrounded by tegument proteins and a glycoprotein-rich lipid bilayer. Non-infectious L-particles are composed mainly of envelope and tegument proteins and are devoid of capsids and viral DNA. L-particles were first described in the early nineties and from then on investigated for their formation and role during virus infection. The development and secretion of L-particles occur simultaneously to the assembly of complete viral particles. HSV-1 L-particles are assembled by budding of condensed tegument into Golgi-delivered vesicles and are capable of delivering their functional content to non-infected cells. Thereby, HSV-1 L-particles contribute to viral pathogenesis within the infected host by enhancing virion infectivity and providing immune evasion functions. In this review we discuss the emergence of HSV-1 L-particles during virus replication and their biological functions described thus far.

A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

PubMed Central

Ruch, Travis R.; Machamer, Carolyn E.

2012-01-01

The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection. PMID:22570613

Visualizing Herpesvirus Procapsids in Living Cells.

PubMed

Maier, Oana; Sollars, Patricia J; Pickard, Gary E; Smith, Gregory A

2016-11-15

A complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components is present on the initial capsid assembly, the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded green fluorescent protein (GFP) fused in frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kinetics in vitro and approximated wild-type virulence in vivo The fusion also proved to be well tolerated in herpes simplex virus. Viruses encoding GFP-VP24, along with a traditional capsid reporter fusion (pUL25/mCherry), demonstrated that GFP-VP24 was a reliable capsid marker and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection and monitoring of capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation. The family Herpesviridae consists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share structurally related icosahedral capsids that encase the double-stranded DNA (dsDNA) viral genome. The dynamics of capsid assembly and maturation have been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Biochemical and Functional Characterization of the Ebola Virus VP24 Protein: Implications for a Role in Virus Assembly and Budding

PubMed Central

Han, Ziying; Boshra, Hani; Sunyer, J. Oriol; Zwiers, Susan H.; Paragas, Jason; Harty, Ronald N.

2003-01-01

The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding. PMID:12525613

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

PubMed

Adu-Gyamfi, Emmanuel; Soni, Smita P; Xue, Yi; Digman, Michelle A; Gratton, Enrico; Stahelin, Robert V

2013-02-22

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

PubMed

Dietzel, Erik; Anderson, Danielle E; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

2011-07-01

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

Varieties of charge distributions in coat proteins of ssRNA+  viruses

NASA Astrophysics Data System (ADS)

Lošdorfer Božič, Anže; Podgornik, Rudolf

2018-01-01

A major part of the interactions involved in the assembly and stability of icosahedral, positive-sense single-stranded RNA (ssRNA+) viruses is electrostatic in nature, as can be inferred from the strong pH- and salt-dependence of their assembly phase diagrams. Electrostatic interactions do not act only between the capsid coat proteins (CPs), but just as often provide a significant contribution to the interactions of the CPs with the genomic RNA, mediated to a large extent by positively charged, flexible N-terminal tails of the CPs. In this work, we provide two clear and complementary definitions of an N-terminal tail of a protein, and use them to extract the tail sequences of a large number of CPs of ssRNA+  viruses. We examine the pH-dependent interplay of charge on both tails and CPs alike, and show that—in contrast to the charge on the CPs—the net positive charge on the N-tails persists even to very basic pH values. In addition, we note a limit to the length of the wild-type genomes of those viruses which utilize positively charged tails, when compared to viruses without charged tails and similar capsid size. At the same time, we observe no clear connection between the charge on the N-tails and the genome lengths of the viruses included in our study.

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

PubMed

Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

2016-02-01

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Coarse-grained Simulations of Viral Assembly

NASA Astrophysics Data System (ADS)

Elrad, Oren M.

2011-12-01

The formation of viral capsids is a marvel of natural engineering and design. A large number (from 60 to thousands) of protein subunits assemble into complete, reproducible structures under a variety of conditions while avoiding kinetic and thermodynamic traps. Small single-stranded RNA viruses not only assemble their coat proteins in this fashion but also package their genome during the self-assembly process. Recent experiments have shown that the coat proteins are competent to assemble not merely around their own genomes but heterologous RNA, synthetic polyanions and even functionalized gold nanoparticles. Remarkably these viruses can even assemble around cargo not commensurate with their native state by adopting different morphologies. Understanding the properties that confer such exquisite precision and flexibility to the assembly process could aid biomedical research in the search for novel antiviral remedies, drug-delivery vehicles and contrast agents used in bioimaging. At the same time, viral assembly provides an excellent model system for the development of a statistical mechanical understanding of biological self-assembly, in the hopes of that we will identify some universal principles that underly such processes. This work consists of computational studies using coarse-grained representations of viral coat proteins and their cargoes. We find the relative strength of protein-cargo and protein-protein interactions has a profound effect on the assembly pathway, in some cases leading to assembly mechanisms that are markedly different from those found in previous work on the assembly of empty capsids. In the case of polymeric cargo, we find the first evidence for a previously theorized mechanism in which the polymer actively participates in recruiting free subunits to the assembly process through cooperative polymer-protein motions. We find that successful assembly is non-monotonic in protein-cargo affinity, such affinity can be detrimental to assembly if it becomes strong enough to stabilize frustrated intermediates that are incompatible with the ground state structure. In cases where the subunits are capable of assembly into different morphologies, we find that maintaining the precise spatial arrangement of subunits seen in the crystal structure is possible even if non-native interactions are disfavored by as little as the thermal energy.

Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging.

PubMed

Logan, Grace; Newman, Joseph; Wright, Caroline F; Lasecka-Dykes, Lidia; Haydon, Daniel T; Cottam, Eleanor M; Tuthill, Tobias J

2017-10-18

Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad spectrum activity. Copyright © 2017 Logan et al.

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus*

PubMed Central

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-01-01

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (NTAIL). XD binding induces NTAIL α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced NTAIL folding, XD-NTAIL binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced NTAIL α-helical folding were created within Box-2 of Edmonston MeV NTAIL. Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-NTAIL binding affinity or reduction/loss of XD-induced NTAIL alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity. PMID:22318731

Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.

PubMed

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-04-06

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.

Particle-based vaccines for HIV-1 infection.

PubMed

Young, Kelly R; Ross, Ted M

2003-06-01

The use of live-attenuated viruses as vaccines has been successful for the control of viral infections. However, the development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge. HIV infects cells of the immune system and results in a severe immunodeficiency. In addition, the ability of the virus to adapt to immune pressure and the ability to reside in an integrated form in host cells present hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes from different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immunity, however, a live-attenuated vaccine for HIV is problematic. The possibility of a live-attenuated vaccine to revert to a pathogenic form or recombine with a wild-type or defective virus in an infected individual is a drawback to this approach. Therefore, these vaccines are currently only being tested in non-human primate models. Live-attenuated vaccines are effective in stimulating immunity, however challenged animals rarely clear viral infection and the degree of attenuation directly correlates with the protection of animals from disease. Another particle-based vaccine approach for HIV involves the use of virus-like particles (VLPs). VLPs mimic the viral particle without causing an immunodeficiency disease. HIV-like particles (HIV-LP) are defined as self-assembling, non-replicating, nonpathogenic, genomeless particles that are similar in size and conformation to intact virions. A variety of VLPs for both HIV and SIV are currently in pre-clinical and clinical trials. This review focuses on the current knowledge regarding the immunogenicity and safety of particle-based vaccine strategies for HIV-1.

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.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

PubMed Central

Milrot, Elad; Mutsafi, Yael; Ben-Dor, Shifra; Levin, Yishai; Savidor, Alon; Kartvelishvily, Elena

2016-01-01

ABSTRACT The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed “viral factories,” where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as “production lines” of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed “viral factories,” are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles. PMID:27581975

Chaperones in hepatitis C virus infection

PubMed Central

Khachatoorian, Ronik; French, Samuel W

2016-01-01

The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses. PMID:26783419

Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages.

PubMed

Kant, Ravi; Rayaprolu, Vamseedhar; McDonald, Kaitlyn; Bothner, Brian

2018-06-01

The beauty, symmetry, and functionality of icosahedral virus capsids has attracted the attention of biologists, physicists, and mathematicians ever since they were first observed. Viruses and protein cages assemble into functional architectures in a range of sizes, shapes, and symmetries. To fulfill their biological roles, these structures must self-assemble, resist stress, and are often dynamic. The increasing use of icosahedral capsids and cages in materials science has driven the need to quantify them in terms of structural properties such as rigidity, stiffness, and viscoelasticity. In this study, we employed Quartz Crystal Microbalance with Dissipation technology (QCM-D) to characterize and compare the mechanical rigidity of different protein cages and viruses. We attempted to unveil the relationships between rigidity, radius, shell thickness, and triangulation number. We show that the rigidity and triangulation numbers are inversely related to each other and the comparison of rigidity and radius also follows the same trend. Our results suggest that subunit orientation, protein-protein interactions, and protein-nucleic acid interactions are important for the resistance to deformation of these complexes, however, the relationships are complex and need to be explored further. The QCM-D based viscoelastic measurements presented here help us elucidate these relationships and show the future prospect of this technique in the field of physical virology and nano-biotechnology.

A minimal kinetic model for a viral DNA packaging machine.

PubMed

Yang, Qin; Catalano, Carlos Enrique

2004-01-20

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.

Three-Dimensionally Engineered Normal Human Lung Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.; McCarthy, M.; Lin, Y-H.; Deatly, A. M.

2008-01-01

In vitro three-dimensional (3D) human lung epithelio-mesenchymal tissue-like assemblies (3D hLEM TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and the detection of membrane bound glycoproteins over time confirm productive infection with the virus. Therefore, we assert TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host s immune system.

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

[Research progress on ebola virus glycoprotein].

PubMed

Ding, Guo-Yong; Wang, Zhi-Yu; Gao, Lu; Jiang, Bao-Fa

2013-03-01

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT.

PubMed

van Leeuwen, Hans; Okuwaki, Mitsuru; Hong, Rui; Chakravarti, Debabrata; Nagata, Kyosuke; O'Hare, Peter

2003-09-01

Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

Complete genome sequence of a novel genotype of squash mosaic virus

USDA-ARS?s Scientific Manuscript database

Complete genome sequence of a novel genotype of Squash mosaic virus (SqMV) infecting squash plants in Spain was obtained using deep sequencing of small ribonucleic acids and assembly. The low nucleotide sequence identities, with 87-88% on RNA1 and 84-86% on RNA2 to known SqMV isolates, suggest a new...

Cytopathogenesis of Vesicular Stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner

USDA-ARS?s Scientific Manuscript database

Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our ...

Viral reprogramming of the Daxx histone H3.3 chaperone during early Epstein-Barr virus infection.

PubMed

Tsai, Kevin; Chan, Lilian; Gibeault, Rebecca; Conn, Kristen; Dheekollu, Jayaraju; Domsic, John; Marmorstein, Ronen; Schang, Luis M; Lieberman, Paul M

2014-12-01

Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive cochaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using coimmunoprecipitation and size-exclusion chromatography with highly purified components. FRAP (fluorescence recovery after photobleaching) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence combined with fluorescence in situ hybridization that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression. Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent antiviral repressive chromatin and establish chromatin structure permissive for viral gene expression and the establishment of latent infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Integrated Nanosystems Templated by Self-assembled Virus Capsids

NASA Astrophysics Data System (ADS)

Stephanopoulos, Nicholas

This dissertation presents the synthesis and modeling of multicomponent nanosystems templated by self-assembled virus capsids. The design principles, synthesis, analysis, and future directions for these capsid-based materials are presented. Chapter 1 gives an overview of the literature on the application of virus capsids in constructing nanomaterials. The uses of capsids in three main areas are considered: (1) as templates for inorganic materials or nanoparticles; (2) as vehicles for biological applications like medical imaging and treatment; and (3) as scaffolds for catalytic materials. In light of this introduction, an overview of the material in this dissertation is described. Chapters 2-4 all describe integrated nanosystems templated by bacteriophage MS2, a spherical icosahedral virus capsid. MS2 possesses an interior and exterior surface that can be modified orthogonally using bioconjugation chemistry to create multivalent, multicomponent constructs with precise localization of components attached to the capsid proteins. Chapter 2 describes the use of MS2 to synthesize a photocatalytic construct by modifying the internal surface with sensitizing chromophores and the external surface with a photocatalytic porphyrin. The chromophores absorbed energy that the porphyrin could not, and transferred it to the porphyrin via FRET through the protein shell. The porphyrin was then able to utilize the energy to carry out photocatalysis at new wavelengths. In Chapter 3, porphyrins were installed on the interior surface of MS2 and DNA aptamers specific for Jurkat leukemia T cells on the exterior surface. The dual-modified capsids were able to bind to Jurkat cells, and upon illumination the porphyrins generated singlet oxygen to kill them selectively over non-targeted cells. Chapter 4 explores integrating MS2 with DNA origami in order to arrange the capsids at larger length scales. Capsids modified with fluorescent dyes inside and single-stranded DNA outside were able to bind to origami tiles bearing complementary DNA probes. The tiles could then be used to arrange the capsids in a one-dimensional array with dimensions far exceeding those of individual MS2 particles. In Chapter 5, the use of a different capsid, that of the tobacco mosaic virus (TMV) is described. The defect tolerance of light harvesting systems built using TMV as a scaffold was investigated using a kinetic Monte Carlo model to simulate the energy transfer processes. The results of the simulation were used to understand and explain experimental results obtained from the system.

Challenging Students' Intuitions—the Influence of a Tangible Model of Virus Assembly on Students' Conceptual Reasoning About the Process of Self-Assembly

NASA Astrophysics Data System (ADS)

Larsson, Caroline; Tibell, Lena A. E.

2015-10-01

A well-ordered biological complex can be formed by the random motion of its components, i.e. self-assemble. This is a concept that incorporates issues that may contradict students' everyday experiences and intuitions. In previous studies, we have shown that a tangible model of virus self-assembly, used in a group exercise, helps students to grasp the process of self-assembly and in particular the facet "random molecular collision". The present study investigates how and why the model and the group exercise facilitate students' learning of this particular facet. The data analysed consist of audio recordings of six group exercises ( n = 35 university students) and individual semi-structured interviews ( n = 5 university students). The analysis is based on constructivist perspectives of learning, a combination of conceptual change theory and learning with external representations. Qualitative analysis indicates that perceived counterintuitive aspects of the process created a cognitive conflict within learners. The tangible model used in the group exercises facilitated a conceptual change in their understanding of the process. In particular, the tangible model appeared to provide cues and possible explanations and functioned as an "eye-opener" and a "thinking tool". Lastly, the results show signs of emotions also being important elements for successful accommodation.

Functional requirements of the yellow fever virus capsid protein.

PubMed

Patkar, Chinmay G; Jones, Christopher T; Chang, Yu-hsuan; Warrier, Ranjit; Kuhn, Richard J

2007-06-01

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

Real-time visualization of perforin nanopore assembly.

PubMed

Leung, Carl; Hodel, Adrian W; Brennan, Amelia J; Lukoyanova, Natalya; Tran, Sharon; House, Colin M; Kondos, Stephanie C; Whisstock, James C; Dunstone, Michelle A; Trapani, Joseph A; Voskoboinik, Ilia; Saibil, Helen R; Hoogenboom, Bart W

2017-05-01

Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.

Real-time visualization of perforin nanopore assembly

NASA Astrophysics Data System (ADS)

Leung, Carl; Hodel, Adrian W.; Brennan, Amelia J.; Lukoyanova, Natalya; Tran, Sharon; House, Colin M.; Kondos, Stephanie C.; Whisstock, James C.; Dunstone, Michelle A.; Trapani, Joseph A.; Voskoboinik, Ilia; Saibil, Helen R.; Hoogenboom, Bart W.

2017-05-01

Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.

Air breathing direct methanol fuel cell

DOEpatents

Ren, Xiaoming; Gottesfeld, Shimshon

2002-01-01

An air breathing direct methanol fuel cell is provided with a membrane electrode assembly, a conductive anode assembly that is permeable to air and directly open to atmospheric air, and a conductive cathode assembly that is permeable to methanol and directly contacting a liquid methanol source. Water loss from the cell is minimized by making the conductive cathode assembly hydrophobic and the conductive anode assembly hydrophilic.

TOOL ASSEMBLY WITH BI-DIRECTIONAL BEARING

DOEpatents

Longhurst, G.E.

1961-07-11

A two-direction motion bearing which is incorporated in a refueling nuclear fuel element trsnsfer tool assembly is described. A plurality of bi- directional bearing assembliesare fixed equi-distantly about the circumference of the transfer tool assembly to provide the tool assembly with a bearing surface- for both axial and rotational motion. Each bi-directional bearing assembly contains a plurality of circumferentially bulged rollers mounted in a unique arrangement which will provide a bearing surface for rotational movement of the tool assembly within a bore. The bi-direc tional bearing assembly itself is capable of rational motion and thus provides for longitudinal movement of the tool assembly.

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

PubMed Central

Gerber, Pehuén Pereyra; Cabrini, Mercedes; Jancic, Carolina; Paoletti, Luciana; Banchio, Claudia; von Bilderling, Catalina; Sigaut, Lorena; Pietrasanta, Lía I.; Duette, Gabriel; Freed, Eric O.; de Saint Basile, Genevieve; Moita, Catarina Ferreira; Moita, Luis Ferreira; Amigorena, Sebastian; Benaroch, Philippe; Geffner, Jorge

2015-01-01

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication. PMID:25940347

Tunable assembly of amyloid-forming peptides into nanosheets as a retrovirus carrier.

PubMed

Dai, Bin; Li, Dan; Xi, Wenhui; Luo, Fang; Zhang, Xiang; Zou, Man; Cao, Mi; Hu, Jun; Wang, Wenyuan; Wei, Guanghong; Zhang, Yi; Liu, Cong

2015-03-10

Using and engineering amyloid as nanomaterials are blossoming trends in bionanotechnology. Here, we show our discovery of an amyloid structure, termed "amyloid-like nanosheet," formed by a key amyloid-forming segment of Alzheimer's Aβ. Combining multiple biophysical and computational approaches, we proposed a structural model for the nanosheet that is formed by stacking the amyloid fibril spines perpendicular to the fibril axis. We further used the nanosheet for laboratorial retroviral transduction enhancement and directly visualized the presence of virus on the nanosheet surface by electron microscopy. Furthermore, based on our structural model, we designed nanosheet-forming peptides with different functionalities, elucidating the potential of rational design for amyloid-based materials with novel architecture and function.

Assembly of Hepatitis Delta Virus: Particle Characterization, Including the Ability To Infect Primary Human Hepatocytes▿

PubMed Central

Gudima, Severin; He, Yiping; Meier, Anja; Chang, Jinhong; Chen, Rongji; Jarnik, Michal; Nicolas, Emmanuelle; Bruss, Volker; Taylor, John

2007-01-01

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells. PMID:17229685

The C Terminus of the Large Tegument Protein pUL36 Contains Multiple Capsid Binding Sites That Function Differently during Assembly and Cell Entry of Herpes Simplex Virus

PubMed Central

Schipke, Julia; Pohlmann, Anja; Diestel, Randi; Binz, Anne; Rudolph, Kathrin; Nagel, Claus-Henning; Bauerfeind, Rudolf

2012-01-01

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating. PMID:22258258

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

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

Ju, Huanyu; Wei, Na; Wang, Qian

Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particlesmore » (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.« less

Lipids of Archaeal Viruses

PubMed Central

Roine, Elina; Bamford, Dennis H.

2012-01-01

Archaeal viruses represent one of the least known territory of the viral universe and even less is known about their lipids. Based on the current knowledge, however, it seems that, as in other viruses, archaeal viral lipids are mostly incorporated into membranes that reside either as outer envelopes or membranes inside an icosahedral capsid. Mechanisms for the membrane acquisition seem to be similar to those of viruses infecting other host organisms. There are indications that also some proteins of archaeal viruses are lipid modified. Further studies on the characterization of lipids in archaeal viruses as well as on their role in virion assembly and infectivity require not only highly purified viral material but also, for example, constant evaluation of the adaptability of emerging technologies for their analysis. Biological membranes contain proteins and membranes of archaeal viruses are not an exception. Archaeal viruses as relatively simple systems can be used as excellent tools for studying the lipid protein interactions in archaeal membranes. PMID:23049284

Zika virus-like particle (VLP) based vaccine

PubMed Central

Boigard, Hélène; Alimova, Alexandra; Martin, George R.; Katz, Al; Gottlieb, Paul

2017-01-01

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development. PMID:28481898

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

A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses

PubMed Central

Priye, Aashish; Bird, Sara W.; Light, Yooli K.; Ball, Cameron S.; Negrete, Oscar A.; Meagher, Robert J.

2017-01-01

Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable “LAMP box” supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device’s utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most. PMID:28317856

Structural transitions in Cowpea chlorotic mottle virus (CCMV)

NASA Astrophysics Data System (ADS)

Liepold, Lars O.; Revis, Jennifer; Allen, Mark; Oltrogge, Luke; Young, Mark; Douglas, Trevor

2005-12-01

Viral capsids act as molecular containers for the encapsulation of genomic nucleic acid. These protein cages can also be used as constrained reaction vessels for packaging and entrapment of synthetic cargos. The icosahedral Cowpea chlorotic mottle virus (CCMV) is an excellent model for understanding the encapsulation and packaging of both genomic and synthetic materials. High-resolution structural information of the CCMV capsid has been invaluable for evaluating structure-function relationships in the assembled capsid but does not allow insight into the capsid dynamics. The dynamic nature of the CCMV capsid might play an important role in the biological function of the virus. The CCMV capsid undergoes a pH and metal ion dependent reversible structural transition where 60 separate pores in the capsid open or close, exposing the interior of the protein cage to the bulk medium. In addition, the highly basic N-terminal domain of the capsid, which is disordered in the crystal structure, plays a significant role in packaging the viral cargo. Interestingly, in limited proteolysis and mass spectrometry experiments the N-terminal domain is the first part of the subunit to be cleaved, confirming its dynamic nature. Based on our fundamental understanding of the capsid dynamics in CCMV, we have utilized these aspects to direct packaging of a range of synthetic materials including drugs and inorganic nanoparticles.

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology

PubMed Central

Schwarz, B.; Uchida, M.; Douglas, T.

2016-01-01

Within biology, molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nanosystems that exist at the interface of living organisms and nonliving biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. Composed of self-assembling protein subunits, VLPs provide both a model for studying materials’ assembly at the nanoscale and useful building blocks for materials design. The robustness and degree of understanding of many VLP structures allow for the ready use of these systems as versatile nanoparticle platforms for the conjugation of active molecules or as scaffolds for the structural organization of chemical processes. Lastly the prevalence of viruses in all domains of life has led to unique activities of VLPs in biological systems most notably the immune system. Here we discuss recent efforts to apply VLPs in a wide variety of applications with the aim of highlighting how the common structural elements of VLPs have led to their emergence as paradigms for the understanding and design of biological nanomaterials. PMID:28057256

Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy.

PubMed

Ferrer-Miralles, Neus; Rodríguez-Carmona, Escarlata; Corchero, José Luis; García-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

2015-06-01

Lack of targeting and improper biodistribution are major flaws in current drug-based therapies that prevent reaching high local concentrations of the therapeutic agent. Such weaknesses impose the administration of high drug doses, resulting in undesired side effects, limited efficacy and enhanced production costs. Currently, missing nanosized containers, functionalized for specific cell targeting will be then highly convenient for the controlled delivery of both conventional and innovative drugs. In an attempt to fill this gap, health-focused nanotechnologies have put under screening a growing spectrum of materials as potential components of nanocages, whose properties can be tuned during fabrication. However, most of these materials pose severe biocompatibility concerns. We review in this study how proteins, the most versatile functional macromolecules, can be conveniently exploited and adapted by conventional genetic engineering as efficient building blocks of fully compatible nanoparticles for drug delivery and how selected biological activities can be recruited to mimic viral behavior during infection. Although engineering of protein self-assembling is still excluded from fully rational approaches, the exploitation of protein nano-assemblies occurring in nature and the direct manipulation of protein-protein contacts in bioinspired constructs open intriguing possibilities for further development. These methodologies empower the construction of new and potent vehicles that offer promise as true artificial viruses for efficient and safe nanomedical applications.

Human Retroviruses: Methods and Protocols

PubMed Central

Zhao, Gongpu; Zhang, Peijun

2015-01-01

Summary After virus fusion with a target cell, the viral core is released into the host cell cytoplasm and undergoes a controlled disassembly process, termed uncoating, before or as reverse transcription takes place. The cellular protein TRIM5α is a host cell restriction factor that blocks HIV-1 infection in rhesus macaque cells by targeting the viral capsid and inducing premature uncoating. The molecular mechanism of the interaction between capsid and TRIM5α remains unclear. Here, we describe an approach that utilizes cryo-electron microscopy (cryoEM) to examine the structural changes exerted on HIV-1 capsid (CA) assembly by TRIM5α binding. The TRIM5α interaction sites on CA assembly were further dissected by combining cryoEM with pair-wise cysteine mutations that crosslink CA either within a CA hexamer or between CA hexamers. Based on the structural information from cryoEM and crosslinking results from in vitro CA assemblies and purified intact HIV-1 cores, we demonstrate that direct binding of TRIM5α CC-SPRY domains to the viral capsid results in disruption and fragmentation of the surface lattice of HIV-1 capsid, specifically at inter-hexamer interfaces. The method described here can be easily adopted to study other important interactions in multi-protein complexes. PMID:24158810

Photonics and plasmonics go viral: self-assembly of hierarchical metamaterials

DOE PAGES

Wen, Amy M.; Podgornik, Rudolf; Strangi, Giuseppe; ...

2015-03-05

Sizing and shaping of mesoscale architectures with nanoscale features is a key opportunity to produce the next generation of higher-performing products and at the same time unveil completely new phenomena. This review article discusses recent advances in the design of novel photonic and plasmonic structures using a biology-inspired design. The proteinaceous capsids from viruses have long been discovered as platform technologies enabling unique applications in nanotechnology, materials, bioengineering, and medicine. In the context of materials applications, the highly organized structures formed by viral capsid proteins provide a 3D scaffold for the precise placement of plasmon and gain materials. Based onmore » their highly symmetrical structures, virus-based nanoparticles have a high propensity to self-assemble into higher-order crystalline structures, yielding hierarchical hybrid materials. Recent advances in the field have led to the development of virus-based light harvesting systems, plasmonic structures for application in high-performance metamaterials, binary nanoparticle lattices, and liquid crystalline arrays for sensing or display technologies. In conclusion, there is still much that could be explored in this area, and we foresee that this is only the beginning of great technological advances in virus-based materials for plasmonics and photonics applications.« less

Interaction of chiral rafts in self-assembled colloidal membranes

NASA Astrophysics Data System (ADS)

Xie, Sheng; Hagan, Michael F.; Pelcovits, Robert A.

2016-03-01

Colloidal membranes are monolayer assemblies of rodlike particles that capture the long-wavelength properties of lipid bilayer membranes on the colloidal scale. Recent experiments on colloidal membranes formed by chiral rodlike viruses showed that introducing a second species of virus with different length and opposite chirality leads to the formation of rafts—micron-sized domains of one virus species floating in a background of the other viruses [Sharma et al., Nature (London) 513, 77 (2014), 10.1038/nature13694]. In this article we study the interaction of such rafts using liquid crystal elasticity theory. By numerically minimizing the director elastic free energy, we predict the tilt angle profile for both a single raft and two rafts in a background membrane, and the interaction between two rafts as a function of their separation. We find that the chiral penetration depth in the background membrane sets the scale for the range of the interaction. We compare our results with the experimental data and find good agreement for the strength and range of the interaction. Unlike the experiments, however, we do not observe a complete collapse of the data when rescaled by the tilt angle at the raft edge.

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

PubMed

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

2012-05-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay.

PubMed

Kim, Sun-Il; Wu, Yuanzheng; Kim, Ka-Lyun; Kim, Geun-Joong; Shin, Hyun-Jae

2013-06-01

An efficient method for Pichia cell disruption that employs an aminopropyl magnesium phyllosilicate (AMP) clay-assisted glass beads mill is presented. AMP clay is functionalized nanocomposite resembling the talc parent structure Si8Mg6O20(OH)4 that has been proven to permeate the bacterial membrane and cause cell lysis. The recombinant capsid protein of cowpea chlorotic mottle virus (CCMV) expressed in Pichia pastoris GS115 was used as demonstration system for their ability of self-assembly into icosahedral virus-like particles (VLPs). The total protein concentration reached 4.24 mg/ml after 4 min treatment by glass beads mill combined with 0.2 % AMP clay, which was 11.2 % higher compared to glass beads mill only and the time was half shortened. The stability of purified CCMV VLPs illustrated AMP clay had no influence on virus assembly process. Considering the tiny amount added and simple approach of AMP clay, it could be a reliable method for yeast cell disruption.

Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

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

Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.

2013-10-01

Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using pointmore » mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.« less

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Levin, Aviad; Steenbergen, Rineke H.; Pang, Daniel; Shields, Justin; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2013-01-01

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly. PMID:24204278

The draft genome sequence of the ferret (Mustela putorius furo) facilitates study of human respiratory disease.

PubMed

Peng, Xinxia; Alföldi, Jessica; Gori, Kevin; Eisfeld, Amie J; Tyler, Scott R; Tisoncik-Go, Jennifer; Brawand, David; Law, G Lynn; Skunca, Nives; Hatta, Masato; Gasper, David J; Kelly, Sara M; Chang, Jean; Thomas, Matthew J; Johnson, Jeremy; Berlin, Aaron M; Lara, Marcia; Russell, Pamela; Swofford, Ross; Turner-Maier, Jason; Young, Sarah; Hourlier, Thibaut; Aken, Bronwen; Searle, Steve; Sun, Xingshen; Yi, Yaling; Suresh, M; Tumpey, Terrence M; Siepel, Adam; Wisely, Samantha M; Dessimoz, Christophe; Kawaoka, Yoshihiro; Birren, Bruce W; Lindblad-Toh, Kerstin; Di Palma, Federica; Engelhardt, John F; Palermo, Robert E; Katze, Michael G

2014-12-01

The domestic ferret (Mustela putorius furo) is an important animal model for multiple human respiratory diseases. It is considered the 'gold standard' for modeling human influenza virus infection and transmission. Here we describe the 2.41 Gb draft genome assembly of the domestic ferret, constituting 2.28 Gb of sequence plus gaps. We annotated 19,910 protein-coding genes on this assembly using RNA-seq data from 21 ferret tissues. We characterized the ferret host response to two influenza virus infections by RNA-seq analysis of 42 ferret samples from influenza time-course data and showed distinct signatures in ferret trachea and lung tissues specific to 1918 or 2009 human pandemic influenza virus infections. Using microarray data from 16 ferret samples reflecting cystic fibrosis disease progression, we showed that transcriptional changes in the CFTR-knockout ferret lung reflect pathways of early disease that cannot be readily studied in human infants with cystic fibrosis disease.