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Sample records for herpesvirus viral protein

  1. Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

    PubMed Central

    Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah

    2016-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further

  2. Equine herpesvirus type 1 tegument protein VP22 is not essential for pathogenicity in a hamster model, but is required for efficient viral growth in cultured cells.

    PubMed

    Okada, Ayaka; Izume, Satoko; Ohya, Kenji; Fukushi, Hideto

    2015-10-01

    VP22 is a major tegument protein of Equine herpesvirus type 1 (EHV-1) that is a conserved protein among alphaherpesviruses. However, the roles of VP22 differ among each virus, and the roles of EHV-1 VP22 are still unclear. Here, we constructed an EHV-1 VP22 deletion mutant and a revertant virus to clarify the role of VP22. We found that EHV-1 VP22 was required for efficient viral growth in cultured cells, but not for virulence in a hamster model.

  3. Kaposi's Sarcoma-Associated Herpesvirus Encodes a Viral Deubiquitinase▿

    PubMed Central

    González, Carlos M.; Wang, Ling; Damania, Blossom

    2009-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically linked to Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease. Like other herpesviruses, KSHV can exist in either a lytic or a latent phase during its life cycle. We report that the lytic protein encoded by KSHV open reading frame 64 (Orf64) is a viral deubiquitinase (DUB) enzyme capable of deubiquitinating cellular proteins in vitro and in vivo. Orf64 DUB activity is effective against lysine 48 (K48)- and lysine 63 (K63)-linked ubiquitin chains. Thus, KSHV Orf64 is a viral DUB that does not show specificity toward K48 or K63 ubiquitin linkages. Orf64 DUB activity lies within the first 205 residues of the protein, and deubiquitination is dependent on a cysteine at position 29, since mutation of this residue ablated this activity. Cell fractionation studies revealed that the N terminus and the full-length protein localized to both the nuclear and cytoplasmic compartments. The function of Orf64 was tested by short interfering RNA (siRNA) knockdown studies on latently infected cells that were induced into lytic replication. We found that depletion of Orf64 by siRNA resulted in decreased viral lytic transcription and lytic protein expression. These experiments indicate that Orf64 plays a role in KSHV lytic replication. PMID:19640989

  4. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  5. Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins.

    PubMed

    Sztuba-Solinska, Joanna; Rausch, Jason W; Smith, Rodman; Miller, Jennifer T; Whitby, Denise; Le Grice, Stuart F J

    2017-04-05

    Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.

  6. Selective peptide inhibitors of antiapoptotic cellular and viral Bcl-2 proteins lead to cytochrome c release during latent Kaposi's sarcoma-associated herpesvirus infection.

    PubMed

    Burrer, Christine M; Foight, Glenna W; Keating, Amy E; Chan, Gary C

    2016-01-04

    Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with B-cell lymphomas including primary effusion lymphoma and multicentric Castleman's disease. KSHV establishes latency within B cells by modulating or mimicking the antiapoptotic Bcl-2 family of proteins to promote cell survival. Our previous BH3 profiling analysis, a functional assay that assesses the contribution of Bcl-2 proteins towards cellular survival, identified two Bcl-2 proteins, cellular Mcl-1 and viral KsBcl-2, as potential regulators of mitochondria polarization within a latently infected B-cell line, Bcbl-1. In this study, we used two novel peptide inhibitors identified in a peptide library screen that selectively bind KsBcl-2 (KL6-7_Y4eK) or KsBcl-2 and Mcl-1 (MS1) in order to decipher the relative contribution of Mcl-1 and KsBcl-2 in maintaining mitochondrial membrane potential. We found treatment with KL6-7_Y4eK and MS1 stimulated a similar amount of cytochrome c release from mitochondria isolated from Bcbl-1 cells, indicating that inhibition of KsBcl-2 alone is sufficient for mitochondrial outer membrane permiabilzation (MOMP) and thus apoptosis during a latent B cell infection. In turn, this study also identified and provides a proof-of-concept for the further development of novel KsBcl-2 inhibitors for the treatment of KSHV-associated B-cell lymphomas via the targeting of latently infected B cells.

  7. Herpesvirus Late Gene Expression: A Viral-Specific Pre-initiation Complex Is Key

    PubMed Central

    Gruffat, Henri; Marchione, Roberta; Manet, Evelyne

    2016-01-01

    During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E), and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the transcription start site (TSS). Moreover, in the case of the β- and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies (β and γ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors – especially ICP4 – play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein. PMID:27375590

  8. Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50/Rta Protein Activates the Entire Viral Lytic Cycle in the HH-B2 Primary Effusion Lymphoma Cell Line†

    PubMed Central

    Gradoville, Lyndle; Gerlach, Jennifer; Grogan, Elizabeth; Shedd, Duane; Nikiforow, Sarah; Metroka, Craig; Miller, George

    2000-01-01

    Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells. PMID:10846108

  9. A gamma-2 herpesvirus nucleocytoplasmic shuttle protein interacts with importin alpha 1 and alpha 5.

    PubMed

    Goodwin, D J; Whitehouse, A

    2001-06-08

    Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin alpha isoforms 1 and 5. In addition, the binding of ORF 57 to importin alpha was mediated by the importin alpha hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin alpha into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.

  10. Dominant-Negative Proteins in Herpesviruses – From Assigning Gene Function to Intracellular Immunization

    PubMed Central

    Mühlbach, Hermine; Mohr, Christian A.; Ruzsics, Zsolt; Koszinowski, Ulrich H.

    2009-01-01

    Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN) proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies. PMID:21994555

  11. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  12. Detection of Viral DNA and Immune Responses to the Human Herpesvirus 6 101-Kilodalton Virion Protein in Patients with Multiple Sclerosis and in Controls

    PubMed Central

    Tejada-Simon, Maria V.; Zang, Ying C. Q.; Hong, Jian; Rivera, Victor M.; Killian, James M.; Zhang, Jingwu Z.

    2002-01-01

    Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients. PMID:12021348

  13. Molecular biology of human herpesvirus 8: novel functions and virus-host interactions implicated in viral pathogenesis and replication.

    PubMed

    Cousins, Emily; Nicholas, John

    2014-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the second identified human gammaherpesvirus. Like its relative Epstein-Barr virus, HHV-8 is linked to B-cell tumors, specifically primary effusion lymphoma and multicentric Castleman's disease, in addition to endothelial-derived KS. HHV-8 is unusual in its possession of a plethora of "accessory" genes and encoded proteins in addition to the core, conserved herpesvirus and gammaherpesvirus genes that are necessary for basic biological functions of these viruses. The HHV-8 accessory proteins specify not only activities deducible from their cellular protein homologies but also novel, unsuspected activities that have revealed new mechanisms of virus-host interaction that serve virus replication or latency and may contribute to the development and progression of virus-associated neoplasia. These proteins include viral interleukin-6 (vIL-6), viral chemokines (vCCLs), viral G protein-coupled receptor (vGPCR), viral interferon regulatory factors (vIRFs), and viral antiapoptotic proteins homologous to FLICE (FADD-like IL-1β converting enzyme)-inhibitory protein (FLIP) and survivin. Other HHV-8 proteins, such as signaling membrane receptors encoded by open reading frames K1 and K15, also interact with host mechanisms in unique ways and have been implicated in viral pathogenesis. Additionally, a set of micro-RNAs encoded by HHV-8 appear to modulate expression of multiple host proteins to provide conditions conducive to virus persistence within the host and could also contribute to HHV-8-induced neoplasia. Here, we review the molecular biology underlying these novel virus-host interactions and their potential roles in both virus biology and virus-associated disease.

  14. Molecular Biology of Human Herpesvirus 8: Novel Functions and Virus–Host Interactions Implicated in Viral Pathogenesis and Replication

    PubMed Central

    Cousins, Emily; Nicholas, John

    2014-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), is the second identified human gammaherpesvirus. Like its relative Epstein-Barr virus, HHV-8 is linked to B-cell tumors, specifically primary effusion lymphoma and multicentric Castleman’s disease, in addition to endothelial-derived KS. HHV-8 is unusual in its possession of a plethora of “accessory” genes and encoded proteins in addition to the core, conserved herpesvirus and gammaherpesvirus genes that are necessary for basic biological functions of these viruses. The HHV-8 accessory proteins specify not only activities deducible from their cellular protein homologies but also novel, unsuspected activities that have revealed new mechanisms of virus–host interaction that serve virus replication or latency and may contribute to the development and progression of virus-associated neoplasia. These proteins include viral interleukin-6 (vIL-6), viral chemokines (vCCLs), viral G protein–coupled receptor (vGPCR), viral interferon regulatory factors (vIRFs), and viral antiapoptotic proteins homologous to FLICE (FADD-like IL-1β converting enzyme)-inhibitory protein (FLIP) and survivin. Other HHV-8 proteins, such as signaling membrane receptors encoded by open reading frames K1 and K15, also interact with host mechanisms in unique ways and have been implicated in viral pathogenesis. Additionally, a set of micro-RNAs encoded by HHV-8 appear to modulate expression of multiple host proteins to provide conditions conducive to virus persistence within the host and could also contribute to HHV-8-induced neoplasia. Here, we review the molecular biology underlying these novel virus–host interactions and their potential roles in both virus biology and virus-associated disease. PMID:24008302

  15. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    PubMed Central

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population. PMID:9060668

  16. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    PubMed

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-04-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

  17. Global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes.

    PubMed

    Davis, Zoe H; Verschueren, Erik; Jang, Gwendolyn M; Kleffman, Kevin; Johnson, Jeffrey R; Park, Jimin; Von Dollen, John; Maher, M Cyrus; Johnson, Tasha; Newton, William; Jäger, Stefanie; Shales, Michael; Horner, Julie; Hernandez, Ryan D; Krogan, Nevan J; Glaunsinger, Britt A

    2015-01-22

    Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers, and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle.

  18. A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

    PubMed Central

    Sun, Ren; Lin, Su-Fang; Gradoville, Lyndle; Yuan, Yan; Zhu, Fanxiu; Miller, George

    1998-01-01

    Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases. PMID:9724796

  19. Kaposi's Sarcoma-associated Herpesvirus Hijacks RNA Polymerase II to Create a Viral Transcriptional Factory.

    PubMed

    Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel; Izumiya, Yoshihiro

    2017-03-22

    Locally concentrated nuclear factors ensure efficient binding to the DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable pre-initiation complexes. Here we have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around KSHV genomes in the host cell nucleus. Using immunofluorescent labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate-early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At single cell level, we found that not all episomes were uniformly transcribed following stimuli. However, those episomes that were being transcribed, would spontaneously aggregate to form transcriptional "factories", which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular genes transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B-cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. 3D imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed

  20. Immunoreceptor tyrosine-based activation motif-dependent signaling by Kaposi's sarcoma-associated herpesvirus K1 protein: effects on lytic viral replication.

    PubMed

    Lagunoff, M; Lukac, D M; Ganem, D

    2001-07-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 gene encodes a polypeptide bearing an immunoreceptor tyrosine-based activation motif (ITAM) that is constitutively active for ITAM-based signal transduction. Although ectopic overexpression of K1 in cultured fibroblasts can lead to growth transformation, in vivo this gene is primarily expressed in lymphoid cells undergoing lytic infection. Here we have examined function of K1 in the setting of lytic replication, through the study of K1 mutants lacking functional ITAMs. Expression of such mutants in BJAB cells cotransfected with wild-type K1 results in dramatic inhibition of K1 signal transduction, as judged by impaired activation of Syk kinase and phospholipase C-gamma2 as well as by diminished expression of a luciferase reporter gene dependent upon K1-induced calcium and Ras signaling. Thus, the mutants behave as dominantly acting inhibitors of K1 function. To assess the role of K1 in lytic replication, we introduced these K1 mutants into BCBL-1 cells, a B-cell lymphoma line latently infected with KSHV, and induced lytic replication by ectopic expression of the KSHV ORF50 transactivator. Expression of lytic cycle genes was diminished up to 80% in the presence of a K1 dominant negative mutant. These inhibitory effects could be overridden by tetradecanoyl phorbol acetate treatment, indicating that inhibition was not due to irreversible cell injury and suggesting that other signaling events could bypass the block. We conclude that ITAM-dependent signaling by K1 is not absolutely required for lytic reactivation but functions to modestly augment lytic replication in B cells, the natural reservoir of KSHV.

  1. Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation

    PubMed Central

    Gelgor, Anastasia; Kalt, Inna; Bergson, Shir; Brulois, Kevin F.; Jung, Jae U.

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. IMPORTANCE The present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression. PMID:25740992

  2. Viruses and viral proteins.

    PubMed

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R N

    2014-11-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

  3. Viruses and viral proteins

    PubMed Central

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R. N.

    2014-01-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes. PMID:25485129

  4. Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR).

    PubMed

    Hebia-Fellah, Imen; Léauté, Anne; Fiéni, Francis; Zientara, Stéphan; Imbert-Marcille, Berthe-Marie; Besse, Bernard; Fortier, Guillaume; Pronost, Stephane; Miszczak, Fabien; Ferry, Bénédicte; Thorin, Chantal; Pellerin, Jean-Louis; Bruyas, Jean-François

    2009-06-01

    In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test. EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p<0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs. There was a significant difference (p<0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing. In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.

  5. APOBEC3 Proteins in Viral Immunity

    PubMed Central

    Stavrou, Spyridon; Ross, Susan R.

    2015-01-01

    Apolipoprotein B Editing Complex (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have also been implicated in the control of other viruses such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus and retrotransposons. While their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. Here we review the data regarding the various steps in the innate and adaptive immune response to virus infection in which APOBEC3 proteins have been implicated. PMID:26546688

  6. A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread.

    PubMed

    Gill, Michael B; Edgar, Rachel; May, Janet S; Stevenson, Philip G

    2008-03-19

    Viruses lack self-propulsion. To move in multi-cellular hosts they must therefore manipulate infected cells. Herpesviruses provide an archetype for many aspects of host manipulation, but only for alpha-herpesviruses in is there much information about they move. Other herpesviruses are not necessarily the same. Here we show that Murine gamma-herpesvirus-68 (MHV-68) induces the outgrowth of long, branched plasma membrane fronds to create an intercellular network for virion traffic. The fronds were actin-based and RhoA-dependent. Time-lapse imaging showed that the infected cell surface became highly motile and that virions moved on the fronds. This plasma membrane remodelling was driven by the cytoplasmic tail of gp48, a MHV-68 glycoprotein previously implicated in intercellular viral spread. The MHV-68 ORF58 was also required, but its role was simply transporting gp48 to the plasma membrane, since a gp48 mutant exported without ORF58 did not require ORF58 to form membrane fronds either. Together, gp48/ORF58 were sufficient to induce fronds in transfected cells, as were the homologous BDLF2/BMRF2 of Epstein-Barr virus. Gp48/ORF58 therefore represents a conserved module by which gamma-herpesviruses rearrange cellular actin to increase intercellular contacts and thereby promote their spread.

  7. Human serum antibodies to a major defined epitope of human herpesvirus 8 small viral capsid antigen.

    PubMed

    Tedeschi, R; De Paoli, P; Schulz, T F; Dillner, J

    1999-04-01

    The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

  8. A single viral gene determines lethal cross-species neurovirulence of baboon herpesvirus HVP2.

    PubMed

    Black, Darla; Ohsawa, Kazutaka; Tyler, Shaun; Maxwell, Lara; Eberle, R

    2014-03-01

    Alpha-herpesviruses can produce more severe infections in non-natural host species than in their natural host. Isolates of the baboon alpha-herpesvirus Papiine herpesvirus 2 (HVP2) are either very neurovirulent in mice (subtype nv) or non-virulent (subtype ap), but no such difference is evident in the natural baboon host. Comparative genome sequencing was used to identify subtype-specific sequence differences (SSDs) between HVP2nv and HVP2ap isolates. Some genes were identified that despite exhibiting sequence variation among isolates did not have any SSDs, while other genes had comparatively high levels of SSDs. Construction of genomic recombinants between HVP2nv and HVP2ap isolates mapped the mouse neurovirulence determinant to within three genes. Construction of gene-specific recombinants demonstrated that the UL39 ORF is responsible for determining the lethal neurovirulence phenotype of HVP2 in mice. These results demonstrate that differences in a single viral gene can determine the severity of herpesvirus infection in a non-natural host species.

  9. Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans.

    PubMed

    Sandalova, Elena; Laccabue, Diletta; Boni, Carolina; Tan, Anthony T; Fink, Katja; Ooi, Eng Eong; Chua, Robert; Shafaeddin Schreve, Bahar; Ferrari, Carlo; Bertoletti, Antonio

    2010-08-19

    Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2(low)) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-gamma during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.

  10. Fluorescent Tagging and Cellular Distribution of the Kaposi's Sarcoma-Associated Herpesvirus ORF45 Tegument Protein

    PubMed Central

    Bergson, Shir; Kalt, Inna; Itzhak, Inbal; Brulois, Kevin F.; Jung, Jae U.

    2014-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress. IMPORTANCE The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions. PMID:25165104

  11. CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.

    PubMed

    Li, Da-Jiang; Verma, Dinesh; Mosbruger, Tim; Swaminathan, Sankar

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as

  12. Human herpesvirus 8-associated neoplasms: the roles of viral replication and antiviral treatment

    PubMed Central

    Gantt, Soren; Casper, Corey

    2014-01-01

    Purpose of review In this review, we highlight the importance of human herpesvirus 8 (HHV-8) lytic replication and the potential for antiviral therapies to prevent or treat HHV-8-related neoplasms. Recent findings Dieases caused by HHV-8 infection include Kaposi sarcoma (KS), multicentric Castleman disease (MCD), and primary effusion lymphoma (PEL), which occur primarily in patients with HIV infection. KS is the most common AIDS-associated malignancy worldwide. MCD and PEL occur less commonly but, like KS, are associated with poor treatment outcomes. Like all herpesviruses, HHV-8 is capable of either latent or lytic infection of cells. Although HHV-8 infection of tumor cells is predominately latent, accumulating data point to the importance of both lytic phase viral gene products and production of infectious virus. Antiviral agents that target herpesvirus DNA synthesis, such as ganciclovir, inhibit HHV-8 lytic replication and can prevent KS. Several HIV protease inhibitors may interfere with tumor growth and angiogenesis, and one PI, nelfinavir, directly inhibits HHV-8 replication in vitro. Summary Controlled trials are indicated to determine the clinical utility of antiviral suppression of HHV-8 replication, and identify the optimal antiretroviral regimens, for the prevention and treatment of KS. PMID:21666458

  13. Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

    PubMed

    Chinchar, V G; Logue, O; Antao, A; Chinchar, G D

    1998-06-19

    Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

  14. Crystal Structure of Human Herpesvirus 6B Tegument Protein U14

    PubMed Central

    Tang, Huamin; Kawabata, Akiko; Mahmoud, Nora F.; Khanlari, Zahra; Hamada, Daizo; Tsuruta, Hiroki; Mori, Yasuko

    2016-01-01

    The tegument protein U14 of human herpesvirus 6B (HHV-6B) constitutes the viral virion structure and is essential for viral growth. To define the characteristics and functions of U14, we determined the crystal structure of the N-terminal domain of HHV-6B U14 (U14-NTD) at 1.85 Å resolution. U14-NTD forms an elongated helix-rich fold with a protruding β hairpin. U14-NTD exists as a dimer exhibiting broad electrostatic interactions and a network of hydrogen bonds. This is first report of the crystal structure and dimerization of HHV-6B U14. The surface of the U14-NTD dimer reveals multiple clusters of negatively- and positively-charged residues that coincide with potential functional sites of U14. Three successive residues, L424, E425 and V426, which relate to viral growth, reside on the β hairpin close to the dimer's two-fold axis. The hydrophobic side-chains of L424 and V426 that constitute a part of a hydrophobic patch are solvent-exposed, indicating the possibility that the β hairpin region is a key functional site of HHV-6 U14. Structure-based sequence comparison suggests that U14-NTD corresponds to the core fold conserved among U14 homologs, human herpesvirus 7 U14, and human cytomegalovirus UL25 and UL35, although dimerization appears to be a specific feature of the U14 group. PMID:27152739

  15. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    PubMed Central

    Diner, Benjamin A.; Lum, Krystal K.; Toettcher, Jared E.

    2016-01-01

    ABSTRACT The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. PMID:27935834

  16. Herpesvirus saimiri.

    PubMed Central

    Fickenscher, H; Fleckenstein, B

    2001-01-01

    Herpesvirus saimiri (saimiriine herpesvirus 2) is the classical prototype of the gamma(2)-herpesviruses or rhadinoviruses, which also contains a human member, the Kaposi's sarcoma-associated herpesvirus. The T-lymphotropic Herpesvirus saimiri establishes specific replicative and persistent conditions in different primate host species. Virtually all squirrel monkeys (Saimiri sciureus) are persistently infected with this virus. In its natural host, the virus does not cause disease, whereas it induces fatal acute T-cell lymphoma in other monkey species after experimental infection. The virus can be isolated by cocultivation of permissive epithelial cells with peripheral blood cells from naturally infected squirrel monkeys and from susceptible New World monkeys during the virus-induced disease. Tumour-derived and in vitro-transformed T-cell lines from New World monkeys release virus particles. Herpesvirus ateles is a closely related virus of spider monkeys (Ateles spp.) and has similar pathogenic properties to Herpesvirus saimiri in other New World primate species. Similar to other rhadinoviruses, the genome of Herpesvirus saimiri harbours a series of virus genes with pronounced homology to cellular counterparts including a D-type cyclin, a G-protein-coupled receptor, an interleukin-17, a superantigen homologue, and several inhibitors of the complement cascade and of different apoptosis pathways. Preserved function has been demonstrated for most of the homologues of cellular proteins. These viral functions are mostly dispensable for the transforming and pathogenic capability of the virus. However, they are considered relevant for the apathogenic persistence of Herpesvirus saimiri in its natural host. A terminal region of the non-repetitive coding part of the virus genome is essential for pathogenicity and T-cell transformation. Based on the pathogenic phenotypes and the different alleles of this variable region, the virus strains have been assigned to three subgroups

  17. Replication of simian herpesvirus SA8 and identification of viral polypeptides in infected cells.

    PubMed Central

    Eberle, R; Hilliard, J K

    1984-01-01

    The replication of the simian herpesvirus SA8 in Vero cells was examined. The time course of replication of the simian herpesvirus SA8 was found to be similar to that of the herpes simplex viruses. Infectious progeny virions were first detectable by 6 h postinfection and were readily released into the extracellular fluids beginning at 9 h postinfection. All cell lines tested, with the exception of Madin-Darby canine kidney cells, were permissive for SA8. Analysis of SA8-infected cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed over 40 infected cell polypeptides ranging in molecular weight from 158,000 to less than 10,000. Of these proteins, 23 were present in virions. Three classes of infected cell polypeptides could be identified based on the kinetics of their synthesis. Post-translational processing of several SA8-induced proteins was also observed in pulse-chase experiments. Six distinct SA8-specific glycoproteins ranging from 118,000 to 19,500 daltons were also identified in infected cells. Of these glycoproteins, five were present in virions. Images PMID:6708170

  18. Locating herpesvirus Bcl-2 homologs in the specificity landscape of anti-apoptotic Bcl-2 proteins

    PubMed Central

    Foight, Glenna Wink; Keating, Amy E.

    2015-01-01

    Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2 and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2, but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9 nM for binding to KSBcl-2 and >1000-fold specificity over human Bcl-2 proteins, and a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners. PMID:26009469

  19. A Point Mutation in a Herpesvirus Co-Determines Neuropathogenicity and Viral Shedding

    PubMed Central

    Franz, Mathias; Goodman, Laura B.; Van de Walle, Gerlinde R.; Osterrieder, Nikolaus; Greenwood, Alex D.

    2017-01-01

    A point mutation in the DNA polymerase gene in equine herpesvirus type 1 (EHV-1) is one determinant for the development of neurological disease in horses. Three recently conducted infection experiments using domestic horses and ponies failed to detect statistically significant differences in viral shedding between the neuropathogenic and non-neuropathogenic variants. These results were interpreted as suggesting the absence of a consistent selective advantage of the neuropathogenic variant and therefore appeared to be inconsistent with a systematic increase in the prevalence of neuropathogenic strains. To overcome potential problems of low statistical power related to small group sizes in these infection experiments, we integrated raw data from all three experiments into a single statistical analysis. The results of this combined analysis showed that infection with the neuropathogenic EHV-1 variant led to a statistically significant increase in viral shedding. This finding is consistent with the idea that neuropathogenic strains could have a selective advantage and are therefore systematically increasing in prevalence in domestic horse populations. However, further studies are required to determine whether a selective advantage indeed exists for neuropathogenic strains. PMID:28075374

  20. Human and viral interleukin-6 and other cytokines in Kaposi sarcoma herpesvirus-associated multicentric Castleman disease

    PubMed Central

    Uldrick, Thomas S.; Wang, Victoria; Aleman, Karen; Wyvill, Kathleen M.; Marshall, Vickie; Pittaluga, Stefania; O’Mahony, Deirdre; Whitby, Denise; Tosato, Giovanna; Steinberg, Seth M.; Little, Richard F.

    2013-01-01

    Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a polyclonal B-cell lymphoproliferative disorder. Human (h) IL-6 and a KSHV-encoded homolog, viral IL-6, have been hypothesized to contribute to its pathogenesis, but their relative contributions to disease activity is not well understood. We prospectively characterized KSHV viral load (VL), viral (v) and hIL-6, and other cytokines during KSHV-MCD flare and remission in 21 patients with 34 flares and 20 remissions. KSHV-VL, vIL-6, hIL-6, IL-10, and to a lesser extent TNF-α, and IL-1β were each elevated during initial flares compared with remission. Flares fell into 3 distinct IL-6 profiles: those associated with elevations of vIL6-only (2 flares, 6%), hIL-6 elevations only (17 flares, 50%), and elevations in both hIL-6 and vIL-6 (13 flares, 38%). Compared with hIL-6–only flares, flares with elevated hIL-6 plus vIL-6 exhibited higher C-reactive protein (CRP) (P = .0009); worse hyponatremia (P = .02); higher KSHV VL (P = .016), and higher IL-10 (P = .012). This analysis shows vIL-6 and hIL-6 can independently or together lead to KSHV-MCD flares, and suggests that vIL-6 and hIL-6 may jointly contribute to disease severity. These findings have implications for the development of novel KSHV-MCD therapies targeting IL-6 and its downstream signaling. This trial was registered at clinicaltrials.gov as #NCT099073. PMID:24174627

  1. Identification and Characterization of the Orf49 Protein of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    González, Carlos M.; Wong, Emily L.; Bowser, Brian S.; Hong, Gregory K.; Kenney, Shannon; Damania, Blossom

    2006-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common neoplasm among human immunodeficiency virus-positive individuals. Like other herpesviruses, KSHV is able to establish a predominantly latent, life-long infection in its host. The KSHV lytic cycle can be triggered by a number of stimuli that induce the expression of the key lytic switch protein, the replication and transcription activator (RTA) encoded by Orf50. The expression of Rta is necessary and sufficient to trigger the full lytic program resulting in the ordered expression of viral proteins, release of viral progeny, and host cell death. We have characterized an unknown open reading frame, Orf49, which lies adjacent and in the opposite orientation to Orf50. Orf49 is expressed during the KSHV lytic cycle and shows early transcription kinetics. We have mapped the 5′ and 3′ ends of the unspliced Orf49 transcript, which encodes a 30-kDa protein that is localized to both the nucleus and the cytoplasm. Interestingly, we found that Orf49 was able to cooperate with Rta to activate several KSHV lytic promoters containing AP-1 sites. The Orf49-encoded protein was also able to induce transcriptional activation through c-Jun but not the ATF1, ATF2, or CREB transcription factor. We found that Orf49 could induce phosphorylation and activation of the transcription factor c-Jun, the Jun N-terminal kinase (JNK), and p38. Our data suggest that Orf49 functions to activate the JNK and p38 pathways during the KSHV lytic cycle. PMID:16501115

  2. Alternative Capture of Noncoding RNAs or Protein-Coding Genes by Herpesviruses to Alter Host T-Cell Function

    PubMed Central

    Guo, Yang Eric; Riley, Kasandra J.; Iwasaki, Akiko; Steitz, Joan A.

    2014-01-01

    SUMMARY In marmoset T cells transformed by Herpesvirus saimiri (HVS), a viral U-rich noncoding RNA, HSUR 1, specifically mediates degradation of host microRNA-27 (miR-27). High-throughput sequencing of RNA after crosslinking immunoprecipitation (HITS-CLIP) identified mRNAs targeted by miR-27 as enriched in the T-cell receptor (TCR) signaling pathway, including GRB2. Accordingly, transfection of miR-27 into human T cells attenuates TCR-induced activation of mitogen-activated protein kinases (MAPKs) and induction of CD69. MiR-27 also robustly regulates SEMA7A and IFN-γ, key modulators and effectors of T-cell function. Knockdown or ectopic expression of HSUR 1 alters levels of these proteins in virally-transformed cells. Two other T-lymphotropic γ-herpesviruses, AlHV-1 and OvHV-2, do not produce a noncoding RNA to downregulate miR-27, but instead encode homologs of miR-27 target genes. Thus, oncogenic γ-herpesviruses have evolved diverse strategies to converge on common targets in host T cells. PMID:24725595

  3. Going Viral with Fluorescent Proteins.

    PubMed

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  4. Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes

    PubMed Central

    Hill, James M.; Quenelle, Debra C.; Cardin, Rhonda D.; Vogel, Jodi L.; Clement, Christian; Bravo, Fernando J.; Foster, Timothy P.; Bosch-Marce, Marta; Raja, Priya; Lee, Jennifer S.; Bernstein, David I.; Krause, Philip R.; Knipe, David M.; Kristie, Thomas M.

    2015-01-01

    The high prevalence of Herpesviruses in the population and the maintenance of lifelong latent reservoirs are challenges to the control of herpetic diseases, despite the availability of antiviral pharmaceuticals that target viral DNA replication. In addition to oral and genital lesions, herpes simplex virus infections and recurrent reactivations from the latent pool can result in severe pathology including neonatal infection and mortality, blindness due to ocular keratitis, and viral-induced complications in immunosuppressed individuals. Herpesviruses, like their cellular hosts, are subject to the regulatory impacts of chromatin and chromatin modulation machinery that promotes or suppresses gene expression. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 and JMJD2s. Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and a block to infection and reactivation in vitro. Here, the concept of epigenetic suppression of viral infection is demonstrated in three animal models of herpes simplex virus infection and disease. Inhibition of LSD1 via treatment of animals with the monoamine oxidase inhibitor tranylcypromine results in suppression of viral lytic infection, subclinical shedding, and reactivation from latency in vivo. Phenotypic suppression is correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Given the expanding development of epipharmaceuticals, this approach has substantial potential for anti-herpetic treatments with distinct advantages over the present pharmaceutical options. PMID:25473037

  5. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    PubMed

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

  6. Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

    PubMed Central

    Liang, Qiming; Chang, Brian; Lee, Patrick; Brulois, Kevin F.; Ge, Jianning; Shi, Mude; Rodgers, Mary A.; Feng, Pinghui; Oh, Byung-Ha; Liang, Chengyu

    2015-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication. PMID:25740994

  7. Concatemeric intermediates of equine herpesvirus type 1 DNA replication contain frequent inversions of adjacent long segments of the viral genome.

    PubMed

    Slobedman, B; Simmons, A

    1997-03-17

    In common with other alpha-herpesviruses, the genome of equine herpesvirus type-1 (EHV-1) comprises covalently linked long and short unique sequences of DNA, each flanked by inverted repeats. Equimolar amounts of two genomic isomers, generated by free inversion of the short segment, relative to the long segment, are packaged into EHV-1 virions. In contrast with herpes simplex virus (HSV), inversion of genomic long segments has not been described. In the current work, the structures of high molecular weight intermediates of EHV-1 DNA replication were studied by field inversion gel electrophoresis. It is shown that adjacent long segments of the viral genome are frequently inverted in concatemeric intermediates of EHV-1 DNA replication. Further, like HSV concatemers, high molecular weight intermediates of EHV-1 replication are flanked exclusively by the long segment of the viral genome. Hence, despite the fact that only two, rather than four, isomers of EHV-1 DNA are packaged into virions, the intermediates of EHV-1 DNA replication closely resemble those of herpes simplex virus type 1 in structure. These data have implications relating to the mechanisms involved in packaging of alpha-herpesvirus DNA.

  8. Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.

    PubMed

    Lewis, J B; Thompson, Y G; Feng, X; Holden, V R; O'Callaghan, D; Caughman, G B

    1997-04-14

    The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

  9. Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity

    PubMed Central

    Ariza, Maria Eugenia; Glaser, Ronald; Williams, Marshall V.

    2014-01-01

    We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses. PMID:25309527

  10. Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity.

    PubMed

    Ariza, Maria Eugenia; Glaser, Ronald; Williams, Marshall V

    2014-01-01

    We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses.

  11. Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein

    PubMed Central

    Wu, Jian-jun; Li, Wenwei; Shao, Yaming; Avey, Denis; Fu, Bishi; Gillen, Joseph; Hand, Travis; Ma, Siming; Liu, Xia; Miley, Wendell; Konrad, Andreas; Neipel, Frank; Stürzl, Michael; Whitby, Denise; Li, Hong; Zhu, Fanxiu

    2015-01-01

    SUMMARY Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS- and DNA-binding. Moreover, ORF52 homologues in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52-null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing. PMID:26320998

  12. Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein.

    PubMed

    Wu, Jian-jun; Li, Wenwei; Shao, Yaming; Avey, Denis; Fu, Bishi; Gillen, Joseph; Hand, Travis; Ma, Siming; Liu, Xia; Miley, Wendell; Konrad, Andreas; Neipel, Frank; Stürzl, Michael; Whitby, Denise; Li, Hong; Zhu, Fanxiu

    2015-09-09

    Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS binding and DNA binding. Moreover, ORF52 homologs in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52 null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing.

  13. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis.

    PubMed

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W; Lira, Sergio A; Levine, Beth

    2016-03-15

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.

  14. Microarray chip based identification of a mixed infection of bovine herpesvirus 1 and bovine viral diarrhea 2 from Indian cattle.

    PubMed

    Ratta, Barkha; Yadav, Brijesh Singh; Pokhriyal, Mayank; Saxena, Meeta; Sharma, Bhaskar

    2014-01-01

    Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.

  15. Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi’s Sarcoma-Associated Herpesvirus for Viral Latency

    PubMed Central

    Zhang, Liming; Li, Yuhong; Feng, Yanling; Xu, Jianqing; Wang, Bin; Yuan, Zhenghong; Robertson, Erle S.; Cai, Qiliang

    2017-01-01

    Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi’s sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV. PMID:28099521

  16. Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi's Sarcoma-Associated Herpesvirus for Viral Latency.

    PubMed

    Wang, Chong; Zhu, Caixia; Wei, Fang; Gao, Shujun; Zhang, Liming; Li, Yuhong; Feng, Yanling; Tong, Yin; Xu, Jianqing; Wang, Bin; Yuan, Zhenghong; Robertson, Erle S; Cai, Qiliang

    2017-01-01

    Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV.

  17. Protein kinase CK2 phosphorylation regulates the interaction of Kaposi's sarcoma-associated herpesvirus regulatory protein ORF57 with its multifunctional partner hnRNP K

    PubMed Central

    Malik, Poonam; Clements, J. Barklie

    2004-01-01

    ORF57 protein of Kaposi's sarcoma-associated herpesvirus has a counterpart in all herpesvirus of mammals and birds and regulates gene expression at transcriptional and post-transcriptional levels. ORF57 was capable of self-interaction and bound a rapidly migrating form of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional cellular protein involved in gene expression. In virus infected cell extracts, ORF57 was present in a complex with hnRNP K that had protein kinase CK2 activity, and was phosphorylated by CK2. Different regions of ORF57 bound both catalytic α/α′ and regulatory β subunits of CK2. CK2 modification enhanced the ORF57–hnRNP K interaction, and may regulate the presence and activities of components in the complex. We suggest that ORF57 and hnRNP K interaction may modulate ORF57-mediated regulation of viral gene expression. Herpesviral ORF57 (Rhadinovirus) and ICP27 (Simplexvirus) proteins both interact with hnRNP K and CK2 implying that adaptation of the ancestral hnRNP K and CK2 to associate with viral regulatory ancestor protein likely pre-dates divergence of these Herpesviridae genera that occurred 200 million years ago. PMID:15486205

  18. Kaposi's sarcoma-associated herpesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to induce vascular endothelial growth factor expression.

    PubMed

    Shin, Young C; Joo, Chul-Hyun; Gack, Michaela U; Lee, Hye-Ra; Jung, Jae U

    2008-03-15

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive alpha-subunit and a constitutively expressed beta-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1 alpha subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1 alpha subunit led to the HIF-1 alpha stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double alpha-helix motifs, was sufficient not only for binding to HIF-1 alpha but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1 alpha protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis.

  19. Epigenotypes of latent herpesvirus genomes.

    PubMed

    Minarovits, J

    2006-01-01

    Epigenotypes are modified cellular or viral genotypes which differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Restricted expression of latent, episomal herpesvirus genomes is also due to epigenetic modifications. There is no virus production (lytic viral replication, associated with the expression of all viral genes) in tight latency. In vitro experiments demonstrated that DNA methylation could influence the activity of latent (and/or crucial lytic) promoters of prototype strains belonging to the three herpesvirus subfamilies (alpha-, beta-, and gamma-herpesviruses). In vivo, however, DNA methylation is not a major regulator of herpes simplex virus type 1 (HSV-1, a human alpha-herpesvirus) latent gene expression in neurons of infected mice. In these cells, the promoter/enhancer region of latency-associated transcripts (LATs) is enriched with acetyl histone H3, suggesting that histone modifications may control HSV-1 latency in terminally differentiated, quiescent neurons. Epstein-Barr virus (EBV, a human gamma-herpesvirus) is associated with a series of neoplasms. Latent, episomal EBV genomes are subject to host cell-dependent epigenetic modifications (DNA methylation, binding of proteins and protein complexes, histone modifications). The distinct viral epigenotypes are associated with distinct EBV latency types, i.e., cell type-specific usage of latent EBV promoters controlling the expression of latent, growth transformation-associated EBV genes. The contribution of major epigenetic mechanisms to the regulation of latent EBV promoters is variable. DNA methylation contributes to silencing of Wp and Cp (alternative promoters for transcripts coding for the nuclear antigens EBNA 1-6) and LMP1p, LMP2Ap, and LMP2Bp (promoters for transcripts encoding transmembrane proteins). DNA methylation does not control, however, Qp (a promoter for EBNA1 transcripts only) in lymphoblastoid cell lines (LCLs), although in vitro

  20. Evaluation of a viral microarray based on simultaneous extraction and amplification of viral nucleotide acid for detecting human herpesviruses and enteroviruses.

    PubMed

    Liu, Yi; Duan, Chunhong; Zhang, Chunxiu; Yang, Xiaomeng; Zhao, Yan; Dong, Rui; Zhou, Jiajing; Gai, Zhongtao

    2015-01-01

    In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5'-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses' detection.

  1. Intercellular trafficking and protein delivery by a herpesvirus structural protein.

    PubMed

    Elliott, G; O'Hare, P

    1997-01-24

    We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.

  2. Effects of Preinfection With Bovine Viral Diarrhea Virus on Immune Cells From the Lungs of Calves Inoculated With Bovine Herpesvirus 1.1.

    PubMed

    Risalde, M A; Molina, V; Sánchez-Cordón, P J; Romero-Palomo, F; Pedrera, M; Gómez-Villamandos, J C

    2015-07-01

    The aim of this work was to study the interstitial aggregates of immune cells observed in pulmonary parenchyma of calves preinfected with bovine viral diarrhea virus and challenged later with bovine herpesvirus 1. In addition, the intent of this research was to clarify the role of bovine viral diarrhea virus in local cell-mediated immunity and potentially in predisposing animals to bovine respiratory disease complex. Twelve Friesian calves, aged 8 to 9 months, were inoculated with noncytopathic bovine viral diarrhea virus genotype 1. Ten were subsequently challenged with bovine herpesvirus 1 and euthanized at 1, 2, 4, 7, or 14 days postinoculation. The other 2 calves were euthanized prior to the second inoculation. Another cohort of 10 calves was inoculated only with bovine herpesvirus 1 and then were euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Pulmonary lesions were evaluated in all animals, while quantitative and biosynthetic changes in immune cells were concurrently examined immunohistochemically to compare coinfected calves and calves challenged only with bovine herpesvirus 1. Calves preinfected with bovine viral diarrhea virus demonstrated moderate respiratory clinical signs and histopathologic evidence of interstitial pneumonia with aggregates of mononuclear cells, which predominated at 4 days postinoculation. Furthermore, this group of animals was noted to have a suppression of interleukin-10 and associated alterations in the Th1-driven cytokine response in the lungs, as well as inhibition of the response of CD8+ and CD4+ T lymphocytes against bovine herpesvirus 1. These findings suggest that bovine viral diarrhea virus preinfection could affect the regulation of the immune response as modulated by regulatory T cells, as well as impair local cell-mediated immunity to secondary respiratory pathogens.

  3. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

    PubMed

    Leigh, Kendra E; Sharma, Mayuri; Mansueto, My Sam; Boeszoermenyi, Andras; Filman, David J; Hogle, James M; Wagner, Gerhard; Coen, Donald M; Arthanari, Haribabu

    2015-07-21

    Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

  4. Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

    PubMed Central

    Kruger, Ernesto Renato; Penha, Tania Regina; Stoffelo, Daura Regina Eira; Roehe, Paulo Michel; Ribeiro, Magda Costa; Soccol, Vanete Thomaz

    2015-01-01

    Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus . This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after Hind III digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes Hind III and Bam HI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of Bam HI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group. PMID:26221118

  5. Functional characterization of viral tumor necrosis factor receptors encoded by cyprinid herpesvirus 3 (CyHV3) genome.

    PubMed

    Yi, Yang; Qi, Hemei; Yuan, Jimin; Wang, Rui; Weng, Shaoping; He, Jianguo; Dong, Chuanfu

    2015-08-01

    Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection.

  6. Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein

    PubMed Central

    Kim, Yuri; Kwon, Eun-Kyung; Jeon, Ju-Hong; So, Insuk; Kim, In-Gyu; Choi, Myung-Sik; Kim, Ik-Sang; Choi, Joong-Kook; Jung, Jae Ung

    2012-01-01

    Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS. PMID:22012462

  7. Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus).

    PubMed

    Stanton, Jeffrey J; Zong, Jian-Chao; Eng, Crystal; Howard, Lauren; Flanagan, Joe; Stevens, Martina; Schmitt, Dennis; Wiedner, Ellen; Graham, Danielle; Junge, Randall E; Weber, Martha A; Fischer, Martha; Mejia, Alicia; Tan, Jie; Latimer, Erin; Herron, Alan; Hayward, Gary S; Ling, Paul D

    2013-03-01

    Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

  8. Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

    PubMed Central

    Chang, Tzu-Hsuan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-01-01

    The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. PMID:27698494

  9. Regulation of the Abundance of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2.

    PubMed

    Chang, Tzu-Hsuan; Wang, Shie-Shan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-10-01

    The switch between latency and the lytic cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490-535) and PARS-II (aa 590-650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.

  10. Experimental intranasal infection of equine herpesvirus 9 (EHV-9) in suckling hamsters: kinetics of viral transmission and inflammation in the nasal cavity and brain.

    PubMed

    El-Habashi, Nagwan; El-Nahass, El-Shaymaa; Fukushi, Hideto; Hibi, Daisuke; Sakai, Hiroki; Sasseville, Vito; Yanai, Tokuma

    2010-05-01

    Equine herpesvirus 9 (EHV-9), the newest member of the equine herpesvirus family, is a highly neurotropic herpesvirus that induces encephalitis in a variety of animals. To access transmission of EHV-9 in the nasal cavity and brain, a suckling hamster model was developed so that precise sagittal sections of nasal and cranial cavities including the brain could be processed, which proved useful in detecting viral transmission as well as extension of pathological lesions. Suckling hamsters were inoculated intranasally with EHV-9, and were sacrificed at 6, 12, 18, 24, 36, 48, and 60 h post inoculation (PI). Sagittal sections of the entire head, including nasal and cranial cavities including the brain, were made to assess viral kinetics and identify the progress of the neuropathological lesions. At 12 to 24 h PI the virus attached to and propagated in the olfactory epithelium, and infected adjacent epithelial cells. At 48 h PI, immunohistochemistry for EHV-9 viral antigen showed that virus had extended from the site of infection into the olfactory bulb and olfactory nerve. These results indicate that EHV-9 rapidly invades the brain via the olfactory route after experimental intranasal infection.

  11. Evaluation of reproductive protection against bovine viral diarrhea virus and bovine herpesvirus-1 afforded by annual revaccination with modified-live viral or combination modified-live/killed viral vaccines after primary vaccination with modified-live viral vaccine.

    PubMed

    Walz, Paul H; Givens, M Daniel; Rodning, Soren P; Riddell, Kay P; Brodersen, Bruce W; Scruggs, Daniel; Short, Thomas; Grotelueschen, Dale

    2017-02-15

    The objective of this study was to compare reproductive protection in cattle against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) provided by annual revaccination with multivalent modified-live viral (MLV) vaccine or multivalent combination viral (CV) vaccine containing temperature-sensitive modified-live BoHV-1 and killed BVDV when MLV vaccines were given pre-breeding to nulliparous heifers. Seventy-five beef heifers were allocated into treatment groups A (n=30; two MLV doses pre-breeding, annual revaccination with MLV vaccine), B (n=30; two MLV doses pre-breeding, annual revaccination with CV vaccine) and C (n=15; saline in lieu of vaccine). Heifers were administered treatments on days 0 (weaning), 183 (pre-breeding), 366 (first gestation), and 738 (second gestation). After first calving, primiparous cows were bred, with pregnancy assessment on day 715. At that time, 24 group A heifers (23 pregnancies), 23 group B heifers (22 pregnancies), and 15 group C heifers (15 pregnancies) were commingled with six persistently infected (PI) cattle for 16days. Ninety-nine days after PI removal, cows were intravenously inoculated with BoHV-1. All fetuses and live offspring were assessed for BVDV and BoHV-1. Abortions occurred in 3/23 group A cows, 1/22 group B cows, and 11/15 group C cows. Fetal infection with BVDV or BoHV-1 occurred in 4/23 group A offspring, 0/22 group B offspring, and 15/15 group C offspring. This research demonstrates efficacy of administering two pre-breeding doses of MLV vaccine with annual revaccination using CV vaccine to prevent fetal loss due to exposure to BVDV and BoHV-1.

  12. Kaposi’s Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor

    PubMed Central

    Li, Mengtao; Lee, Heuiran; Guo, Jie; Neipel, Frank; Fleckenstein, Bernhard; Ozato, Keiko; Jung, Jae U.

    1998-01-01

    Interferons (IFNs) are a family of multifunctional cytokines with antiviral activities. The K9 open reading frame of Kaposi’s sarcoma-associated herpesvirus (KSHV) exhibits significant homology with cellular IFN regulatory factors (IRFs). We have investigated the functional consequence of K9 expression in IFN-mediated signal transduction. Expression of K9 dramatically repressed transcriptional activation induced by IFN-α, -β, and -γ. Further, it induced transformation of NIH 3T3 cells, resulting in morphologic changes, focus formation, and growth in reduced-serum conditions. The expression of antisense K9 in KSHV-infected BCBL-1 cells consistently increased IFN-mediated transcriptional activation but drastically decreased the expression of certain KSHV genes. Thus, the K9 gene of KSHV encodes the first virus-encoded IRF (v-IRF) which functions as a repressor for cellular IFN-mediated signal transduction. In addition, v-IRF likely plays an important role in regulating KSHV gene expression. These results suggest that KSHV employs an unique mechanism to antagonize IFN-mediated antiviral activity by harboring a functional v-IRF. PMID:9620998

  13. Use of plasma human herpesvirus-8 viral load measurement: evaluation of practice in three UK HIV treatment centres.

    PubMed

    Nugent, D B; Webster, D; Mabayoje, D; Chung, E; El Bouzidi, K; O'Sullivan, A; Ainsworth, J; Miller, R F

    2017-02-01

    A retrospective audit of plasma human herpesvirus-8 (HHV-8) viral load testing was performed in three HIV treatment centres over 24 months. Reasons for testing (360 tests) were: symptoms of systemic inflammatory response syndrome (SIRS) (fever, lymphadenopathy and raised inflammatory markers); monitoring in known HHV-8 pathology other than Kaposi sarcoma (KS); investigation of known/suspected KS, and other/no reason. Of patients with multicentric Castleman disease (MCD), 14/16 (88%) had detectable plasma HHV-8, as did 27/45 (60%) with biopsy proven or clinically confirmed KS, and 6/19 (32%) with lymphoma. Neither of the two patients with MCD and no detectable HHV-8 had SIRS symptoms at the time of the test. There was wide variation between centres in the indications prompting HHV-8 testing, with a more conservative approach resulting in a higher proportion of positive results. Measuring plasma HHV-8 in the absence of SIRS symptoms, established HHV-8 disease monitoring, or confirmed/suspected KS is unlikely to yield detectable HHV-8 thus allowing potential cost savings.

  14. Seroprevalence and risk factors associated with bovine herpesvirus 1 and bovine viral diarrhea virus in North-Eastern Mexico

    PubMed Central

    Segura-Correa, J.C.; Zapata-Campos, C.C.; Jasso-Obregón, J.O.; Martinez-Burnes, J.; López-Zavala, R.

    2016-01-01

    Bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) are well known etiological agents of cattle that produce important economic losses due to reproductive failures and calf mortality, as well as enteric and respiratory disease. Tamaulipas is located northeast of Mexico, an important cattle production and the principal exporter of calf and heifer to the United States. The objectives of this study were to estimate the seroprevalence of BoHV-1 and of BVDV, and to determine the effects of risk factors on these infections. Blood samples of cattle from 57 farms from rural districts of Tamaulipas were collected. The samples were tested for antibodies against BoHV-1 and BVDV using commercial ELISA kits. Data on potential risk factors were obtained using a questionnaire administered to the farmer at the time the blood samples were taken. The seroprevalences for BoHV-1 and BVDV were 64.4% and 47.8%, respectively. In the logistic regression analysis, the significant risk factors were rural district, herd size and cattle introduced to the farm. This study confirms the high seroprevalence of BoHV-1 and BVDV in unvaccinated cattle in Tamaulipas, Mexico. The results of this study could be used for the development of BoHV-1 and BVDV prevention and control program in North-Eastern, Mexico. PMID:27622156

  15. Seroprevalence and risk factors associated with bovine herpesvirus 1 and bovine viral diarrhea virus in North-Eastern Mexico.

    PubMed

    Segura-Correa, J C; Zapata-Campos, C C; Jasso-Obregón, J O; Martinez-Burnes, J; López-Zavala, R

    2016-01-01

    Bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) are well known etiological agents of cattle that produce important economic losses due to reproductive failures and calf mortality, as well as enteric and respiratory disease. Tamaulipas is located northeast of Mexico, an important cattle production and the principal exporter of calf and heifer to the United States. The objectives of this study were to estimate the seroprevalence of BoHV-1 and of BVDV, and to determine the effects of risk factors on these infections. Blood samples of cattle from 57 farms from rural districts of Tamaulipas were collected. The samples were tested for antibodies against BoHV-1 and BVDV using commercial ELISA kits. Data on potential risk factors were obtained using a questionnaire administered to the farmer at the time the blood samples were taken. The seroprevalences for BoHV-1 and BVDV were 64.4% and 47.8%, respectively. In the logistic regression analysis, the significant risk factors were rural district, herd size and cattle introduced to the farm. This study confirms the high seroprevalence of BoHV-1 and BVDV in unvaccinated cattle in Tamaulipas, Mexico. The results of this study could be used for the development of BoHV-1 and BVDV prevention and control program in North-Eastern, Mexico.

  16. Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation

    PubMed Central

    Zhang, Kuan; Brownlie, Robert; Snider, Marlene

    2016-01-01

    ABSTRACT VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. IMPORTANCE In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other

  17. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-06

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy.

  18. Characterization of human herpesvirus 6A/B U94 as ATPase, helicase, exonuclease and DNA-binding proteins

    PubMed Central

    Trempe, Frédéric; Gravel, Annie; Dubuc, Isabelle; Wallaschek, Nina; Collin, Vanessa; Gilbert-Girard, Shella; Morissette, Guillaume; Kaufer, Benedikt B.; Flamand, Louis

    2015-01-01

    Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration. PMID:25999342

  19. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  20. A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA Proteins

    PubMed Central

    Krausze, Joern; Richter, Ulrike; Adler, Heiko; Fedorov, Roman; Pietrek, Marcel; Rückert, Jessica; Ritter, Christiane; Schulz, Thomas F.; Lührs, Thorsten

    2013-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence. PMID:24146614

  1. Molecular piracy of Kaposi's sarcoma associated herpesvirus.

    PubMed

    Choi, J; Means, R E; Damania, B; Jung, J U

    2001-01-01

    Kaposi's Sarcoma associated Herpesvirus (KSHV) is the most recently discovered human tumor virus and is associated with the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and Multicentric Casttleman's disease. KSHV contains numerous open reading frames with striking homology to cellular genes. These viral gene products play a variety of roles in KSHV-associated pathogenesis by disrupting cellular signal transduction pathways, which include interferon-mediated anti-viral responses, cytokine-regulated cell growth, apoptosis, and cell cycle control. In this review, we will attempt to cover our understanding of how viral proteins deregulate cellular signaling pathways, which ultimately contribute to the conversion of normal cells to cancerous cells.

  2. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    PubMed

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  3. Viral Meningitis

    MedlinePlus

    ... have severe illness from viral meningitis. Causes Non-polio enteroviruses are the most common cause of viral ... following viruses spread by visiting CDC’s websites: Non-polio enteroviruses Mumps virus Herpesviruses, including Epstein-Barr virus , ...

  4. HCMV gB shares structural and functional properties with gB proteins from other herpesviruses

    SciTech Connect

    Sharma, Sapna; Wisner, Todd W.; Johnson, David C.; Heldwein, Ekaterina E.

    2013-01-20

    Glycoprotein B (gB) facilitates HCMV entry into cells by binding receptors and mediating membrane fusion. The crystal structures of gB ectodomains from HSV-1 and EBV are available, but little is known about the HCMV gB structure. Using multiangle light scattering and electron microscopy, we show here that HCMV gB ectodomain is a trimer with the overall shape similar to HSV-1 and EBV gB ectodomains. HCMV gB ectodomain forms rosettes similar to rosettes formed by EBV gB and the postfusion forms of other viral fusogens. Substitution of several bulky hydrophobic residues within the putative fusion loops with more hydrophilic residues reduced rosette formation and abolished cell fusion. We propose that like gB proteins from HSV-1 and EBV, HCMV gB has two internal hydrophobic fusion loops that likely interact with target membranes. Our work establishes structural and functional similarities between gB proteins from three subfamilies of herpesviruses.

  5. Bovine herpesvirus 1 regulatory proteins are detected in trigeminal ganglionic neurons during the early stages of stress-induced escape from latency.

    PubMed

    Kook, Insun; Doster, Alan; Jones, Clinton

    2015-10-01

    Bovine herpesvirus 1 (BHV-1) establishes latency in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency. Within 90 min after latently infected calves are treated with dexamethasone, two BHV-1 regulatory proteins, BHV-1-infected cell protein 0 (bICP0) and viral protein 16 (VP16), are expressed in the same neuron. In this study, we demonstrate that VP16 and bICP0 can be detected at 22 and 33 min after dexamethasone (DEX) treatment of latently infected calves. However, we were unable to discern whether VP16 or bICP0 was expressed at early times after reactivation. VP16+ neurons consistently express the glucocorticoid receptor suggesting corticosteroid-mediated activation of its receptor rapidly stimulates reactivation from latency.

  6. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

    PubMed

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S

    2017-03-15

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular

  7. Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease

    PubMed Central

    Williams, Marshall V.; Cox, Brandon; Ariza, Maria Eugenia

    2016-01-01

    The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person’s lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein–Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer. PMID:28036046

  8. Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease.

    PubMed

    Williams, Marshall V; Cox, Brandon; Ariza, Maria Eugenia

    2016-12-28

    The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person's lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein-Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer.

  9. Activation of the Kaposi's Sarcoma-Associated Herpesvirus Major Latency Locus by the Lytic Switch Protein RTA (ORF50)

    PubMed Central

    Matsumura, Satoko; Fujita, Yuriko; Gomez, Evan; Tanese, Naoko; Wilson, Angus C.

    2005-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma cells but can be induced to enter full lytic replication by exposure to a variety of chemical inducing agents or by expression of the KSHV-encoded replication and transcription activator (RTA) protein. During latency, only a few viral genes are expressed, and these include the three genes of the so-called latency transcript (LT) cluster: v-FLIP (open reading frame 71 [ORF71]), v-cyclin (ORF72), and latency-associated nuclear antigen (ORF73). During latency, all three open reading frames are transcribed from a common promoter as part of a multicistronic mRNA. Subsequent alternative mRNA splicing and internal ribosome entry allows for the expression of each protein. Here, we show that transcription of LT cassette mRNA can be induced by RTA through the activation of a second promoter (LTi) immediately downstream of the constitutively active promoter (LTc). We identified a minimal cis-regulatory region, which overlaps with the promoter for the bicistronic K14/v-GPCR delayed early gene that is transcribed in the opposite direction. In addition to a TATA box at −30 relative to the LTi mRNA start sites, we identified three separate RTA response elements that are also utilized by the K14/v-GPCR promoter. Interestingly, LTi is unresponsive to sodium butyrate, a potent inducer of lytic replication. This suggests there is a previously unrecognized class of RTA-responsive promoters that respond to direct, but not indirect, induction of RTA. These studies highlight the fact that induction method can influence the precise program of viral gene expression during early events in reactivation and also suggest a mechanism by which RTA contributes to establishment of latency during de novo infections. PMID:15956592

  10. Viral load of equine herpesviruses 2 and 5 in nasal swabs of actively racing Standardbred trotters: Temporal relationship of shedding to clinical findings and poor performance.

    PubMed

    Back, Helena; Ullman, Karin; Treiberg Berndtsson, Louise; Riihimäki, Miia; Penell, Johanna; Ståhl, Karl; Valarcher, Jean-François; Pringle, John

    2015-09-30

    The equine gamma herpesviruses 2 and 5 (EHV-2 and -5) have frequently been observed in the equine population and until recently presumed low to nonpathogenic. However, recent reports linking presence of equine gamma herpesviruses with clinical signs of mild to severe lung disease, suggest that the role of these viruses in respiratory disease and poor performance syndrome is still unclear. Moreover, baseline data regarding the temporal pattern of shedding of EHV-2 and EHV-5 within stables and within individual actively racing horses have been lacking. In a prospective longitudinal study, we followed elite racing Standardbred trotters at monthly intervals for 13 months, to investigate whether the amount of EHV-2 and EHV-5 shedded in nasal secretions varied over time within and between individual horses. Sixty-six elite horses were investigated by analyzing nasal swabs and serum samples, a health check and evaluation of athletic performance monthly during the study period. Nasal swabs were analyzed with two newly developed qPCR assays for EHV-2 and EHV-5, respectively. Of 663 samples, 197 (30%) were positive for EHV-2 and 492 (74%) positive for EHV-5. Furthermore, 176 (27%) of the samples were positive for both EHV-2 and EHV-5 simultaneously. There was considerable variation in the amount and frequency of shedding of EHV-2 and EHV-5 within and between individual horses. Viral load varied seasonally, but neither EHV-2 nor EHV-5 viral peaks were associated with clinical respiratory disease and/or poor performance in racing Standardbred trotters.

  11. Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

    PubMed Central

    Chatterjee, Deboeeta; Chandran, Bala

    2012-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is etiologically associated with three neoplastic syndromes: Kaposi sarcoma and the uncommon HIV-associated B-cell lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. The incidence of the latter B-cell pathology has been increasing in spite of antiretroviral therapy; its association with lytic virus replication has prompted interest in therapeutic strategies aimed at this phase of the virus life cycle. We designed and expressed a recombinant immunotoxin (2014-PE38) targeting the gpK8.1A viral glycoprotein expressed on the surface of the virion and infected cells. We show that this immunotoxin selectively kills KSHV-infected cells in dose-dependent fashion, resulting in major reductions of infectious virus release. The immunotoxin and ganciclovir, an inhibitor of viral DNA replication, showed marked reciprocal potentiation of antiviral activities. These results suggest that the immunotoxin, alone or in combination, may represent a new approach to treat diseases associated with KSHV lytic replication. PMID:22377676

  12. Bovine viral diarrhoea, bovine herpesvirus and parainfluenza-3 virus infection in three cattle herds in Egypt in 2000.

    PubMed

    Aly, N M; Shehab, G G; Abd el-Rahim, I H A

    2003-12-01

    This study reported field outbreaks of bovine viral diarrhoea virus (BVDV) infection, either alone or mixed with bovine herpesvirus-1 (BHV-1) and/or parainfluenza-3 virus (PI-3V) in Egypt during 2000. In Lower Egypt, young calves in three cattle herds in El-Minufiya Province, El-Fayoum Province and in governmental quarantine in El-Behira Province, showed symptoms of enteritis, either alone or accompanied by respiratory manifestations. The affected herds were visited and the diseased animals were clinically examined. Many epidemiological aspects, such as morbidities, mortalities and case fatalities, as well as the abortive rate, were calculated. Ethylenediamine tetra-acetic acid-blood samples, sterile nasal swabs and serum samples were obtained for virological and serological diagnosis. The laboratory investigations revealed that the main cause of calf mortalities in the three herds was infection with BVDV, either alone, as on the El-Minufiya farm, or mixed with PI-3V, as on the El-Fayoum farm, or mixed with both BHV-1 and PI-3V, as in the herd in governmental quarantine in El-Behira Province. A total of nine dead calves from the three herds were submitted for thorough post-mortem examination. Tissue samples from recently dead calves were obtained for immunohistochemical and histopathological studies. The most prominent histopathological findings were massive degeneration, necrosis and erosions of the lining epithelium of the alimentary tract. Most of the lymphoreticular organs were depleted of lymphocytes. In pneumonic cases, bronchopneumonia and atypical interstitial pneumonia were evident. The present study suggested that the immunosuppressive effect of BVDV had predisposed the animals to secondary infection with BHV-1 and PI-3V. This study concluded that concurrent infection with BVDV, BHV-1 and PI-3V should be considered as one of the infectious causes of pneumoenteritis and, subsequently, the high morbidities and mortalities among young calves in Egypt

  13. Real-time PCR detection of Human Herpesvirus 1-5 in patients lacking clinical signs of a viral CNS infection

    PubMed Central

    2011-01-01

    Background Infections of the central nervous system (CNS) with herpes- or enterovirus can be self-limiting and benign, but occasionally result in severe and fatal disease. The polymerase chain reaction (PCR) has revolutionized the diagnostics of viral pathogens, and by multiple displacement amplification (MDA) prior to real-time PCR the sensitivity might be further enhanced. The aim of this study was to investigate if herpes- or enterovirus can be detected in cerebrospinal fluid (CSF) from patients without symptoms. Methods Cerebrospinal fluid (CSF) samples from 373 patients lacking typical symptoms of viral CNS infection were analysed by real-time PCR targeting herpesviruses or enteroviruses with or without prior MDA. Results In total, virus was detected in 17 patients (4%). Epstein-Barr virus (EBV) was most commonly detected, in general from patients with other conditions (e.g. infections, cerebral hemorrhage). MDA satisfactorily amplified viral DNA in the absence of human nucleic acids, but showed poor amplification capacity for viral DNA in CSF samples, and did not increase the sensitivity for herpes virus-detection with our methodology. Conclusions Viral pathogens are rarely detected in CSF from patients without signs of CNS infection, supporting the view that real-time PCR is a highly specific method to detect symptomatic CNS-infection caused by these viruses. However, EBV may be subclinically reactivated due to other pathological conditions in the CNS. PMID:21849074

  14. Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other.

    PubMed

    Said, Abdelrahman; Azab, Walid; Damiani, Armando; Osterrieder, Nikolaus

    2012-08-01

    Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.

  15. The Role of PI3K/Akt in Human Herpesvirus Infection: from the Bench to the Bedside

    PubMed Central

    Liu, XueQiao; Cohen, Jeffrey I.

    2015-01-01

    The phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway regulates several key cellular functions including protein synthesis, cell growth, glucose metabolism, and inflammation. Many viruses have evolved mechanisms to manipulate this signaling pathway to ensure successful virus replication. The human herpesviruses undergo both latent and lytic infection, but differ in cell tropism, growth kinetics, and disease manifestations. Herpesviruses express multiple proteins that target the PI3K/Akt cell signaling pathway during the course of their life cycle to facilitate viral infection, replication, latency, and reactivation. Rare human genetic disorders with mutations in either the catalytic or regulatory subunit of PI3K that result in constitutive activation of the protein predispose to severe herpesvirus infections as well as to virus-associated malignancies. Inhibiting the PI3K/Akt pathway or its downstream proteins using drugs already approved for other diseases can block herpesvirus lytic infection and may reduce malignancies associated with latent herpesvirus infections. PMID:25798530

  16. Kaposi's Sarcoma-associated herpesvirus lytic switch protein stimulates DNA binding of RBP-Jk/CSL to activate the Notch pathway.

    PubMed

    Carroll, Kyla Driscoll; Bu, Wei; Palmeri, Diana; Spadavecchia, Sophia; Lynch, Stephen J; Marras, Salvatore A E; Tyagi, Sanjay; Lukac, David M

    2006-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

  17. Glycoprotein C of equine herpesvirus 4 plays a role in viral binding to cell surface heparan sulfate.

    PubMed

    Azab, Walid; Tsujimura, Koji; Maeda, Ken; Kobayashi, Kyousuke; Mohamed, Yassir Mahgoub; Kato, Kentaro; Matsumura, Tomio; Akashi, Hiroomi

    2010-07-01

    Heparan sulfate moieties of cell surface proteoglycans serve as receptors for several herpesviruses. For herpes simplex virus 1, pseudorabies virus and equine herpesvirus 1, glycoprotein C (gC) homologues have been shown to mediate the binding to cell surface heparan sulfate. However, the role of gC in equine herpesvirus 4 (EHV-4) infection has not yet been analyzed. Using pull-down assay, we first determined that EHV-4 gC as well as gB are heparin-binding glycoproteins. To study the role of gC in EHV-4 infection, we constructed a gC-deletion mutant, WA79DeltagC, where the kanamycin resistant gene was inserted instead of the open reading frame encoding gC. We found that soluble heparin was capable of blocking both wild-type EHV-4 and WA79DeltagC infection of fetal horse kidney. Furthermore, pretreatment of cells with heparinase reduces considerably the ability of both viruses to adsorb to these cells and to form plaques. Similar results were obtained when cellular glycosaminoglycan synthesis was inhibited by chlorate treatment. In addition, we did find that gC protects EHV-4 from complement-mediated neutralization. These results suggest that, like other herpesviruses, EHV-4 gC plays a role in the interaction of the virus with cellular heparan sulfate. Moreover, gC can protect the virus from complement-mediated neutralization.

  18. Superresolution imaging of viral protein trafficking

    PubMed Central

    Salka, Kyle; Bhuvanendran, Shivaprasad; Yang, David

    2015-01-01

    The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses. PMID:25724304

  19. RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

    PubMed

    Batra, Ranjan; Stark, Thomas J; Clark, Elizabeth; Belzile, Jean-Philippe; Wheeler, Emily C; Yee, Brian A; Huang, Hui; Gelboin-Burkhart, Chelsea; Huelga, Stephanie C; Aigner, Stefan; Roberts, Brett T; Bos, Tomas J; Sathe, Shashank; Donohue, John Paul; Rigo, Frank; Ares, Manuel; Spector, Deborah H; Yeo, Gene W

    2016-12-01

    Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

  20. Detection of cyprinid herpesvirus 2 in peripheral blood cells of silver crucian carp, Carassius auratus gibelio (Bloch), suggests its potential in viral diagnosis.

    PubMed

    Wang, H; Xu, Lj; Lu, Lq

    2016-02-01

    Epidemics caused by cyprinid herpesvirus 2 (CyHV-2) in domestic cyprinid species have been reported in both European and Asian countries. Although the mechanisms remain unknown, acute CyHV-2 infections generally result in high mortality, and the surviving carps become chronic carriers displaying no external clinical signs. In this study, in situ hybridization analysis showed that CyHV-2 tended to infect peripheral blood cells during either acute or chronic infections in silver crucian carp, Carassius auratus gibelio (Bloch). Laboratory challenge experiments coupled with real-time PCR quantification assays further indicated that steady-state levels of the viral genomic copy number in fish serum exhibited a typical 'one-step' growth curve post-viral challenge. Transcriptional expression of open reading frames (ORF) 121, which was selected due to its highest transcriptional levels in almost all tested tissues, was monitored to represent the replication kinetics of CyHV-2 in peripheral blood cells. Similar kinetic curve of active viral gene transcription in blood cells was obtained as that of serum viral load, indicating that CyHV-2 replicated in peripheral blood cells as well as in other well-characterized tissues. This study should pave the way for designing non-invasive and cost-effective serum diagnostic methods for quick detection of CyHV-2 infection.

  1. The role of Kaposi sarcoma-associated herpesvirus in the pathogenesis of Kaposi sarcoma.

    PubMed

    Gramolelli, Silvia; Schulz, Thomas F

    2015-01-01

    Kaposi sarcoma (KS) is an unusual vascular tumour caused by an oncogenic-herpesvirus, Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV 8). KS lesions are characterized by an abundant inflammatory infiltrate, the presence of KSHV-infected endothelial cells that show signs of aberrant differentiation, as well as faulty angiogenesis/ vascularization. Here we discuss the molecular mechanisms that lead to the development of these histological features of KS, with an emphasis on the viral proteins that are responsible for their development.

  2. Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California.

    PubMed

    Pusterla, Nicola; Wilson, W David; Mapes, Samantha; Finno, Carrie; Isbell, Diane; Arthur, Rick M; Ferraro, Gregory L

    2009-02-01

    The objective of this study was to determine viral loads, strain (neuropathogenic versus non-neuropathogenic) and state (lytic, non-replicating, latent) of equine herpesvirus-1 (EHV-1) by real-time polymerase chain reaction (PCR) in the blood and nasopharyngeal secretions of adult horses following natural exposure. The index case, a 4-year-old Thoroughbred gelding with confirmed EHV-1 myeloencephalopathy, as well as potentially exposed horses, were sampled over a period of 3 weeks. The study population comprised of 39 adult Thoroughbred horses and 35 adult "pony" and outrider horses of various breeds housed at a racetrack in Northern California. Blood samples and nasopharyngeal secretions (NPS) from all horses were tested on several occasions for EHV-1 DNA viral loads, targeting the glycoprotein B (gB) gene, viral strain, targeting the ORF 30 gene, and transcriptional activity of EHV-1, targeting the gB gene and latency-associated transcripts (LATs). Viral loads and transcriptional activity of the gB gene declined rapidly in the index case following antiviral treatment. The prevalence of EHV-1 infection in NPS determined by PCR slowly decreased over the 22 day study period from 25% to 14%. The initial surveillance showed multiple clusters of exposure, one associated with the index case and two related to horses that had recently returned from a different racetrack. Viral strain differentiation showed that only two horses (the index case and a neighboring horse) were infected with only a neuropathogenic strain, while all other horses were infected with either a non-neuropathogenic strain or were dually infected with both neuropathogenic and non-neuropathogenic strains. In most cases, the virus was present in either a lytic or a non-replicating form, while latent virus was found in blood and NPS much less frequently. The molecular approach used in this study showed promise for assessing the risk of exposing other horses to EHV-1 and for studying viral kinetics in

  3. Complex alternative cytoplasmic protein isoforms of the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 generated through noncanonical translation initiation.

    PubMed

    Toptan, Tuna; Fonseca, Lidia; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2013-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) protein is constitutively expressed in all KSHV-infected cells, as well as in all forms of KSHV-associated malignancies. LANA1 is a multifunctional KSHV oncoprotein containing multiple repeat sequences that is important for viral episome maintenance and the regulation of cellular and viral gene expression. We characterize here multiple LANA1 isoforms and show that ∼50% of LANA1 is naturally generated as N-terminally truncated shoulder proteins that are detected on SDS-PAGE as faster-migrating shoulder bands designated LANA1(S). Higher-molecular-weight LANA1(S) isoforms initiate downstream at noncanonical sites within the N-terminal region, whereas lower-molecular-weight LANA1(S) isoforms initiate downstream within the central repeat 1 domain. LANA1(S) proteins lack an N-terminal nuclear localization signal motif, and some isoforms differ from full-length, canonical LANA1 by localizing to perinuclear and cytoplasmic sites. Although LANA1 has until now been assumed to be solely active in the nucleus, this finding indicates that this major KSHV oncoprotein may have cytoplasmic activities as well. KSHV overcomes its limited genetic coding capacity by generating alternatively initiated protein isoforms that may have distinct biological functions.

  4. Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome.

    PubMed

    Huang, Yan; Hidalgo-Bravo, Alberto; Zhang, Enjie; Cotton, Victoria E; Mendez-Bermudez, Aaron; Wig, Gunjan; Medina-Calzada, Zahara; Neumann, Rita; Jeffreys, Alec J; Winney, Bruce; Wilson, James F; Clark, Duncan A; Dyer, Martin J; Royle, Nicola J

    2014-01-01

    Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.

  5. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    PubMed Central

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-01-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA. PMID:27599637

  6. The equine herpesvirus 1 gene 63 RING finger protein partially complements Vmw110, its herpes simplex virus type 1 counterpart.

    PubMed

    Everett, R; Orr, A; Elliott, M

    1995-09-01

    All alpha herpesviruses of known DNA sequence have been found to encode a protein with similarities to immediate early protein Vmw110 (ICP0) of herpes simplex virus type 1 (HSV-1). The conserved portion of this family of proteins is a characteristic zinc binding module, known as a RING finger or C3HC4 domain. Examples of RING finger domains occur in many other proteins of diverse evolutionary origin and function. Recently, the solution structure of the equine herpesvirus 1 (EHV-1) RING finger protein, encoded by gene 63, has been solved. To investigate whether this structure could be considered to be a paradigm of herpesvirus RING domains, we have constructed a recombinant HSV-1 which expresses the EHV-1 gene 63 protein (EHVg63) in place of Vmw110. Comparison of the growth properties of the recombinant with those of wild-type and Vmw110-defective viruses indicates that EHVg63 is able to fulfil partially, but not completely, the roles of Vmw110 during virus growth in tissue culture.

  7. VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling

    PubMed Central

    Afroz, Sharmin; Brownlie, Robert; Fodje, Michel

    2016-01-01

    ABSTRACT The UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo. In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication

  8. Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis▿

    PubMed Central

    Ward, Katherine N.; Leong, Hoe Nam; Thiruchelvam, Anton D.; Atkinson, Claire E.; Clark, Duncan A.

    2007-01-01

    The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log10 copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log10 copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log10 copies/ml) or whole blood (≥6.0 log10 copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log10 copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log10 copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood. PMID:17229866

  9. Prevalence of exposure to bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) in Irish dairy herds.

    PubMed

    Sayers, R G; Byrne, N; O'Doherty, E; Arkins, S

    2015-06-01

    Bovine viral diarrhoea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) are contagious bovine viral agents. The objectives of this study were to use quarterly bulk milk and 'spot' testing of unvaccinated youngstock to establish the national prevalence of exposure to BVDV and/or BoHV-1 in Irish dairy herds. Seasonality of bulk milk ELISA results was also examined. From a geographically representative population of 305 dairy herds, 88% and 80% of herds yielded mean annual positive bulk milk readings for BVDV and BoHV-1, respectively. Of these, 61% were vaccinated against BVDV and 12% against BoHV-1. A total of 2171 serum samples from weanlings having a mean age of 291 days yielded 543 (25%) seropositive for BVDV, and 117 (5.4%) seropositive for BoHV-1. A significant seasonal trend in bulk milk antibody ELISA readings and herd status was recorded for BVDV, with more herds categorised as positive in the latter half of the year.

  10. Gene for the major antigenic structural protein (p100) of human herpesvirus 6.

    PubMed Central

    Neipel, F; Ellinger, K; Fleckenstein, B

    1992-01-01

    A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions. The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera. The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa. Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus. Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed. The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera. They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus. Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6. Images PMID:1374813

  11. Molecular piracy of chemokine receptors by herpesviruses.

    PubMed

    Murphy, P M

    1994-01-01

    To succeed as a biological entity, viruses must exploit normal cellular functions and elude the host immune system; they often do so by molecular mimicry. One way that mimicry may occur is when viruses copy and modify host genes. The best studied examples of this are the oncogenes of RNA retroviruses, but a growing number of examples are also known for DNA viruses. So far they all come from just two groups of DNA viruses, the herpesviruses and poxviruses, and the majority of examples are for genes whose products regulate immune responses, such as cytokines, cytokine receptors, and complement control proteins. This review will focus on human and herpesvirus receptors for chemokines, a family of leukocyte chemoattractant and activating factors that are thought to be important mediators of inflammation. Although the biological roles of the viral chemokine receptor homologues are currently unknown, their connection to specific sets of chemokines has suggested a number of possible functions.

  12. Herpesvirus BACs: past, present, and future.

    PubMed

    Warden, Charles; Tang, Qiyi; Zhu, Hua

    2011-01-01

    The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

  13. Experimental infection of European flat oyster Ostrea edulis with ostreid herpesvirus 1 microvar (OsHV-1μvar): Mortality, viral load and detection of viral transcripts by in situ hybridization.

    PubMed

    López Sanmartín, Monserrat; Power, Deborah M; de la Herrán, Roberto; Navas, José I; Batista, Frederico M

    2016-06-02

    Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called μvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1μvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1μvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1μvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1μvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals' using in situ hybridization.

  14. Two Subclasses of Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle Promoters Distinguished by Open Reading Frame 50 Mutant Proteins That Are Deficient in Binding to DNA

    PubMed Central

    Chang, Pey-Jium; Shedd, Duane; Miller, George

    2005-01-01

    A transcriptional activator encoded in open reading frame 50 (ORF50) of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome initiates the viral lytic cycle. Here we classify four lytic cycle genes on the basis of several characteristics of the ORF50 response elements (ORF50 REs) in their promoters: nucleotide sequence homology, the capacity to bind ORF50 protein in vitro, the ability to bind the cellular protein RBP-Jκ in vitro, and the capacity to confer activation by DNA binding-deficient mutants of ORF50 protein. ORF50 expressed in human cells binds the promoters of PAN and K12 but does not bind ORF57 or vMIP-1 promoters. Conversely, the RBP-Jκ protein binds ORF57 and vMIP-1 but not PAN or K12 promoters. DNA binding-deficient mutants of ORF50 protein differentiate these two subclasses of promoters in reporter assays; the PAN and K12 promoters cannot be activated, while the ORF57 and vMIP-1 promoters are responsive. Although DNA binding-deficient mutants of ORF50 protein are defective in activating direct targets, they are nonetheless capable of activating the lytic cascade of KSHV. Significantly, DNA binding-deficient ORF50 mutants are competent to autostimulate expression of endogenous ORF50 and to autoactivate ORF50 promoter reporters. The experiments show that ORF50 protein activates downstream targets by at least two distinct mechanisms: one involves direct binding of ORF50 REs in promoter DNA; the other mechanism employs interactions with the RBP-Jκ cellular protein bound to promoter DNA in the region of the ORF50 RE. The DNA binding-deficient mutants allow classification of ORF50-responsive genes and will facilitate study of the several distinct mechanisms of activation of KSHV lytic cycle genes that are under the control of ORF50 protein. PMID:15994769

  15. Two subclasses of Kaposi's sarcoma-associated herpesvirus lytic cycle promoters distinguished by open reading frame 50 mutant proteins that are deficient in binding to DNA.

    PubMed

    Chang, Pey-Jium; Shedd, Duane; Miller, George

    2005-07-01

    A transcriptional activator encoded in open reading frame 50 (ORF50) of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome initiates the viral lytic cycle. Here we classify four lytic cycle genes on the basis of several characteristics of the ORF50 response elements (ORF50 REs) in their promoters: nucleotide sequence homology, the capacity to bind ORF50 protein in vitro, the ability to bind the cellular protein RBP-Jkappa in vitro, and the capacity to confer activation by DNA binding-deficient mutants of ORF50 protein. ORF50 expressed in human cells binds the promoters of PAN and K12 but does not bind ORF57 or vMIP-1 promoters. Conversely, the RBP-Jkappa protein binds ORF57 and vMIP-1 but not PAN or K12 promoters. DNA binding-deficient mutants of ORF50 protein differentiate these two subclasses of promoters in reporter assays; the PAN and K12 promoters cannot be activated, while the ORF57 and vMIP-1 promoters are responsive. Although DNA binding-deficient mutants of ORF50 protein are defective in activating direct targets, they are nonetheless capable of activating the lytic cascade of KSHV. Significantly, DNA binding-deficient ORF50 mutants are competent to autostimulate expression of endogenous ORF50 and to autoactivate ORF50 promoter reporters. The experiments show that ORF50 protein activates downstream targets by at least two distinct mechanisms: one involves direct binding of ORF50 REs in promoter DNA; the other mechanism employs interactions with the RBP-Jkappa cellular protein bound to promoter DNA in the region of the ORF50 RE. The DNA binding-deficient mutants allow classification of ORF50-responsive genes and will facilitate study of the several distinct mechanisms of activation of KSHV lytic cycle genes that are under the control of ORF50 protein.

  16. A neurotropic herpesvirus infecting the gastropod, abalone, shares ancestry with oyster herpesvirus and a herpesvirus associated with the amphioxus genome

    PubMed Central

    2010-01-01

    Background With the exception of the oyster herpesvirus OsHV-1, all herpesviruses characterized thus far infect only vertebrates. Some cause neurological disease in their hosts, while others replicate or become latent in neurological tissues. Recently a new herpesvirus causing ganglioneuritis in abalone, a gastropod, was discovered. Molecular analysis of new herpesviruses, such as this one and others, still to be discovered in invertebrates, will provide insight into the evolution of herpesviruses. Results We sequenced the genome of a neurotropic virus linked to a fatal ganglioneuritis devastating parts of a valuable wild abalone fishery in Australia. We show that the newly identified virus forms part of an ancient clade with its nearest relatives being a herpesvirus infecting bivalves (oyster) and, unexpectedly, one we identified, from published data, apparently integrated within the genome of amphioxus, an invertebrate chordate. Predicted protein sequences from the abalone virus genome have significant similarity to several herpesvirus proteins including the DNA packaging ATPase subunit of (putative) terminase and DNA polymerase. Conservation of amino acid sequences in the terminase across all herpesviruses and phylogenetic analysis using the DNA polymerase and terminase proteins demonstrate that the herpesviruses infecting the molluscs, oyster and abalone, are distantly related. The terminase and polymerase protein sequences from the putative amphioxus herpesvirus share more sequence similarity with those of the mollusc viruses than with sequences from any of the vertebrate herpesviruses analysed. Conclusions A family of mollusc herpesviruses, Malacoherpesviridae, that was based on a single virus infecting oyster can now be further established by including a distantly related herpesvirus infecting abalone, which, like many vertebrate viruses is neurotropic. The genome of Branchiostoma floridae (amphioxus) provides evidence for the existence of a herpesvirus

  17. Structural framework for DNA translocation via the viral portal protein

    PubMed Central

    Lebedev, Andrey A; Krause, Margret H; Isidro, Anabela L; Vagin, Alexei A; Orlova, Elena V; Turner, Joanne; Dodson, Eleanor J; Tavares, Paulo; Antson, Alfred A

    2007-01-01

    Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave' of positional and conformational changes propagating sequentially along this belt. PMID:17363899

  18. Illuminating structural proteins in viral "dark matter" with metaproteomics.

    PubMed

    Brum, Jennifer R; Ignacio-Espinoza, J Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M; Roux, Simon; VerBerkmoes, Nathan C; Rich, Virginia I; Sullivan, Matthew B

    2016-03-01

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.

  19. ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus.

    PubMed

    Kumar, Binod; Dutta, Dipanjan; Iqbal, Jawed; Ansari, Mairaj Ahmed; Roy, Arunava; Chikoti, Leela; Pisano, Gina; Veettil, Mohanan Valiya; Chandran, Bala

    2016-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and-III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of

  20. ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Kumar, Binod; Dutta, Dipanjan; Iqbal, Jawed; Ansari, Mairaj Ahmed; Roy, Arunava; Chikoti, Leela; Pisano, Gina; Veettil, Mohanan Valiya; Chandran, Bala

    2016-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of

  1. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  2. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  3. RNA-protein interaction methods to study viral IRES elements.

    PubMed

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Lozano, Gloria; Diaz-Toledano, Rosa; Martínez-Salas, Encarnación

    2015-12-01

    Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA-protein interactions on IRES activity.

  4. Piracy on the molecular level: human herpesviruses manipulate cellular chemotaxis.

    PubMed

    Cornaby, Caleb; Tanner, Anne; Stutz, Eric W; Poole, Brian D; Berges, Bradford K

    2016-03-01

    Cellular chemotaxis is important to tissue homeostasis and proper development. Human herpesvirus species influence cellular chemotaxis by regulating cellular chemokines and chemokine receptors. Herpesviruses also express various viral chemokines and chemokine receptors during infection. These changes to chemokine concentrations and receptor availability assist in the pathogenesis of herpesviruses and contribute to a variety of diseases and malignancies. By interfering with the positioning of host cells during herpesvirus infection, viral spread is assisted, latency can be established and the immune system is prevented from eradicating viral infection.

  5. Structural Proteomics of Herpesviruses

    PubMed Central

    Leroy, Baptiste; Gillet, Laurent; Vanderplasschen, Alain; Wattiez, Ruddy

    2016-01-01

    Herpesviruses are highly prevalent viruses associated with numerous pathologies both in animal and human populations. Until now, most of the strategies used to prevent or to cure these infections have been unsuccessful because these viruses have developed numerous immune evasion mechanisms. Therefore, a better understanding of their complex lifecycle is needed. In particular, while the genome of numerous herpesviruses has been sequenced, the exact composition of virions remains unknown for most of them. Mass spectrometry has recently emerged as a central method and has permitted fundamental discoveries in virology. Here, we review mass spectrometry-based approaches that have recently allowed a better understanding of the composition of the herpesvirus virion. In particular, we describe strategies commonly used for proper sample preparation and fractionation to allow protein localization inside the particle but also to avoid contamination by nonstructural proteins. A collection of other important data regarding post-translational modifications or the relative abundance of structural proteins is also described. This review also discusses the poorly studied importance of host proteins in herpesvirus structural proteins and the necessity to develop a quantitative workflow to better understand the dynamics of the structural proteome. In the future, we hope that this collaborative effort will assist in the development of new strategies to fight these infections. PMID:26907323

  6. Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59.

    PubMed

    Majerciak, Vladimir; Uranishi, Hiroaki; Kruhlak, Michael; Pilkington, Guy R; Massimelli, Maria Julia; Bear, Jenifer; Pavlakis, George N; Felber, Barbara K; Zheng, Zhi-Ming

    2011-02-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

  7. Full trans-activation mediated by the immediate-early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence.

    PubMed

    Kim, Seong K; Shakya, Akhalesh K; O'Callaghan, Dennis J

    2016-01-04

    The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt -89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS).

  8. Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene.

    PubMed

    Lee, Hye-Ra; Mitra, Jaba; Lee, Stacy; Gao, Shou-Jiang; Oh, Tae-Kwang; Kim, Myung Hee; Ha, Taekjip; Jung, Jae U

    2015-10-21

    Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.

  9. Assessing ubiquitination of viral proteins: lessons from flavivirus NS5

    PubMed Central

    Taylor, R. Travis; Best, Sonja M.

    2011-01-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains. PMID:21855635

  10. Assessing ubiquitination of viral proteins: Lessons from flavivirus NS5.

    PubMed

    Taylor, R Travis; Best, Sonja M

    2011-10-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains.

  11. Molecular piracy: manipulation of the ubiquitin system by Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Fujimuro, Masahiro; Hayward, S Diane; Yokosawa, Hideyoshi

    2007-01-01

    Ubiquitination, one of several post-translational protein modifications, plays a key role in the regulation of cellular events, including protein degradation, signal transduction, endocytosis, protein trafficking, apoptosis and immune responses. Ubiquitin attachment at the lysine residue of cellular factors acts as a signal for endocytosis and rapid degradation by the 26S proteasome. It has recently been observed that viruses, especially oncogenic herpesviruses, utilise molecular piracy by encoding their own proteins to interfere with regulation of cell signalling. Kaposi's sarcoma- associated herpesvirus (KSHV) manipulates the ubiquitin system to facilitate cell proliferation, anti-apoptosis and evasion from immunity. In this review, we will describe the strategies used by KSHV at distinct stages of the viral life-cycle to control the ubiquitin system and promote oncogenesis and viral persistence.

  12. Potential Role for a β-Catenin Coactivator (High-Mobility Group AT-Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1.

    PubMed

    Zhu, Liqian; Workman, Aspen; Jones, Clinton

    2017-03-01

    The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/β-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148-3159, 2016). In the present study, a β-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and β-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing β-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and β-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate β-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing β-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate β-catenin-dependent transcription. Since the canonical Wnt/β-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and β-catenin may be important for certain aspects of the latency-reactivation cycle.IMPORTANCE The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant

  13. Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Chen, Wuguo; Dittmer, Dirk P.

    2011-01-01

    The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA. PMID:21734034

  14. Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle

    PubMed Central

    Loureiro, Maria Eugenia; D’Antuono, Alejandra; Levingston Macleod, Jesica M.; López, Nora

    2012-01-01

    The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein) and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article. PMID:23170177

  15. Have NEC Coat, Will Travel: Structural Basis of Membrane Budding During Nuclear Egress in Herpesviruses.

    PubMed

    Bigalke, J M; Heldwein, E E

    2017-01-01

    Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. Furthermore, unlike other DNA viruses that replicate in the nucleus, herpesviruses do not exit it by passing through the nuclear pores or by rupturing the nuclear envelope. Instead, herpesviruses have a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. This makes them some of the very few known viruses that bud into the nuclear envelope. The envelope acquired during nuclear budding does not end up in the mature viral particle but instead allows the capsid to translocate from the nucleus into the cytosol. The viral nuclear egress complex (NEC) is a critical player in the nuclear egress, yet its function and mechanism have remained enigmatic. Recent studies have demonstrated that the NEC buds membranes without the help of other proteins by forming a honeycomb coat, which established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane. This review discusses our current understanding of the NEC budding mechanism, with the emphasis on studies that illuminated the structure of the NEC coat and its role in capsid budding during herpesvirus nuclear escape.

  16. Have NEC Coat, Will Travel: Structural Basis of Membrane Budding During Nuclear Egress in Herpesviruses

    PubMed Central

    Bigalke, J.M.; Heldwein, E.E.

    2017-01-01

    Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. Furthermore, unlike other DNA viruses that replicate in the nucleus, herpesviruses do not exit it by passing through the nuclear pores or by rupturing the nuclear envelope. Instead, herpesviruses have a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. This makes them some of the very few known viruses that bud into the nuclear envelope. The envelope acquired during nuclear budding does not end up in the mature viral particle but instead allows the capsid to translocate from the nucleus into the cytosol. The viral nuclear egress complex (NEC) is a critical player in the nuclear egress, yet its function and mechanism have remained enigmatic. Recent studies have demonstrated that the NEC buds membranes without the help of other proteins by forming a honeycomb coat, which established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane. This review discusses our current understanding of the NEC budding mechanism, with the emphasis on studies that illuminated the structure of the NEC coat and its role in capsid budding during herpesvirus nuclear escape. PMID:28057257

  17. Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus.

    PubMed Central

    Smith, R F; Smith, T F

    1989-01-01

    By using amino acid sequence patterns (motifs) diagnostic of conserved regions within the catalytic domains of protein kinases, homologous open reading frames of three herpesviruses were identified as protein kinase-related genes. The three sequences, herpes simplex virus gene UL13, varicella-zoster virus gene 47, and Epstein-Barr virus gene BGLF4, resemble serine/threonine kinases rather than tyrosine kinases. PMID:2535748

  18. Controlled Assembly of Viral Surface Proteins into Biological Nanoparticles

    NASA Astrophysics Data System (ADS)

    Nakatani-Webster, Eri

    In recent years, therapeutic use of engineered particles on the 1-1,000 nm scale has gained popularity; these nanoparticles have been developed for use in drug delivery, gene therapy, vaccine preparation, and diagnostics. Often, viral proteins are utilized in the design of such species, and outlined here are completed studies on the in vitro assembly of nanoparticles derived from two very different viral systems. The incorporation of the human immunodeficiency virus (HIV) envelope glycoprotein precursor gp160 into phospholipid bilayer nanodiscs is discussed as a potential platform for vaccine design; efforts were successful, however yield currently limits the practical application of this approach. The utility of bacteriophage lambda procapsids and virus-like particles in therapeutic nanoparticle design is also outlined, as are efforts toward the structural and thermodynamic characterization of a urea-triggered capsid maturation event. It is demonstrated that lambda virus-like particles can be assembled from purified capsid and scaffolding proteins, and that these particles undergo urea-triggered maturation and in vitro decoration protein addition similar to that seen in lambda procapsids. The studies on lambda provided materials for the further development of nanoparticles potentially useful in a clinical setting, as well as shedding light on critical viral assembly and maturation events as they may take place in vivo.

  19. Molecular and virological evidence of viral activation from chromosomally integrated human herpesvirus 6A in a patient with X-linked severe combined immunodeficiency.

    PubMed

    Endo, Akifumi; Watanabe, Ken; Ohye, Tamae; Suzuki, Kyoko; Matsubara, Tomoyo; Shimizu, Norio; Kurahashi, Hiroki; Yoshikawa, Tetsushi; Katano, Harutaka; Inoue, Naoki; Imai, Kohsuke; Takagi, Masatoshi; Morio, Tomohiro; Mizutani, Shuki

    2014-08-15

    It has been unclear whether chromosomally integrated human herpesvirus 6 (ciHHV-6) can be activated with pathogenic effects on the human body. We present molecular and virological evidence of ciHHV-6A activation in a patient with X-linked severe combined immunodeficiency. These findings have significant implications for the management of patients with ciHHV-6.

  20. Ovine herpesvirus 2 glycoproteins B, H, and L are required for cell-to-cell membrane fusion and viral glycoprotein Ov8 significantly enhances membrane fusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no...

  1. Cementing proteins provide extra mechanical stabilization to viral cages

    NASA Astrophysics Data System (ADS)

    Hernando-Pérez, M.; Lambert, S.; Nakatani-Webster, E.; Catalano, C. E.; de Pablo, P. J.

    2014-07-01

    The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of ‘decoration’ proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann’s constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment.

  2. In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas.

    PubMed

    Martenot, Claire; Segarra, Amélie; Baillon, Laury; Faury, Nicole; Houssin, Maryline; Renault, Tristan

    2016-05-01

    Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection. Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the

  3. Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Lee, H; Guo, J; Li, M; Choi, J K; DeMaria, M; Rosenzweig, M; Jung, J U

    1998-09-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

  4. Characterization of the bipartite nuclear localization signal of protein LANA2 from Kaposi's sarcoma-associated herpesvirus.

    PubMed Central

    Muñoz-Fontela, Cesar; Rodríguez, Estefanía; Nombela, Cesar; Arroyo, Javier; Rivas, Carmen

    2003-01-01

    LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355-440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity. PMID:12767255

  5. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  6. Kaposi's sarcoma-associated herpesvirus-G protein-coupled receptor-expressing endothelial cells exhibit reduced migration and stimulated chemotaxis by chemokine inverse agonists.

    PubMed

    Couty, Jean-Pierre; Lupu-Meiri, Monica; Oron, Yoram; Gershengorn, Marvin C

    2009-06-01

    A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposi's sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon gamma-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.

  7. Low prevalence of human herpesvirus-6 and varicella zoster virus in blood of multiple sclerosis patients, irrespective of inflammatory status or disease progression.

    PubMed

    Hon, Gloudina M; Erasmus, Rajiv T; Matsha, Tandi

    2014-08-01

    Herpesviruses, including human herpesvirus-6 and varicella zoster virus, have been implicated in the disease aetiology of multiple sclerosis. These viruses are capable of reactivation, reminiscent of the relapsing-remitting nature of multiple sclerosis. However, viral DNA has also been reported present in healthy controls, often at similar prevalence rates. This study aimed to determine whether prevalence could be associated with different stages of activity of the disease as well as the inflammatory status of the patients. Polymerase chain reaction assays were used to screen for human herpesvirus-6 and varicella zoster virus DNA in blood from 31 Caucasian patients with multiple sclerosis and 30 healthy age, sex and race matched control subjects. The patients were screened for inflammation using C-reactive protein as a marker and were also categorized according to their remitting/relapsing status. Results were positive for human herpesvirus-6 in blood from only one patient (3.2%) and human herpesvirus-6 DNA was not present in any control subjects. Varicella zoster virus was not detected in either the patients or control subjects. Similar to some other studies we saw an absence or very low viral positivity in blood from both patients and controls. These findings were irrespective of relapse episodes, increased inflammatory status or duration of the disease. Results therefore do not support a causative role for either human herpesvirus-6 or varicella zoster virus in the disease aetiology of multiple sclerosis, but rather that prevalence in patients may be linked to that of the general population.

  8. Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression

    PubMed Central

    Gjyshi, Olsi; Roy, Arunava; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan

    2015-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines

  9. Detection of a Gene Cluster That Is Dispensable for Human Herpesvirus 6 Replication and Latency

    PubMed Central

    Kondo, Kazuhiro; Nozaki, Hideo; Shimada, Kazuya; Yamanishi, Koichi

    2003-01-01

    The U3-U7 gene cluster of human herpesvirus 6 (HHV-6) was replaced with an enhanced green fluorescent protein-puromycin gene cassette containing the cytomegalovirus major immediate-early promoter. Neither viral replication in T cells nor latency and reactivation in macrophages was impaired. During HHV-6 latency, the cytomegalovirus promoter used the transcription start sites employed in cytomegalovirus latency. PMID:12970461

  10. Assembly of multilayer films incorporating a viral protein cage architecture.

    PubMed

    Suci, Peter A; Klem, Michael T; Arce, Fernando T; Douglas, Trevor; Young, Mark

    2006-10-10

    Protein cage architectures such as viral capsids, heat shock proteins, ferritins, and DNA-binding proteins are nanoscale modular subunits that can be used to expand the structural and functional range of composite materials. Here, layer-by-layer (LbL) assembly was used to incorporate cowpea chlorotic mottle virus (CCMV) into multilayer films. Three types of multilayer films were prepared. In the first type, ionic interactions were employed to assemble CCMV into triple layers. In the second type, complementary biological interactions (streptavidin/biotin) were used for this purpose. In a third variation of LbL assembly, complementary biological interactions were employed to produce nanotextured films that exhibit in-plane order over a micron scale without the need to adsorb onto a prepatterned template.

  11. Understanding Viral Transmission Behavior via Protein Intrinsic Disorder Prediction: Coronaviruses

    PubMed Central

    Goh, Gerard Kian-Meng; Dunker, A. Keith; Uversky, Vladimir N.

    2012-01-01

    Besides being a common threat to farm animals and poultry, coronavirus (CoV) was responsible for the human severe acute respiratory syndrome (SARS) epidemic in 2002–4. However, many aspects of CoV behavior, including modes of its transmission, are yet to be fully understood. We show that the amount and the peculiarities of distribution of the protein intrinsic disorder in the viral shell can be used for the efficient analysis of the behavior and transmission modes of CoV. The proposed model allows categorization of the various CoVs by the peculiarities of disorder distribution in their membrane (M) and nucleocapsid (N). This categorization enables quick identification of viruses with similar behaviors in transmission, regardless of genetic proximity. Based on this analysis, an empirical model for predicting the viral transmission behavior is developed. This model is able to explain some behavioral aspects of important coronaviruses that previously were not fully understood. The new predictor can be a useful tool for better epidemiological, clinical, and structural understanding of behavior of both newly emerging viruses and viruses that have been known for a long time. A potentially new vaccine strategy could involve searches for viral strains that are characterized by the evolutionary misfit between the peculiarities of the disorder distribution in their shells and their behavior. PMID:23097708

  12. Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Wilson, Stuart A.; Kalra, Priti; Golovanov, Alexander P.

    2014-01-01

    The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions. PMID:24550725

  13. Isolation and characterization of a herpesvirus from feral pigeons in China.

    PubMed

    Zhao, Panpan; Ma, Jian; Guo, Ying; Tian, Li; Guo, Guangyang; Zhang, Kexin; Xing, Mingwei

    2015-12-01

    A herpesvirus was isolated during a diagnostic investigation of severe cases of conjunctivitis in feral pigeons (Columba livia f. domestica). Isolates of the virus were recovered from throat swabs of the pigeons followed by inoculation of the swab samples in chicken embryo fibroblasts. Pigeons inoculated with the isolated virus had similar clinical signs to those observed in naturally infected birds. Transmission electron microscopy revealed viral structures with typical herpesvirus morphology. Polymerase chain reaction amplification, using herpesvirus-identifying primers resulted in an amplicon of the expected size for herpesvirus. Sequencing of these amplicons and database comparisons identified the herpesvirus UL30 homologue. Phylogenetic reconstructions suggested that the isolated herpesvirus belongs to the Mardivirus genus of Alphaherpesvirinae. Using the current herpesvirus nomenclature conventions, the authors propose that the herpesvirus be named Columbid herpesvirus-1 Heilongjiang.

  14. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

  15. The Pacific Ocean virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology.

    PubMed

    Hurwitz, Bonnie L; Sullivan, Matthew B

    2013-01-01

    Bacteria and their viruses (phage) are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90%) of viral sequences are functionally 'unknown' making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased (i) from deep to surface waters, (ii) from winter to summer, (iii) and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown.

  16. Toll-like receptor sensing of human herpesvirus infection

    PubMed Central

    West, John A.; Gregory, Sean M.; Damania, Blossom

    2012-01-01

    Toll-like receptors (TLRs) are evolutionarily conserved pathogen sensors that constitute the first line of defense in the human immune system. Herpesviruses are prevalent throughout the world and cause significant disease in the human population. Sensing of herpesviruses via TLRs has only been documented in the last 10 years and our understanding of the relationship between these sentinels of the immune system and herpesvirus infection has already provided great insight into how the host cell responds to viral infection. This report will summarize the activation and modulation of TLR signaling in the context of human herpesvirus infections. PMID:23061052

  17. Bovine herpesvirus tegument protein VP22 enhances thymidine kinase/ganciclovir suicide gene therapy for neuroblastomas compared to herpes simplex virus VP22.

    PubMed

    Qiu, Zhaohua; Harms, Jerome S; Zhu, Jun; Splitter, Gary A

    2004-04-01

    Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.

  18. Anguillid Herpesvirus 1 Transcriptome

    PubMed Central

    van Beurden, Steven J.; Gatherer, Derek; Kerr, Karen; Galbraith, Julie; Herzyk, Pawel; Peeters, Ben P. H.; Rottier, Peter J. M.; Engelsma, Marc Y.

    2012-01-01

    We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5′ untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5′ and 3′ ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5′ untranslated exons or, because of alternative splicing, 5′ untranslated and 5′ translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome. PMID:22787220

  19. A codon-shuffling method to prevent reversion during production of replication-defective herpesvirus stocks: Implications for herpesvirus vaccines.

    PubMed

    Li, Gang; Ward, Charles; Yeasmin, Rukhsana; Skiena, Steven; Krug, Laurie T; Forrest, J Craig

    2017-03-13

    Herpesviruses establish life-long chronic infections that place infected hosts at risk for severe disease. Herpesvirus genomes readily undergo homologous recombination (HR) during productive replication, often leading to wild-type (WT) reversion during complementation of replication-defective and attenuated viruses via HR with the helper gene provided in trans. To overcome this barrier, we developed a synthetic-biology approach based on a technique known as codon shuffling. Computer-assisted algorithms redistribute codons in a helper gene, thereby eliminating regions of homology, while enabling manipulation of factors such as codon-pair bias and CpG content to effectively titrate helper-gene protein levels. We apply this technique to rescue the replication of a murine gammaherpesvirus engineered with a mutation in the major immediate-early transactivator protein RTA. Complementation with codon-shuffled RTA constructs did not yield any WT revertant virus, a sharp contrast to WT virus contamination frequently observed during complementation with an unmodified helper gene. We further demonstrate the importance of eliminating WT virus contamination in an animal model of gammaherpesvirus lethality. We propose complementation by codon shuffling as a means to produce replication-defective or attenuated viruses. This method has immediate utility for investigating roles of essential genes in viral replication and will better enable future development of herpesvirus vaccines.

  20. A codon-shuffling method to prevent reversion during production of replication-defective herpesvirus stocks: Implications for herpesvirus vaccines

    PubMed Central

    Li, Gang; Ward, Charles; Yeasmin, Rukhsana; Skiena, Steven; Krug, Laurie T.; Forrest, J. Craig

    2017-01-01

    Herpesviruses establish life-long chronic infections that place infected hosts at risk for severe disease. Herpesvirus genomes readily undergo homologous recombination (HR) during productive replication, often leading to wild-type (WT) reversion during complementation of replication-defective and attenuated viruses via HR with the helper gene provided in trans. To overcome this barrier, we developed a synthetic-biology approach based on a technique known as codon shuffling. Computer-assisted algorithms redistribute codons in a helper gene, thereby eliminating regions of homology, while enabling manipulation of factors such as codon-pair bias and CpG content to effectively titrate helper-gene protein levels. We apply this technique to rescue the replication of a murine gammaherpesvirus engineered with a mutation in the major immediate-early transactivator protein RTA. Complementation with codon-shuffled RTA constructs did not yield any WT revertant virus, a sharp contrast to WT virus contamination frequently observed during complementation with an unmodified helper gene. We further demonstrate the importance of eliminating WT virus contamination in an animal model of gammaherpesvirus lethality. We propose complementation by codon shuffling as a means to produce replication-defective or attenuated viruses. This method has immediate utility for investigating roles of essential genes in viral replication and will better enable future development of herpesvirus vaccines. PMID:28287622

  1. Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits beta interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels

    PubMed Central

    da Silva, Leticia Frizzo; Gaudreault, Natasha; Jones, Clinton

    2011-01-01

    Bovine herpesvirus 1 (BHV-1), an alpha-herpesvirinae subfamily member, establishes a life-long latent infection in sensory neurons. Periodically, BHV-1 reactivates from latency, infectious virus is spread, and consequently virus transmission occurs. BHV-1 acute infection causes upper respiratory track infections and conjunctivitis in infected cattle. As a result of transient immunesuppression, BHV-1 infections can also lead to life-threatening secondary bacterial pneumonia that is referred to as bovine respiratory disease. The infected cell protein 0 (bICP0) encoded by BHV-1 reduces human beta-interferon (IFN-β) promoter activity, in part, by inducing degradation of interferon response factor 3 (IRF3) and interacting with IRF7. In contrast to humans, cattle contain three IFN-β genes. All three bovine IFN-β proteins have anti-viral activity: but each IFN-β gene has a distinct transcriptional promoter. We have recently cloned and characterized the three bovine IFN-β promoters. Relative to the human IFN-β promoter, each of the three IFN-β promoters contain differences in the four positive regulatory domains that are required for virus-induced activity. In this study, we demonstrate that bICP0 effectively inhibits bovine IFN-β promoter activity following transfection of low passage bovine cells with interferon response factor 3 (IRF3) or IRF7. A bICP0 mutant that localizes to the cytoplasm inhibits bovine IFN-β promoter activity as efficiently as wt bICP0. The cytoplasmic localized bICP0 protein also induced IRF3 degradation with similar efficiency as wt bICP0. In summary, these studies suggested that cytoplasmic localized bICP0 plays a role in inhibiting the IFN-β response during productive infection. PMID:21689696

  2. Human herpesvirus 8 – A novel human pathogen

    PubMed Central

    Edelman, Daniel C

    2005-01-01

    In 1994, Chang and Moore reported on the latest of the gammaherpesviruses to infect humans, human herpesvirus 8 (HHV-8) [1]. This novel herpesvirus has and continues to present challenges to define its scope of involvement in human disease. In this review, aspects of HHV-8 infection are discussed, such as, the human immune response, viral pathogenesis and transmission, viral disease entities, and the virus's epidemiology with an emphasis on HHV-8 diagnostics. PMID:16138925

  3. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  4. Cyprinid Herpesvirus 3

    PubMed Central

    Michel, Benjamin; Fournier, Guillaume; Lieffrig, François; Costes, Bérénice

    2010-01-01

    The recently designated cyprinid herpesvirus 3 (CyHV-3) is an emerging agent that causes fatal disease in common and koi carp. Since its emergence in the late 1990s, this highly contagious pathogen has caused severe financial losses in common and koi carp culture industries worldwide. In addition to its economic role, recent studies suggest that CyHV-3 may have a role in fundamental research. CyHV-3 has the largest genome among viruses in the order Herpesvirales and serves as a model for mutagenesis of large DNA viruses. Other studies suggest that the skin of teleost fish represents an efficient portal of entry for certain viruses. The effect of temperature on viral replication suggests that the body temperature of its poikilotherm host could regulate the outcome of the infection (replicative vs. nonreplicative). Recent advances with regard to CyHV-3 provide a role for this virus in fundamental and applied research. PMID:21122210

  5. Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences

    PubMed Central

    Green, Lisa; Houck-Loomis, Brian; Yueh, Andrew

    2012-01-01

    Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release. PMID:22718819

  6. Viral Inhibition of the Transporter Associated with Antigen Processing (TAP): A Striking Example of Functional Convergent Evolution

    PubMed Central

    Verweij, Marieke C.; Horst, Daniëlle; Griffin, Bryan D.; Luteijn, Rutger D.; Davison, Andrew J.; Ressing, Maaike E.; Wiertz, Emmanuel J. H. J.

    2015-01-01

    Herpesviruses are large DNA viruses that are highly abundant within their host populations. Even in the presence of a healthy immune system, these viruses manage to cause lifelong infections. This persistence is partially mediated by the virus entering latency, a phase of infection characterized by limited viral protein expression. Moreover, herpesviruses have devoted a significant part of their coding capacity to immune evasion strategies. It is believed that the close coexistence of herpesviruses and their hosts has resulted in the evolution of viral proteins that specifically attack multiple arms of the host immune system. Cytotoxic T lymphocytes (CTLs) play an important role in antiviral immunity. CTLs recognize their target through viral peptides presented in the context of MHC molecules at the cell surface. Every herpesvirus studied to date encodes multiple immune evasion molecules that effectively interfere with specific steps of the MHC class I antigen presentation pathway. The transporter associated with antigen processing (TAP) plays a key role in the loading of viral peptides onto MHC class I molecules. This is reflected by the numerous ways herpesviruses have developed to block TAP function. In this review, we describe the characteristics and mechanisms of action of all known virus-encoded TAP inhibitors. Orthologs of these proteins encoded by related viruses are identified, and the conservation of TAP inhibition is discussed. A phylogenetic analysis of members of the family Herpesviridae is included to study the origin of these molecules. In addition, we discuss the characteristics of the first TAP inhibitor identified outside the herpesvirus family, namely, in cowpox virus. The strategies of TAP inhibition employed by viruses are very distinct and are likely to have been acquired independently during evolution. These findings and the recent discovery of a non-herpesvirus TAP inhibitor represent a striking example of functional convergent evolution

  7. Viral epigenetics.

    PubMed

    Milavetz, Barry I; Balakrishnan, Lata

    2015-01-01

    DNA tumor viruses including members of the polyomavirus, adenovirus, papillomavirus, and herpes virus families are presently the subject of intense interest with respect to the role that epigenetics plays in control of the virus life cycle and the transformation of a normal cell to a cancer cell. To date, these studies have primarily focused on the role of histone modification, nucleosome location, and DNA methylation in regulating the biological consequences of infection. Using a wide variety of strategies and techniques ranging from simple ChIP to ChIP-chip and ChIP-seq to identify histone modifications, nuclease digestion to genome wide next generation sequencing to identify nucleosome location, and bisulfite treatment to MeDIP to identify DNA methylation sites, the epigenetic regulation of these viruses is slowly becoming better understood. While the viruses may differ in significant ways from each other and cellular chromatin, the role of epigenetics appears to be relatively similar. Within the viral genome nucleosomes are organized for the expression of appropriate genes with relevant histone modifications particularly histone acetylation. DNA methylation occurs as part of the typical gene silencing during latent infection by herpesviruses. In the simple tumor viruses like the polyomaviruses, adenoviruses, and papillomaviruses, transformation of the cell occurs via integration of the virus genome such that the virus's normal regulation is disrupted. This results in the unregulated expression of critical viral genes capable of redirecting cellular gene expression. The redirected cellular expression is a consequence of either indirect epigenetic regulation where cellular signaling or transcriptional dysregulation occurs or direct epigenetic regulation where epigenetic cofactors such as histone deacetylases are targeted. In the more complex herpersviruses transformation is a consequence of the expression of the viral latency proteins and RNAs which again can

  8. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  9. Viral proteins that bridge unconnected proteins and components in the human PPI network.

    PubMed

    Rachita, H R; Nagarajaram, H A

    2014-07-29

    Viruses, despite having small genomes and few proteins, make an array of interactions with host proteins as they solely depend on host machinery for their replication and reproduction. Hence, analysis of the Human-Virus Protein-Protein Interaction Network (Hu-Vir PPI network) helps us to gain certain insights into the molecular mechanisms underlying the hijacking of host cell machinery by viruses for their perpetuation. Here we report an analysis of the Human-Virus Bridged PPI Networks that has led us to identify viral articulation points (VAPs) which connect unconnected components of the Human-PPI (Hu-PPI) network. VAPs cross-link peripheral nodes to the giant component of the Hu-PPI network. VAPs interact with a number of relatively lower topologically central human proteins and are conserved among related viruses. The linked nodes comprise of those that are mostly expressed during viral infection, as well as those that are found exclusively in some metabolic pathways, indicating that the novel viral mediation of certain human protein-protein interactions may form the basis for virus-specific tuning of the host machinery. The functional importance of VAPs and their interaction partners in virus replication make them potential drug targets against viral infection. Our investigations also led to the discovery of an example of a Human Endogenous Retrovirus (HERV) encoded protein, syncytin, as an Articulation Point (AP) in the Hu-PPI network, suggesting that VAPs may be retained in a genome if they result in any beneficial function in the host.

  10. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  11. Effect of Acyclovir on Viral Protein Synthesis in Cells Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Furman, Phillip A.; McGuirt, Paul V.

    1983-01-01

    The effect of the antiviral agent 9-(2-hydroxyethoxymethyl)guanine (acyclovir) on herpes simplex virus type 1 protein synthesis during virus replication was examined. Treatment of infected cells with acyclovir markedly affected the amounts of the four major glycosylated and certain non-glycosylated viral polypeptides synthesized; other viral polypeptides were made in normal amounts. The reduced amount of late protein synthesis was most likely due to the inhibition of progeny viral DNA synthesis by acyclovir. Images PMID:6301368

  12. Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

    PubMed

    Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

    2015-11-15

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and

  13. Visualizing Viral Protein Structures in Cells Using Genetic Probes for Correlated Light and Electron Microscopy

    PubMed Central

    Ou, Horng D.; Deerinck, Thomas J.; Bushong, Eric; Ellisman, Mark H.; O’Shea, Clodagh C.

    2015-01-01

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host’s cellular environment, their natural in-situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940’s and subsequent application to cells in the 1950’s. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in-situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining

  14. Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east Africa.

    PubMed Central

    Hofmann-Lehmann, R; Fehr, D; Grob, M; Elgizoli, M; Packer, C; Martenson, J S; O'Brien, S J; Lutz, H

    1996-01-01

    While viral infections and their impact are well studied in domestic cats, only limited information is available on their occurrence in free-ranging lions. The goals of the present study were (i) to investigate the prevalence of antibodies to feline calicivirus (FCV), herpesvirus (FHV), coronavirus (FCoV), parvovirus (FPV), and immunodeficiency virus (FIV) and of feline leukemia virus (FeLV) antigen in 311 serum samples collected between 1984 and 1991 from lions inhabiting Tanzania's national parks and (ii) to evaluate the possible biological importance and the interrelationship of these viral infections. Antibodies to FCV, never reported previously in free-ranging lions, were detected in 70% of the sera. In addition, a much higher prevalence of antibodies to FCoV (57%) was found than was previously reported in Etosha National Park and Kruger National Park. Titers ranged from 25 to 400. FeLV antigen was not detectable in any of the serum samples. FCoV, FCV, FHV, and FIV were endemic in the Serengeti, while a transient elevation of FPV titers pointed to an outbreak of FPV infection between 1985 and 1987. Antibody titers to FPV and FCV were highly prevalent in the Serengeti (FPV, 75%; FCV, 67%) but not in Ngorongoro Crater (FPV, 27%; FCV, 2%). These differences could be explained by the different habitats and biological histories of the two populations and by the well-documented absence of immigration of lions from the Serengeti plains into Ngorongoro Crater after 1965. These observations indicate that, although the pathological potential of these viral infections seemed not to be very high in free-ranging lions, relocation of seropositive animals by humans to seronegative lion populations must be considered very carefully. PMID:8877134

  15. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    PubMed

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  16. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    PubMed

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  17. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation

    PubMed Central

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; De Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-01-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation. PMID:14528266

  18. KSHV Rta Promoter Specification and Viral Reactivation

    PubMed Central

    Guito, Jonathan; Lukac, David M.

    2011-01-01

    Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi’s sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression

  19. Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4.

    PubMed Central

    Whittaker, G R; Riggio, M P; Halliburton, I W; Killington, R A; Allen, G P; Meredith, D M

    1991-01-01

    Monospecific polyclonal antisera raised against VP13/14, a major tegument protein of herpes simplex virus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of Mr 120,000 and 123,000, respectively; these proteins are identical in molecular weight to the corresponding glycoprotein 10 (gp10) of each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we confirmed that anti-VP13/14 and a monoclonal antibody to gp10 reacted with the same protein. Sequence analysis of a lambda gt11 insert of equine herpesvirus 1 gp10 identified an open reading frame in equine herpesvirus 4 with which it showed strong homology; this open reading frame also shared homology with gene UL47 of herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14 and gp10 have protein sequence homology; it also allowed identification of VP13/14 as the gene product of UL47. Images PMID:1850013

  20. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    SciTech Connect

    Sathish, Narayanan; Yuan Yan

    2010-11-25

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-{Delta}65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  1. ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle

    PubMed Central

    Wood, Jennifer J.; Boyne, James R.; Paulus, Christina; Jackson, Brian R.; Nevels, Michael M.

    2016-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle—viral latency and the productive lytic cycle—and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of

  2. Cytoplasm-to-nucleus translocation of a herpesvirus tegument protein during cell division.

    PubMed

    Elliott, G; O'Hare, P

    2000-03-01

    We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G(1) stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.

  3. Graphical representation and mathematical characterization of protein sequences and applications to viral proteins.

    PubMed

    Ghosh, Ambarnil; Nandy, Ashesh

    2011-01-01

    Graphical representation and numerical characterization (GRANCH) of nucleotide and protein sequences is a new field that is showing a lot of promise in analysis of such sequences. While formulation and applications of GRANCH techniques for DNA/RNA sequences started just over a decade ago, analyses of protein sequences by these techniques are of more recent origin. The emphasis is still on developing the underlying technique, but significant results have been achieved in using these methods for protein phylogeny, mass spectral data of proteins and protein serum profiles in parasites, toxicoproteomics, determination of different indices for use in QSAR studies, among others. We briefly mention these in this chapter, with some details on protein phylogeny and viral diseases. In particular, we cover a systematic method developed in GRANCH to determine conserved surface exposed peptide segments in selected viral proteins that can be used for drug and vaccine targeting. The new GRANCH techniques and applications for DNAs and proteins are covered briefly to provide an overview to this nascent field.

  4. SIAH-1 interacts with the Kaposi's sarcoma-associated herpesvirus-encoded ORF45 protein and promotes its ubiquitylation and proteasomal degradation.

    PubMed

    Abada, Rinat; Dreyfuss-Grossman, Tsofia; Herman-Bachinsky, Yifat; Geva, Haim; Masa, Shiri-Rivka; Sarid, Ronit

    2008-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.

  5. SIAH-1 Interacts with the Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORF45 Protein and Promotes Its Ubiquitylation and Proteasomal Degradation▿

    PubMed Central

    Abada, Rinat; Dreyfuss-Grossman, Tsofia; Herman-Bachinsky, Yifat; Geva, Haim; Masa, Shiri-Rivka; Sarid, Ronit

    2008-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection. PMID:18077711

  6. A core viral protein binds host nucleosomes to sequester immune danger signals.

    PubMed

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-07

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

  7. A core viral protein binds host nucleosomes to sequester immune danger signals

    PubMed Central

    Avgousti, Daphne C.; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J.; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C.; Blumenthal, Daniel; Paris, Andrew J.; Reyes, Emigdio D.; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H.; Worthen, G. Scott; Black, Ben E.; Garcia, Benjamin A.; Weitzman, Matthew D.

    2016-01-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling. PMID:27362237

  8. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  9. Chemotherapy targeting by DNA capture in viral protein particles

    PubMed Central

    Agadjanian, Hasmik; Chu, David; Hwang, Jae Youn; Wachsmann-Hogiu, Sebastian; Rentsendorj, Altan; Song, Lei; Valluripalli, Vinod; Lubow, Jay; Ma, Jun; Sharifi, Behrooz; Farkas, Daniel L; Medina-Kauwe, Lali K

    2012-01-01

    Aim This study tests the hypothesis that DNA intercalation and electrophilic interactions can be exploited to noncovalently assemble doxorubicin in a viral protein nanoparticle designed to target and penetrate tumor cells through ligand-directed delivery. We further test whether this new paradigm of doxorubicin targeting shows therapeutic efficacy and safety in vitro and in vivo. Materials & methods We tested serum stability, tumor targeting and therapeutic efficacy in vitro and in vivo using biochemical, microscopy and cytotoxicity assays. Results Self-assembly formed approximately 10-nm diameter serum-stable nanoparticles that can target and ablate HER2+ tumors at >10× lower dose compared with untargeted doxorubicin, while sparing the heart after intravenous delivery. The targeted nanoparticle tested here allows doxorubicin potency to remain unaltered during assembly, transport and release into target cells, while avoiding peripheral tissue damage and enabling lower, and thus safer, drug dose for tumor killing. Conclusion This nanoparticle may be an improved alternative to chemical conjugates and signal-blocking antibodies for tumor-targeted treatment. PMID:22385197

  10. Recombinant protein-based viral disease diagnostics in veterinary medicine.

    PubMed

    Balamurugan, Vinayagamurthy; Venkatesan, Gnanavel; Sen, Arnab; Annamalai, Lakshmanan; Bhanuprakash, Veerakyathappa; Singh, Raj Kumar

    2010-09-01

    Identification of pathogens or antibody response to pathogens in human and animals modulates the treatment strategies for naive population and subsequent infections. Diseases can be controlled and even eradicated based on the epidemiology and effective prophylaxis, which often depends on development of efficient diagnostics. In addition, combating newly emerging diseases in human as well as animal healthcare is challenging and is dependent on developing safe and efficient diagnostics. Detection of antibodies directed against specific antigens has been the method of choice for documenting prior infection. Other than zoonosis, development of inexpensive vaccines and diagnostics is a unique problem in animal healthcare. The advent of recombinant DNA technology and its application in the biotechnology industry has revolutionized animal healthcare. The use of recombinant DNA technology in animal disease diagnosis has improved the rapidity, specificity and sensitivity of various diagnostic assays. This is because of the absence of host cellular proteins in the recombinant derived antigen preparations that dramatically decrease the rate of false-positive reactions. Various recombinant products are used for disease diagnosis in veterinary medicine and this article discusses recombinant-based viral disease diagnostics currently used for detection of pathogens in livestock and poultry.

  11. Herpesviruses that Infect Fish

    PubMed Central

    Hanson, Larry; Dishon, Arnon; Kotler, Moshe

    2011-01-01

    Herpesviruses are host specific pathogens that are widespread among vertebrates. Genome sequence data demonstrate that most herpesviruses of fish and amphibians are grouped together (family Alloherpesviridae) and are distantly related to herpesviruses of reptiles, birds and mammals (family Herpesviridae). Yet, many of the biological processes of members of the order Herpesvirales are similar. Among the conserved characteristics are the virion structure, replication process, the ability to establish long term latency and the manipulation of the host immune response. Many of the similar processes may be due to convergent evolution. This overview of identified herpesviruses of fish discusses the diseases that alloherpesviruses cause, the biology of these viruses and the host-pathogen interactions. Much of our knowledge on the biology of Alloherpesvirdae is derived from research with two species: Ictalurid herpesvirus 1 (channel catfish virus) and Cyprinid herpesvirus 3 (koi herpesvirus). PMID:22163339

  12. A negative element involved in Kaposi's sarcoma-associated herpesvirus-encoded ORF11 gene expression

    SciTech Connect

    Chen, Lei

    2009-01-01

    The ORF11 of the Kaposi's sarcoma-associated herpesvirus (KSHV) is a lytic viral gene with delayed-early expression kinetics. How the ORF11 gene expression is regulated in the KSHV lytic cascade is largely unknown. Here we report that the deletion of the KSHV viral IL-6 gene from the viral genome leads to deregulated ORF11 gene expression. The KSHV-encoded viral IL-6 protein was found not to be essentially involved in the regulation of ORF11, suggesting a potential transcriptional cis-regulation. A negative element was identified downstream of the ORF11 gene, which suppresses the ORF11 basal promoter activity in a position-independent manner.

  13. Tat is a multifunctional viral protein that modulates cellular gene expression and functions.

    PubMed

    Clark, Evan; Nava, Brenda; Caputi, Massimo

    2017-02-07

    The human immunodeficiency virus type I (HIV-1) has developed several strategies to condition the host environment to promote viral replication and spread. Viral proteins have evolved to perform multiple functions, aiding in the replication of the viral genome and modulating the cellular response to the infection. Tat is a small, versatile, viral protein that controls transcription of the HIV genome, regulates cellular gene expression and generates a permissive environment for viral replication by altering the immune response and facilitating viral spread to multiple tissues. Studies carried out utilizing biochemical, cellular, and genomic approaches show that the expression and activity of hundreds of genes and multiple molecular networks are modulated by Tat via multiple mechanisms.

  14. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

    PubMed

    de Munnik, Sabrina M; Kooistra, Albert J; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J; Leurs, Rob; Vischer, Henry F

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

  15. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

    PubMed Central

    de Munnik, Sabrina M.; Kooistra, Albert J.; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74. PMID:25894435

  16. One year duration of immunity of the modified live bovine viral diarrhea virus type 1 and type 2 and bovine herpesvirus-1 fractions of Vista® Once SQ vaccine.

    PubMed

    Purtle, Lisa; Mattick, Debra; Schneider, Corey; Smith, Linda; Xue, Wenzhi; Trigo, Emilio

    2016-03-18

    Three studies were performed to determine the duration of immunity of the bovine viral diarrhea virus type 1 and type 2 (BVDV-1 and BVDV-2) and bovine herpesvirus-1 (BHV-1) fractions of a commercially prepared modified-live vaccine. Vista® Once SQ (Vista®) vaccine contains five modified-live viruses, BVDV-1, BVDV-2, BHV-1, bovine respiratory syncytial virus, and bovine parainfluenza 3 virus, and two modified-live bacteria, Pasteurella multocida and Mannheimia haemolytica. For all three studies, calves were administered a single dose of vaccine or placebo vaccine subcutaneously, and were challenged with one of the three virulent viruses at least one year following vaccination. Calves were evaluated daily following challenge for clinical signs of disease associated with viral infection, nasal swab samples were evaluated for virus shedding, and serum was tested for neutralizing antibodies. Following the BVDV-1 and BVDV-2 challenges, whole blood was evaluated for white blood cell counts, and for the BVDV-2 study, whole blood was also evaluated for platelet counts. Calves vaccinated with BVDV type 1a, were protected from challenge with BVDV type 1b, and had significant reductions in clinical disease, fever, leukopenia, and virus shedding compared to control calves. Vaccinated calves in the BVDV-2 study were protected from clinical disease, mortality, fever, leukopenia, thrombocytopenia, and virus shedding compared to controls. Vaccinated calves in the BHV-1 study were protected from clinical disease and fever, and had significantly reduced duration of nasal virus shedding. These three studies demonstrated that a single administration of the Vista® vaccine to healthy calves induces protective immunity against BVDV-1, BVDV-2 and BHV-1 that lasts at least one year following vaccination.

  17. Access of viral proteins to mitochondria via mitochondria-associated membranes.

    PubMed

    Williamson, Chad D; Colberg-Poley, Anamaris M

    2009-05-01

    By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterised MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

  18. Generation of Recombinant Viral Hemorrhagic Septicemia Virus (rVHSV) Expressing Two Foreign Proteins and Effect of Lengthened Viral Genome on Viral Growth and In Vivo Virulence.

    PubMed

    Kim, Min Sun; Lee, Su Jin; Kim, Dong Soo; Kim, Ki Hong

    2016-04-01

    In this study, a new recombinant VHSV (rVHSV-Arfp-Bgfp) was generated by insertion of a red fluorescent protein (RFP) gene between N and P genes, a green fluorescent protein (GFP) gene between P and M genes of VHSV genome, the expression of each heterologous gene in infected cells, and effects of the lengthened recombinant VHSV's genome on the replication ability and in vivo virulence to olive flounder (Paralichthys olivaceus) fingerlings were compared with previously generated rVHSVs (rVHSV-wild, rVHSV-Arfp, and rVHSV-Brfp). The expression of RFP and GFP in cells infected with rVHSV-Arfp-Bgfp was verified through fluorescent microscopy and FACS analysis. In the viral growth analysis, rVHSV-Arfp and rVHSV-Brfp showed significantly lower viral titers than rVHSV-wild, and the replication of rVHSV-Arfp-Bgfp was significantly decreased compared to that of even rVHSV-Arfp or rVHSV-Brfp. These results suggest that the genome length is a critical factor for the determination of rVHSVs replication efficiency. In the in vivo virulence experiment, the cumulative mortalities of olive flounder fingerlings infected with each rVHSV were inversely proportional to the length of the viral genome, suggesting that decreased viral growth rate due to the lengthened viral genome is accompanied with the decrease of in vivo virulence of rVHSVs. Recombinant viruses expressing multiple foreign antigens can be used for the development of combined vaccines. However, as the present rVHSV-Arfp-Bgfp still possesses an ability to kill hosts (although very weakened), researches on the producing more attenuated viruses or propagation-deficient replicon particles are needed to solve safety-related problems.

  19. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  20. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

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

    SciTech Connect

    Wang, Robert Y.L.; Kuo, Rei-Lin; Ma, Wei-Chieh; Huang, Hsing-I; Yu, Jau-Song; Yen, Sih-Min; Huang, Chi-Ruei; Shih, Shin-Ru

    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 to 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.

  2. Columbid herpesvirus-1 mortality in great horned owls (Bubo virginianus) from Calgary, Alberta.

    PubMed

    Rose, Nicole; Warren, Amy L; Whiteside, Douglas; Bidulka, Julie; Robinson, John H; Illanes, Oscar; Brookfield, Caroline

    2012-03-01

    Four cases of Columbid herpesvirus-1 infection in great horned owls (Bubo virginianus) were identified in Calgary, Alberta. Necropsy findings included severe multifocal hepatic and splenic necrosis, pharyngeal ulceration and necrosis, and gastrointestinal necrosis. Occasional eosinophilic intranuclear viral inclusion bodies were associated with the foci of necrosis and polymerase chain reaction (PCR) testing confirmed a diagnosis of herpesvirus-induced disease. The sequence of a PCR amplicon had 99.7% homology to Columbid herpesvirus-1.

  3. Cleavage of Grb2-Associated Binding Protein 2 by Viral Proteinase 2A during Coxsackievirus Infection

    PubMed Central

    Deng, Haoyu; Fung, Gabriel; Qiu, Ye; Wang, Chen; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2017-01-01

    Coxsackievirus type B3 (CV-B3), an enterovirus associated with the pathogenesis of several human diseases, subverts, or employs the host intracellular signaling pathways to support effective viral infection. We have previously demonstrated that Grb2-associated binding protein 1 (GAB1), a signaling adaptor protein that serves as a platform for intracellular signaling assembly and transduction, is cleaved upon CV-B3 infection, resulting in a gain-of-pro-viral-function via the modification of GAB1-mediated ERK1/2 pathway. GAB2 is a mammalian homolog of GAB1. In this study, we aim to address whether GAB2 plays a synergistic role with GAB1 in the regulation of CV-B3 replication. Here, we reported that GAB2 is also a target of CV-B3-encoded viral proteinase. We showed that GAB2 is cleaved at G238 during CV-B3 infection by viral proteinase 2A, generating two cleaved fragments of GAB2-N1−237 and GAB2-C238−676. Moreover, knockdown of GAB2 significantly inhibits the synthesis of viral protein and subsequent viral progeny production, accompanied by reduced levels of phosphorylated p38, suggesting a pro-viral function for GAB2 linked to p38 activation. Finally, we examined whether the cleavage of GAB2 can promote viral replication as observed for GAB1 cleavage. We showed that expression of neither GAB2-N1−237 nor GAB2-C238−676 results in enhanced viral infectivity, indicating a loss-of-function, rather than a gain-of-function of GAB2 cleavage in mediating virus replication. Taken together, our findings in this study suggest a novel host defense machinery through which CV-B3 infection is limited by the cleavage of a pro-viral protein. PMID:28361043

  4. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures

    PubMed Central

    Rao, Vasudev R.; Eugenin, Eliseo A.; Prasad, Vinayaka R.

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. PMID:26714725

  5. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    PubMed

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  6. Koi herpesvirus encodes and expresses a functional interleukin-10.

    PubMed

    Sunarto, Agus; Liongue, Clifford; McColl, Kenneth A; Adams, Mathew M; Bulach, Dieter; Crane, Mark St J; Schat, Karel A; Slobedman, Barry; Barnes, Andrew C; Ward, Alister C; Walker, Peter J

    2012-11-01

    Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.

  7. Biological roles and functional mechanisms of arenavirus Z protein in viral replication.

    PubMed

    Wang, Jialong; Danzy, Shamika; Kumar, Naveen; Ly, Hinh; Liang, Yuying

    2012-09-01

    Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

  8. Viral Proteins Acquired from a Host Converge to Simplified Domain Architectures

    PubMed Central

    Rappoport, Nadav; Linial, Michal

    2012-01-01

    The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for manipulation of viral

  9. Dynamics of virus shedding and in situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with Fibropapillomatosis

    USGS Publications Warehouse

    Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schettle, Nelli; Ackermann, Mathias

    2015-01-01

    Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

  10. Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.

    PubMed

    Smales, C M; Pepper, D S; James, D C

    2001-01-01

    During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.

  11. The nucleolar protein GLTSCR2 is required for efficient viral replication

    PubMed Central

    Wang, Peng; Meng, Wen; Han, Shi-Chong; Li, Cui-Cui; Wang, Xiao-Jun; Wang, Xiao-Jia

    2016-01-01

    Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2) is a nucleolar protein. In the investigation of the role of GLTSCR2 that played in the cellular innate immune response to viral infection, we found GLTSCR2 supported viral replication of rhabdovirus, paramyxovirus, and coronavirus in cells. Viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm that enabled GLTSCR2 to attenuate type I interferon IFN-β and support viral replication. Cytoplasmic GLTSCR2 was able to interact with retinoic acid-inducible gene I (RIG-I) and the ubiquitin-specific protease 15 (USP15), and the triple interaction induced USP15 activity to remove K63-linked ubiquitination of RIG-I, leading to attenuation of RIG-I and IFN-β. Blocking cytoplasmic translocation of GLTSCR2, by deletion of its nuclear export sequence (NES), abrogated its ability to attenuate IFN-β and support viral replication. GLTSCR2-mediated attenuation of RIG-I and IFN-β led to alleviation of host cell innate immune response to viral infection. Our findings suggested that GLTSCR2 contributed to efficient viral replication, and GLTSCR2 should be considered as a potential target for therapeutic control of viral infection. PMID:27824081

  12. Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression.

    PubMed

    Sun, R; Lin, S F; Staskus, K; Gradoville, L; Grogan, E; Haase, A; Miller, G

    1999-03-01

    Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor

  13. Serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and Pasteurella haemolytica in feedlot calves with respiratory disease: associations with bacteriological and pulmonary cytological variables.

    PubMed Central

    Allen, J W; Viel, L; Bateman, K G; Nagy, E; Røsendal, S; Shewen, P E

    1992-01-01

    Acute and convalescent serum samples were taken from 59 calves with signs of respiratory disease (cases) and 60 clinically normal animals (controls) during their first month in the feedlot. Sera were analyzed for antibodies to bovine parainfluenza 3 (PI3) virus by hemagglutination inhibition, to bovine viral diarrhea (BVD) virus, bovine respiratory syncytial (BRS) virus and bovine herpesvirus 1 (BHV1) by virus neutralization, and to Pasteurella haemolytica by indirect agglutination (PhIA) and cytotoxin neutralization (PhCN) tests. There was minimal evidence of serological activity to BHV1. Serological activity to the other agents occurred commonly and the prevalence of acute titers and their mean values was similar in case and control groups. Mean convalescent PI3 and P. haemolytica (PhIA) titers were higher in controls than cases (p < 0.01) but, otherwise, convalescent titers did not differ between groups. The incidence of seroconversion was similar in both groups for all agents except for PI3 virus which was more frequent in controls than cases (p < 0.0001). There was a positive association between PhIA and CN seroconversion and isolation of P. haemolytica from bronchoalveolar lavage (BAL) fluid (p < 0.1). The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51. Bacteriological and cytological evaluations of the respiratory tract were made using BAL. No associations were evident between serological titers and pulmonary cytology. A multivariate logistic analysis was used to evaluate associations between disease status and serological, bacteriological and cytological data. Cases were positively associated with the presence of neutrophils and Pasteurella multocida in BAL fluid and negatively associated with PI3 virus and PhIA seroconversion. PMID:1335831

  14. Separation and isolation of BTV dsRNA segments and viral proteins.

    PubMed

    Li, Joseph K-K; Huang, I-Jen; Hayama, Emiko

    2012-05-01

    Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

  15. Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

    PubMed Central

    Baquero-Pérez, Belinda; Whitehouse, Adrian

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents. PMID:26587836

  16. Primary structure of the herpesvirus saimiri genome.

    PubMed Central

    Albrecht, J C; Nicholas, J; Biller, D; Cameron, K R; Biesinger, B; Newman, C; Wittmann, S; Craxton, M A; Coleman, H; Fleckenstein, B

    1992-01-01

    This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation. PMID:1321287

  17. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  18. Broad-spectrum non-nucleoside inhibitors of human herpesviruses

    PubMed Central

    McClain, Lora; Zhi, Yun; Cheng, Hoyee; Ghosh, Ayantika; Piazza, Paolo; Yee, Michael B.; Kumar, Santosh; Milosevic, Jadranka; Bloom, David C.; Arav-Boger, Ravit; Kinchington, Paul R.; Yolken, Robert; Nimgaonkar, Vishwajit; D’Aiuto, Leonardo

    2015-01-01

    Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of ‘quiescent’ HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo. PMID:26079681

  19. Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

    PubMed Central

    Haatveit, Hanne Merethe; Wessel, Øystein; Markussen, Turhan; Lund, Morten; Thiede, Bernd; Nyman, Ingvild Berg; Braaen, Stine; Dahle, Maria Krudtaa; Rimstad, Espen

    2017-01-01

    Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection. PMID:28335455

  20. In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models

    PubMed Central

    Nakaya, Yuki; Stavrou, Spyridon; Blouch, Kristin; Tattersall, Peter

    2016-01-01

    ABSTRACT APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo. IMPORTANCE It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo. PMID:27356895

  1. Live cell imaging reveals the relocation of dsRNA binding proteins upon viral infection.

    PubMed

    Barton, Deborah; Roovers, Elke; Gouil, Quentin; C da Fonseca, Guilherme; Reis, Rodrigo S; Jackson, Craig; Overall, Robyn; Fusaro, Adriana; Waterhouse, Peter

    2017-03-15

    Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins, DRB3 and DRB4, are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3 and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRCs) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis, by T-DNA insertion, rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3 and DRB5 are invasion sensors that relocate to nascent VRCs, where they bind to viral RNA and inhibit virus replication.

  2. Illuminating structural proteins in viral “dark matter” with metaproteomics

    PubMed Central

    Brum, Jennifer R.; Ignacio-Espinoza, J. Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M.; Roux, Simon; VerBerkmoes, Nathan C.; Rich, Virginia I.; Sullivan, Matthew B.

    2016-01-01

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional “viral dark matter.” Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world’s oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter. PMID:26884177

  3. Hepatitis C viral protein translation: mechanisms and implications in developing antivirals.

    PubMed

    Hoffman, Brett; Liu, Qiang

    2011-11-01

    Hepatitis C viral protein translation occurs in a cap-independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5'-untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap-dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis-acting viral factors, as well as trans-acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.

  4. Three-Dimensional Structure of the Human Herpesvirus 8 Capsid

    PubMed Central

    Wu, Lijun; Lo, Pierrette; Yu, Xuekui; Stoops, James K.; Forghani, B.; Zhou, Z. Hong

    2000-01-01

    Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first description in 1994. However, its three-dimensional (3D) structure remained heretofore undetermined largely due to difficulties in viral purification. We have used log-phase cultures of body cavity-based lymphoma 1 cells induced with 12-O-tetradecanoylphorbol-13-acetate to obtain HHV-8 capsids for electron cryomicroscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical members of alpha- and betaherpesviruses, respectively. The inner radius of the HHV-8 capsid is identical to that of the HSV-1 capsid but is smaller than that of the HCMV capsid, which is consistent with the relative sizes of the genomes they enclose. While the HHV-8 capsid exhibits many structural similarities to the HSV-1 capsid, our reconstruction shows two major differences: its hexons lack the “horn-shaped” VP26 densities bound to the HSV-1 hexon subunits, and the HHV-8 triplexes appear smaller and less elongated than those of HSV-1. These differences are in excellent agreement with our sequence comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus capsid structure complements previous structural studies on alpha- and betaherpesviruses in providing an account of structural similarities and differences among capsids representing all human herpesvirus subfamilies. PMID:11000237

  5. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

    PubMed

    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

  6. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    PubMed

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  7. Construction of a lytically replicating Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Budt, Matthias; Hristozova, Tsvetana; Hille, Georg; Berger, Katrin; Brune, Wolfram

    2011-10-01

    Karposi's sarcoma-associated herpesvirus (KSHV) is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here, we engineered the cloned KSHV genome, bacterial artificial chromosome 36 (BAC36), to enforce constitutive expression of the main lytic switch regulator, the replication and transcription activator (RTA) (open reading frame 50 [ORF50]). The resulting virus, KSHV-lyt, activated by default the lytic cycle and replicated to high titers in various cells. Using KSHV-lyt, we showed that ORF33 (encoding a tegument protein) is essential for lytic KSHV replication in cell culture, but ORF73 (encoding the latent nuclear antigen [LANA]) is not. Thus, KSHV-lyt should be highly useful to study viral gene function during lytic replication.

  8. Kaposi Sarcoma-associated Herpesvirus: mechanisms of oncogenesis.

    PubMed

    Schulz, Thomas F; Cesarman, Ethel

    2015-10-01

    Kaposi Sarcoma-associated Herpesvirus (KSHV, HHV8) causes three human malignancies, Kaposi Sarcoma (KS), an endothelial tumor, as well as Primary Effusion Lymphoma (PEL) and the plasma cell variant of Multicentric Castleman's Disease (MCD), two B-cell lymphoproliferative diseases. All three cancers occur primarily in the context of immune deficiency and/or HIV infection, but their pathogenesis differs. KS most likely results from the combined effects of an endotheliotropic virus with angiogenic properties and inflammatory stimuli and thus represents an interesting example of a cancer that arises in an inflammatory context. Viral and cellular angiogenic and inflammatory factors also play an important role in the pathogenesis of MCD. In contrast, PEL represents an autonomously growing malignancy that is, however, still dependent on the continuous presence of KSHV and the action of several KSHV proteins.

  9. At the crossroads of autophagy and infection: Noncanonical roles for ATG proteins in viral replication

    PubMed Central

    Solvik, Tina

    2016-01-01

    Autophagy-related (ATG) proteins have increasingly demonstrated functions other than cellular self-eating. In this issue, Mauthe et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602046) conduct an unbiased RNA interference screen of the ATG proteome to reveal numerous noncanonical roles for ATG proteins during viral infection. PMID:27573461

  10. Temporal proteomic analysis and label-free quantification of viral proteins of an invertebrate iridovirus.

    PubMed

    İnce, İkbal Agah; Boeren, Sjef; van Oers, Monique M; Vlak, Just M

    2015-01-01

    Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells, of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection, proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant, but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.

  11. Detection of viral proteins in human cells lines by xeno-proteomics: elimination of the last valid excuse for not testing every cellular proteome dataset for viral proteins.

    PubMed

    Chernobrovkin, Alexey L; Zubarev, Roman A

    2014-01-01

    Cell cultures used routinely in proteomic experiments may contain proteins from other species because of infection, transfection or just contamination. Since infection or contamination may affect the results of a biological experiment, it is important to test the samples for the presence of "alien" proteins. Usually cells are tested only for the most common infections, and most of the existing tests are targeting specific contaminations. Here we describe a three-step procedure for reliable untargeted detection of viral proteins using proteomics data, and recommend this or similar procedure to be applied to every proteomics dataset submitted for publication.

  12. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    SciTech Connect

    Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

    2008-10-10

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) {beta} and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

  13. Lipids as modulators of membrane fusion mediated by viral fusion proteins.

    PubMed

    Teissier, Elodie; Pécheur, Eve-Isabelle

    2007-11-01

    Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.

  14. Induction of lytic cycle replication of Kaposi's sarcoma-associated herpesvirus by herpes simplex virus type 1: involvement of IL-10 and IL-4.

    PubMed

    Qin, Di; Zeng, Yi; Qian, Chao; Huang, Zan; Lv, Zhigang; Cheng, Lin; Yao, Shuihong; Tang, Qiao; Chen, Xiuying; Lu, Chun

    2008-03-01

    Previously, we identified that both human herpesvirus 6 and human immunodeficiency virus type 1 Tat were important cofactors that activated lytic cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we further investigated the potential of herpes simplex virus type 1 (HSV-1) to influence KSHV replication. We demonstrated that HSV-1 was a potentially important factor in the pathogenesis of Kaposi's sarcoma, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV ORF50 and a luciferase reporter assay testing ORF50 promoter-driven luciferase activity. Finally, we discovered that production of human interleukin-10 (IL-10) and IL-4 partially contributed to HSV-1-induced KSHV replication. Our data present the first direct evidence that HSV-1 can activate KSHV lytic replication and suggest a role of HSV-1 in KSHV pathogenesis.

  15. Two Distinct Gamma-2 Herpesviruses in African Green Monkeys: a Second Gamma-2 Herpesvirus Lineage among Old World Primates?

    PubMed Central

    Greensill, Julie; Sheldon, Julie A.; Renwick, Neil M.; Beer, Brigitte E.; Norley, Steve; Goudsmit, Jaap; Schulz, Thomas F.

    2000-01-01

    Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens. PMID:10627572

  16. The Role of F-Box Proteins during Viral Infection

    PubMed Central

    Correa, Régis Lopes; Bruckner, Fernanda Prieto; de Souza Cascardo, Renan; Alfenas-Zerbini, Poliane

    2013-01-01

    The F-box domain is a protein structural motif of about 50 amino acids that mediates protein–protein interactions. The F-box protein is one of the four components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes. Several discoveries have been made on the use of the ubiquitin–proteasome system by viruses of several families to complete their infection cycle. On the other hand, F-box proteins can be used in the defense response by the host. This review describes the role of F-box proteins and the use of the ubiquitin–proteasome system in virus–host interactions. PMID:23429191

  17. Prediction of Interactions between Viral and Host Proteins Using Supervised Machine Learning Methods

    PubMed Central

    Barman, Ranjan Kumar; Saha, Sudipto; Das, Santasabuj

    2014-01-01

    Background Viral-host protein-protein interaction plays a vital role in pathogenesis, since it defines viral infection of the host and regulation of the host proteins. Identification of key viral-host protein-protein interactions (PPIs) has great implication for therapeutics. Methods In this study, a systematic attempt has been made to predict viral-host PPIs by integrating different features, including domain-domain association, network topology and sequence information using viral-host PPIs from VirusMINT. The three well-known supervised machine learning methods, such as SVM, Naïve Bayes and Random Forest, which are commonly used in the prediction of PPIs, were employed to evaluate the performance measure based on five-fold cross validation techniques. Results Out of 44 descriptors, best features were found to be domain-domain association and methionine, serine and valine amino acid composition of viral proteins. In this study, SVM-based method achieved better sensitivity of 67% over Naïve Bayes (37.49%) and Random Forest (55.66%). However the specificity of Naïve Bayes was the highest (99.52%) as compared with SVM (74%) and Random Forest (89.08%). Overall, the SVM and Random Forest achieved accuracy of 71% and 72.41%, respectively. The proposed SVM-based method was evaluated on blind dataset and attained a sensitivity of 64%, specificity of 83%, and accuracy of 74%. In addition, unknown potential targets of hepatitis B virus-human and hepatitis E virus-human PPIs have been predicted through proposed SVM model and validated by gene ontology enrichment analysis. Our proposed model shows that, hepatitis B virus “C protein” binds to membrane docking protein, while “X protein” and “P protein” interacts with cell-killing and metabolic process proteins, respectively. Conclusion The proposed method can predict large scale interspecies viral-human PPIs. The nature and function of unknown viral proteins (HBV and HEV), interacting partners of host protein

  18. HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells

    PubMed Central

    Sainis, Ioannis; Angelidis, Charalambos; Pagoulatos, Gerasimos N.; Lazaridis, Ioannis

    2000-01-01

    SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon γ, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines. PMID:11147964

  19. The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs.

    PubMed

    Heilman, Destin W; Green, Michael R; Teodoro, Jose G

    2005-04-01

    The study of animal viruses has provided extraordinary insights into cell cycle dynamics and tumor biology. The significance of the p53 and Rb tumor suppressor proteins, for example, was discovered due to their interactions with viral oncogenes. In the past several years, investigations with four viral proteins, human immunodeficiency virus type 1 (HIV-1) vpr, adenovirus E4orf4, chicken anemia virus (CAV) apoptin and human T lymphotropic virus type I (HTLV-I) Tax, have indicated that there are also critical viral targets involved in G2/M control. In particular, recent studies with E4orf4 and apoptin have shown that they induce G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Notably, these two viral proteins induce apoptosis selectively in transformed cells in a p53-independent manner; thus pathways affected by these proteins are of significant therapeutic interest. Further investigation of the underlying mechanism of G2/M arrest and subsequent apoptosis induced by viral APC/C inhibitors may shed light on the mechanisms of current cancer therapies and provide the foundation for developing novel therapeutic targets.

  20. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  1. Comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus 1, bovine viral diarrhea virus 2, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine parainfluenza virus 3 in calves fed maternal colostrum or a colostrum-replacement product.

    PubMed

    Chamorro, Manuel F; Walz, Paul H; Haines, Deborah M; Passler, Thomas; Earleywine, Thomas; Palomares, Roberto A; Riddell, Kay P; Galik, Patricia; Zhang, Yijing; Givens, M Daniel

    2014-04-01

    Colostrum-replacement products are an alternative to provide passive immunity to neonatal calves; however, their ability to provide adequate levels of antibodies recognizing respiratory viruses has not been described. The objective of this study was to compare the serum levels of IgG at 2 d of age and the duration of detection of antibodies to bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BHV-1), and bovine parainfluenza virus 3 (BPIV-3) in calves fed maternal colostrum (MC) or a colostrum replacement (CR) at birth. Forty newborn male Holstein calves were assigned to the CR or the MC group. Group CR (n = 20) received 2 packets of colostrum replacement (100 g of IgG per 470-g packet), while group MC (n = 20) received 3.8 L of maternal colostrum. Blood samples for detection of IgG and virus antibodies were collected from each calf at birth, at 2 and 7 d, and monthly until the calves became seronegative. Calves in the MC group had greater IgG concentrations at 2 d of age. The apparent efficiency of absorption of IgG was greater in the MC group than in the CR group, although the difference was not significant. Calves in the CR group had greater concentrations of BVDV neutralizing antibodies during the first 4 mo of life. The levels of antibodies to BRSV, BHV-1, and BPIV-3 were similar in the 2 groups. The mean time to seronegativity was similar for each virus in the 2 groups; however, greater variation was observed in the antibody levels and in the duration of detection of immunity in the MC group than in the CR group. Thus, the CR product provided calves with more uniform levels and duration of antibodies to common bovine respiratory viruses.

  2. Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

    PubMed Central

    Sanderson, C M; McQueen, N L; Nayak, D P

    1993-01-01

    We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins. Images PMID:8380460

  3. An integrated map of HIV-human protein complexes that facilitate viral infection.

    PubMed

    Emig-Agius, Dorothea; Olivieri, Kevin; Pache, Lars; Shih, Hsin Ling; Pustovalova, Olga; Bessarabova, Marina; Young, John A T; Chanda, Sumit K; Ideker, Trey

    2014-01-01

    Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study.

  4. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.

    PubMed

    Montaner, Silvia; Kufareva, Irina; Abagyan, Ruben; Gutkind, J Silvio

    2013-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction. Surprisingly, open reading frames for multiple GPCRs were hijacked in the process of coevolution between Herpesviridae family viruses and their human and mammalian hosts. Virally encoded GPCRs (vGPCRs) evolved as parts of viral genomes, and this evolution allowed the power of host GPCR signaling circuitries to be harnessed in order to ensure viral replicative success. Phylogenetically, vGPCRs are distantly related to human chemokine receptors, although they feature several unique characteristics. Here, we describe the molecular mechanisms underlying vGPCR-mediated viral pathogenesis. These mechanisms include constitutive activity, aberrant coupling to human G proteins and β-arrestins, binding and activation by human chemokines, and dimerization with other GPCRs expressed in infected cells. The likely structural basis for these molecular events is described for the two closest viral homologs of human GPCRs. This information may aid in the development of novel targeted therapeutic strategies against viral diseases.

  5. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

    PubMed Central

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V.; Kann, Michael; Villanueva, Rodrigo A.; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  6. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

    PubMed

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V; Kann, Michael; Villanueva, Rodrigo A; Loyola, Alejandra

    2016-05-13

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.

  7. Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes

    SciTech Connect

    Dresang, Lindsay R.; Teuton, Jeremy R.; Feng, Huichen; Jacobs, Jon M.; Camp, David G.; Purvine, Samuel O.; Gritsenko, Marina A.; Li, Zhihua; Smith, Richard D.; Sugden, Bill; Moore, Patrick S.; Chang, Yuan

    2011-12-20

    Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

  8. Novel approaches and challenges to treatment of CNS viral infections

    PubMed Central

    Nath, Avindra; Tyler, Kenneth L.

    2014-01-01

    Existing and emerging viral CNS infections are major sources of human morbidity and mortality. Treatments of proven efficacy are currently limited predominantly to herpesviruses and human immunodeficiency virus. Development of new therapies has been hampered by the lack of appropriate animal model systems for some important viruses and by the difficulty in conducting human clinical trials for diseases that may be rare, or in the case of arboviral infections, often have variable seasonal and geographic incidence. Nonetheless, many novel approaches to antiviral therapy are available including candidate thiazolide and purazinecarboxamide derivatives with potential broad-spectrum antiviral efficacy. New herpesvirus drugs include viral helicase-primase and terminase inhibitors. The use of antisense oligonucleotides and other strategies to interfere with viral RNA translation has shown efficacy in experimental models of CNS viral disease. Identifying specific molecular targets within viral replication cycles has led to many existing antivirals and will undoubtedly continue to be the basis of future drug design. A promising new area of research involves therapies based on enhanced understanding of host antiviral immune responses. Toll-like receptor agonists, and drugs that inhibit specific cytokines as well as interferon preparations have all shown potential therapeutic efficacy. Passive transfer of virus-specific cytotoxic T-lymphocytes have been used in humans and may provide an effective therapies for some herpesvirus infections and potentially for progressive multifocal leukoencephalopathy. Humanized monoclonal antibodies directed against specific viral proteins have been developed and in several cases evaluated in humans in settings including West Nile virus and HIV infection and in pre-exposure prophylaxis for rabies. PMID:23913580

  9. Presence of viral proteins in drinkable water--sufficient condition to consider water a vector of viral transmission?

    PubMed

    Gutiérrez, M F; Alvarado, M V; Martínez, E; Ajami, N J

    2007-01-01

    In order to determine the role of water as a possible vector for transmission of the most prevalent enteric viruses affecting infantile populations, 226 water samples were collected from Facatativa's (Colombian municipality located 30km away from Bogotá) water works in the years 2000, 2002, and 2005. The samples were clarified and virus was concentrated by filtering and ultrafiltering techniques. The presence of viral protein (VP) was assessed by enzyme immunoassay method (EIA) and viral RNA presence was detected by reverse trascriptase and polymerase chain reaction (RT-PCR). Using these techniques, one sample positive for Astrovirus (HAstV) was found in a sample collected from the river that supplies the aqueduct, two samples positive for Norovirus (NV) from fresh treated potable water and 13 samples positive for Rotavirus (RV), some in water from the plant during treatment and others from treated fresh water. RT-PCR inhibitors were also found in water samples obtained from the plant and in the fresh treated water. No inhibitors were found in the river water. VP, but no nucleic acid, was detected in the water samples at different stages of treatment, thus suggesting that the virus might have been complete and infectious at some stage prior to water purification.

  10. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    PubMed Central

    Julien, Perino; Thielens, Nicole M.; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-01-01

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27. PMID:23518578

  11. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  12. A Herpesviral Lytic Protein Regulates the Structure of Latent Viral Chromatin

    PubMed Central

    Raja, Priya; Lee, Jennifer S.; Pan, Dongli; Pesola, Jean M.; Coen, Donald M.

    2016-01-01

    ABSTRACT Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV), for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs) and microRNAs (miRNAs) as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0) is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo. Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections. PMID:27190217

  13. Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

    PubMed Central

    Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

    2015-01-01

    ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the

  14. Oligomeric viral proteins: small in size, large in presence

    PubMed Central

    Jayaraman, Bhargavi; Smith, Amber M.; Fernandes, Jason D.; Frankel, Alan D.

    2016-01-01

    Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states. PMID:27685368

  15. Analysis and Characterization of the Complete Genome of Tupaia (Tree Shrew) Herpesvirus

    PubMed Central

    Bahr, Udo; Darai, Gholamreza

    2001-01-01

    The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity. PMID:11312357

  16. Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

    PubMed

    Karttunen, Heidi; Savas, Jeffrey N; McKinney, Caleb; Chen, Yu-Hung; Yates, John R; Hukkanen, Veijo; Huang, Tony T; Mohr, Ian

    2014-07-03

    DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

  17. Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

    PubMed Central

    Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

    2015-01-01

    SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

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

    PubMed

    Wang, Robert Y L; Kuo, Rei-Lin; Ma, Wei-Chieh; Huang, Hsing-I; Yu, Jau-Song; Yen, Sih-Min; Huang, Chi-Ruei; Shih, Shin-Ru

    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 to 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.

  19. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

    PubMed Central

    O'Neill, R E; Talon, J; Palese, P

    1998-01-01

    Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex. PMID:9427762

  20. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    SciTech Connect

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian . E-mail: bzheng@hkucc.hku.hk

    2007-07-20

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

  1. Mechanisms of coronavirus cell entry mediated by the viral spike protein.

    PubMed

    Belouzard, Sandrine; Millet, Jean K; Licitra, Beth N; Whittaker, Gary R

    2012-06-01

    Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes-A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

  2. New insights into the expression and functions of the Kaposi's sarcoma-associated herpesvirus long noncoding PAN RNA.

    PubMed

    Conrad, Nicholas K

    2016-01-02

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is a clinically relevant pathogen associated with several human diseases that primarily affect immunocompromised individuals. KSHV encodes a noncoding polyadenylated nuclear (PAN) RNA that is essential for viral propagation and viral gene expression. PAN RNA is the most abundant viral transcript produced during lytic replication. The accumulation of PAN RNA depends on high levels of transcription driven by the Rta protein, a KSHV transcription factor necessary and sufficient for latent-to-lytic phase transition. In addition, KSHV uses several posttranscriptional mechanisms to stabilize PAN RNA. A cis-acting element, called the ENE, prevents PAN RNA decay by forming a triple helix with its poly(A) tail. The viral ORF57 and the cellular PABPC1 proteins further contribute to PAN RNA stability during lytic phase. PAN RNA functions are only beginning to be uncovered, but PAN RNA has been proposed to control gene expression by several different mechanisms. PAN RNA associates with the KSHV genome and may regulate gene expression by recruiting chromatin-modifying factors. Moreover, PAN RNA binds the viral latency-associated nuclear antigen (LANA) protein and decreases its repressive activity by sequestering it from the viral genome. Surprisingly, PAN RNA was found to associate with translating ribosomes, so this noncoding RNA may be additionally used to produce viral peptides. In this review, I highlight the mechanisms of PAN RNA accumulation and describe recent insights into potential functions of PAN RNA.

  3. Effect of metal catalyzed oxidation in recombinant viral protein assemblies

    PubMed Central

    2014-01-01

    Background Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. Results The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. Conclusions Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of

  4. Proteomic Analysis of Pathogenic and Attenuated Alcelaphine Herpesvirus 1▿

    PubMed Central

    Dry, Inga; Haig, David M.; Inglis, Neil F.; Imrie, Lisa; Stewart, James P.; Russell, George C.

    2008-01-01

    The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell. PMID:18353942

  5. Structural biology of the Bcl-2 family and its mimicry by viral proteins

    PubMed Central

    Kvansakul, M; Hinds, M G

    2013-01-01

    Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity

  6. Effect of truncation of the N-terminal region of the viral hemorrhagic septicemia virus (VHSV) P protein on viral replication.

    PubMed

    Park, Ji Sun; Kim, Min Sun; Choi, Seung Hyuk; Kang, Yue Jai; Kim, Ki Hong

    2015-11-01

    The phosphoprotein (P) of viral hemorrhagic septicemia virus (VHSV) plays an essential role in viral replication by interconnecting the L protein and the N protein-RNA complex. In this study, to investigate the role of the N-terminal region of the P protein in viral replication, we mutated the first or the first and second or the first, second, and third ATG codon into TGA stop codons. The respective mutants were named P1, P2, and P3. Recombinant VHSVs containing each mutated P gene (rVHSV-P1, -P2, and -P3) were successfully generated by supplying the intact P protein in trans. The rVHSV-P2 and -P3 were not generated from cells expressing truncated P proteins (P1, P2 or P3 protein), but the rVHSV-P1 produced infectious viruses, even in cells without any P-protein-expressing plasmids. Nucleotide sequence analysis of the P gene of rVHSV-P1 showed that a mutation had occurred that resulted in the fourth amino acid (isoleucine, ATT) being changed to methionine (ATG) without a frameshift (P0.5), suggesting that strong selection pressure might facilitate mutations that are advantageous or essential for virus replication. Infectious rVHSV-P2 and -P3 were produced in cells expressing the P0.5 protein, suggesting that the first three amino acids of the P protein of VHSV are dispensable for viral replication. Furthermore, although the P1 protein was shorter than the P0.5 protein by only two amino acid residues, no viruses were produced when the P1 protein was supplied indicating that the fourth and the fifth amino acid residues are indispensable for normal P protein functions involved in viral replication.

  7. Towards protein-based viral mimetics for cancer therapies.

    PubMed

    Unzueta, Ugutz; Céspedes, María Virtudes; Vázquez, Esther; Ferrer-Miralles, Neus; Mangues, Ramón; Villaverde, Antonio

    2015-05-01

    High resistance and recurrence rates, together with elevated drug clearance, compel the use of maximum-tolerated drug doses in cancer therapy, resulting in high-grade toxicities and limited clinical applicability. Promoting active drug accumulation in tumor tissues would minimize such issues and improve therapeutic outcomes. A new class of therapeutic drugs suitable for the task has emerged based on the concept of virus-mimetic nanocarriers, or 'artificial viruses'. Among the spectrum of materials under exploration in nanocarrier research, proteins offer unparalleled structural and functional versatility for designing virus-like molecular vehicles. By exhibiting 'smart' functions and biomimetic traits, protein-based nanocarriers will be a step ahead of the conventional drug-protein conjugates already in the clinic in ensuring efficient delivery of passenger antitumor drugs.

  8. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.

    PubMed

    Meng, Qiao; Hagemeier, Stacy R; Fingeroth, Joyce D; Gershburg, Edward; Pagano, Joseph S; Kenney, Shannon C

    2010-05-01

    Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC(50)] = 1.5 microM) and ACV (IC(50) = 4.1 microM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC(50) = 19.6 microM) and ACV (IC(50) = 36.4 microM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC(50) = 1.2 microM) and ACV (IC(50) = 2.8 microM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

  9. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    PubMed

    Hedil, Marcio; Kormelink, Richard

    2016-07-23

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  10. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

    PubMed Central

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

  11. The virally encoded killer proteins from Ustilago maydis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a 'killer' phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in ...

  12. Arenavirus budding resulting from viral-protein-associated cell membrane curvature.

    PubMed

    Schley, David; Whittaker, Robert J; Neuman, Benjamin W

    2013-09-06

    Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

  13. Arenavirus budding resulting from viral-protein-associated cell membrane curvature

    PubMed Central

    Schley, David; Whittaker, Robert J.; Neuman, Benjamin W.

    2013-01-01

    Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations. PMID:23864502

  14. The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.

    PubMed

    Stokes, A; Alber, D G; Greensill, J; Amellal, B; Carvalho, R; Taylor, L A; Doel, T R; Killington, R A; Halliburton, I W; Meredith, D M

    1996-01-01

    Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.

  15. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    PubMed

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  16. Role of microRNAs in herpesvirus latency and persistence.

    PubMed

    Grey, Finn

    2015-04-01

    The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.

  17. Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

    PubMed Central

    Cui, Hongguang

    2016-01-01

    ABSTRACT The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. IMPORTANCE Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically

  18. Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

    PubMed Central

    Kuny, Chad V.

    2016-01-01

    ABSTRACT Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner. IMPORTANCE Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally

  19. Multiple origins of viral capsid proteins from cellular ancestors

    PubMed Central

    Koonin, Eugene V.

    2017-01-01

    Viruses are the most abundant biological entities on earth and show remarkable diversity of genome sequences, replication and expression strategies, and virion structures. Evolutionary genomics of viruses revealed many unexpected connections but the general scenario(s) for the evolution of the virosphere remains a matter of intense debate among proponents of the cellular regression, escaped genes, and primordial virus world hypotheses. A comprehensive sequence and structure analysis of major virion proteins indicates that they evolved on about 20 independent occasions, and in some of these cases likely ancestors are identifiable among the proteins of cellular organisms. Virus genomes typically consist of distinct structural and replication modules that recombine frequently and can have different evolutionary trajectories. The present analysis suggests that, although the replication modules of at least some classes of viruses might descend from primordial selfish genetic elements, bona fide viruses evolved on multiple, independent occasions throughout the course of evolution by the recruitment of diverse host proteins that became major virion components. PMID:28265094

  20. Identification and Function of MicroRNAs Encoded by Herpesviruses*

    PubMed Central

    Bai, Zhi-Qiang; Lei, Xiu-Fen; Wang, Lin-Ding; Gao, Shou-Jiang

    2009-01-01

    MicroRNAs (miRNAs) play important roles in eukaryotes, plants and some viruses. It is increasingly clear that miRNAs-encoded by viruses can affect the viral life cycle and host physiology. Viral miRNAs could repress the innate and adaptive host immunity, modulate cellular signaling pathways, and regulate the expression of cellular and viral genes. These functions facilitate viral acute and persistent infections, and have profound effects on the host cell survival and disease progression. Here, we discuss the miRNAs encoded by herpesviruses, and their regulatory roles involved in virus-host interactions. PMID:20084183

  1. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    PubMed Central

    Kudryashova, Elena; Koneru, Pratibha C.; Kvaratskhelia, Mamuka; Strömstedt, Adam A.; Lu, Wuyuan; Kudryashov, Dmitri S.

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural “anti-chaperones”, i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins – the intrinsically low thermodynamic protein stability. PMID:27581352

  2. The Great Escape: Viral Strategies to Counter BST-2/Tetherin

    PubMed Central

    Douglas, Janet L.; Gustin, Jean K.; Viswanathan, Kasinath; Mansouri, Mandana; Moses, Ashlee V.; Früh, Klaus

    2010-01-01

    The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma–associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as “tetherin”. However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses. PMID:20485522

  3. Heat Shock Protein 27 Mediated Signaling in Viral Infection

    PubMed Central

    Rajaiya, Jaya; Yousuf, Mohammad A.; Singh, Gurdeep; Stanish, Heather; Chodosh, James

    2013-01-01

    Heat shock proteins (HSPs) play a critical role in many intracellular processes, including apoptosis and delivery of other proteins to intracellular compartments. Small HSPs have been shown previously to participate in many cellular functions, including IL-8 induction. Human adenovirus infection activates intracellular signaling, involving particularly the c-Src and mitogen-activated protein kinases [Natarajan, K., et al. (2003) J. Immunol. 170, 6234–6243]. HSP27 and MK2 are also phosphorylated, and c-Src, and its downstream targets, p38, ERK1/2, and c-Jun-terminal kinase (JNK), differentially mediate IL-8 and MCP-1 expression. Specifically, activation and translocation of transcription factor NFκB-p65 occurs in a p38-dependent fashion [Rajaiya, J., et al. (2009) Mol. Vision 15, 2879–2889]. Herein, we report a novel role for HSP27 in an association of p38 with NFκB-p65. Immunoprecipitation assays of virus-infected but not mock-infected cells revealed a signaling complex including p38 and NFκB-p65. Transfection with HSP27 short interfering RNA (siRNA) but not scrambled RNA disrupted this association and reduced the level of IL-8 expression. Transfection with HSP27 siRNA also reduced the level of nuclear localization of NFκB-p65 and p38. By use of tagged p38 mutants, we found that amino acids 279–347 of p38 are necessary for the association of p38 with NFκB-p65. These studies strongly suggest that HSP27, p38, and NFκB-p65 form a signalosome in virus-infected cells and influence downstream expression of pro-inflammatory mediators. PMID:22734719

  4. The Role of microRNAs in the Pathogenesis of Herpesvirus Infection

    PubMed Central

    Piedade, Diogo; Azevedo-Pereira, José Miguel

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein–Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis. PMID:27271654

  5. Shutoff of Host Gene Expression in Influenza A Virus and Herpesviruses: Similar Mechanisms and Common Themes.

    PubMed

    Rivas, Hembly G; Schmaling, Summer K; Gaglia, Marta M

    2016-04-16

    The ability to shut off host gene expression is a shared feature of many viral infections, and it is thought to promote viral replication by freeing host cell machinery and blocking immune responses. Despite the molecular differences between viruses, an emerging theme in the study of host shutoff is that divergent viruses use similar mechanisms to enact host shutoff. Moreover, even viruses that encode few proteins often have multiple mechanisms to affect host gene expression, and we are only starting to understand how these mechanisms are integrated. In this review we discuss the multiplicity of host shutoff mechanisms used by the orthomyxovirus influenza A virus and members of the alpha- and gamma-herpesvirus subfamilies. We highlight the surprising similarities in their mechanisms of host shutoff and discuss how the different mechanisms they use may play a coordinated role in gene regulation.

  6. Shutoff of Host Gene Expression in Influenza A Virus and Herpesviruses: Similar Mechanisms and Common Themes

    PubMed Central

    Rivas, Hembly G.; Schmaling, Summer K.; Gaglia, Marta M.

    2016-01-01

    The ability to shut off host gene expression is a shared feature of many viral infections, and it is thought to promote viral replication by freeing host cell machinery and blocking immune responses. Despite the molecular differences between viruses, an emerging theme in the study of host shutoff is that divergent viruses use similar mechanisms to enact host shutoff. Moreover, even viruses that encode few proteins often have multiple mechanisms to affect host gene expression, and we are only starting to understand how these mechanisms are integrated. In this review we discuss the multiplicity of host shutoff mechanisms used by the orthomyxovirus influenza A virus and members of the alpha- and gamma-herpesvirus subfamilies. We highlight the surprising similarities in their mechanisms of host shutoff and discuss how the different mechanisms they use may play a coordinated role in gene regulation. PMID:27092522

  7. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-09

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  8. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

    PubMed

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé; Emiliani, Stéphane; Gallois-Montbrun, Sarah

    2016-12-20

    Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

  9. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells

    PubMed Central

    1981-01-01

    Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells. PMID:6265461

  10. The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

    PubMed Central

    Proft, Alexandra; Spiesschaert, Bart; Izume, Satoko; Taferner, Selina; Lehmann, Maik J.; Azab, Walid

    2016-01-01

    The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells. PMID:27754319

  11. The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation.

    PubMed

    Bergson, Shir; Itzhak, Inbal; Wasserman, Talya; Gelgor, Anastasia; Kalt, Inna; Sarid, Ronit

    2016-12-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.

  12. Infectious Bursal Disease Virus-Host Interactions: Multifunctional Viral Proteins that Perform Multiple and Differing Jobs.

    PubMed

    Qin, Yao; Zheng, Shijun J

    2017-01-14

    Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease caused by IBD virus (IBDV). The consequent immunosuppression increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well. Since the genome of IBDV is relatively small, it has a limited number of proteins inhibiting the cellular antiviral responses and acting as destroyers to the host defense system. Thus, these virulence factors must be multifunctional in order to complete the viral replication cycle in a host cell. Insights into the roles of these viral proteins along with their multiple cellular targets in different pathways will give rise to a rational design for safer and effective vaccines. Here we summarize the recent findings that focus on the virus-cell interactions during IBDV infection at the protein level.

  13. Infectious Bursal Disease Virus-Host Interactions: Multifunctional Viral Proteins that Perform Multiple and Differing Jobs

    PubMed Central

    Qin, Yao; Zheng, Shijun J.

    2017-01-01

    Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease caused by IBD virus (IBDV). The consequent immunosuppression increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well. Since the genome of IBDV is relatively small, it has a limited number of proteins inhibiting the cellular antiviral responses and acting as destroyers to the host defense system. Thus, these virulence factors must be multifunctional in order to complete the viral replication cycle in a host cell. Insights into the roles of these viral proteins along with their multiple cellular targets in different pathways will give rise to a rational design for safer and effective vaccines. Here we summarize the recent findings that focus on the virus–cell interactions during IBDV infection at the protein level. PMID:28098808

  14. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    PubMed Central

    Wu, Nicholas C.; Olson, C. Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q.; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-01-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available. PMID:26132554

  15. Toll-like receptor 2 senses hepatitis C virus core protein but not infectious viral particles

    PubMed Central

    Hoffmann, Marco; Zeisel, Mirjam B.; Jilg, Nikolaus; Paranhos-Baccalà, Glaucia; Stoll-Keller, Françoise; Wakita, Takaji; Hafkemeyer, Peter; Blum, Hubert E.; Barth, Heidi; Henneke, Philipp; Baumert, Thomas F.

    2009-01-01

    Toll-like receptors (TLRs) are pathogen recognition molecules activating the innate immune system. Cell surface expressed TLRs, such as TLR2 and TLR4 have been shown to play an important role in human host defenses against viruses through sensing of viral structural proteins. In this study, we aimed to elucidate whether TLR2 and TLR4 participate in inducing antiviral immunity against hepatitis C virus by sensing viral structural proteins. We studied TLR2 and TLR4 activation by cell-culture derived infectious virions (HCVcc) and serum-derived virions in comparison to purified recombinant HCV structural proteins and enveloped virus-like particles. Incubation of TLR2 or TLR4 transfected cell lines with recombinant core protein resulted in activation of TLR2-dependent signaling. In contrast, neither infectious virions nor enveloped HCV-like particles triggered TLR2 and TLR4 signaling. These findings suggest that monomeric HCV core protein but not intact infectious particles are sensed by TLR2. Impairment of core-TLR interaction in infectious viral particles may contribute to escape from innate antiviral immune responses. PMID:20375602

  16. Phage phenomics: Physiological approaches to characterize novel viral proteins

    ScienceCinema

    Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

    2016-07-12

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  17. Phage phenomics: Physiological approaches to characterize novel viral proteins

    SciTech Connect

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  18. Phage phenomics: Physiological approaches to characterize novel viral proteins

    DOE PAGES

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

  19. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  20. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins.

    PubMed

    Granstedt, Andrea E; Bosse, Jens B; Thiberge, Stephan Y; Enquist, Lynn W

    2013-09-10

    A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.

  1. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    PubMed

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan

    2014-08-01

    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  2. The interaction between claudin-1 and dengue viral prM/M protein for its entry.

    PubMed

    Che, Pulin; Tang, Hengli; Li, Qianjun

    2013-11-01

    Dengue disease is becoming a huge public health concern around the world as more than one-third of the world's population living in areas at risk of infection. In an effort to assess host factors interacting with dengue virus, we identified claudin-1, a major tight junction component, as an essential cell surface protein for dengue virus entry. When claudin-1 was knocked down in Huh 7.5 cells via shRNA, the amount of dengue virus entering host cells was reduced. Consequently, the progeny virus productions were decreased and dengue virus-induced CPE was prevented. Furthermore, restoring the expression of claudin-1 in the knockdown cells facilitated dengue virus entry. The interaction between claudin-1 and dengue viral prM protein was further demonstrated using the pull-down assay. Deletion of the extracellular loop 1 (ECL1) of claudin-1 abolished such interaction, so did point mutations C54A, C64A and I32M on ECL1. These results suggest that the interaction between viral protein prM and host protein claudin-1 was essential for dengue entry. Since host and viral factors involved in virus entry are promising therapeutic targets, determining the essential role of claudin-1 could lead to the discovery of entry inhibitors with attractive therapeutic potential against dengue disease.

  3. High Serum Lipopolysaccharide-Binding Protein Level in Chronic Hepatitis C Viral Infection Is Reduced by Anti-Viral Treatments

    PubMed Central

    Nien, Hsiao-Ching; Hsu, Shih-Jer; Su, Tung-Hung; Yang, Po-Jen; Sheu, Jin-Chuan; Wang, Jin-Town; Chow, Lu-Ping; Chen, Chi-Ling

    2017-01-01

    Background Lipopolysaccharide-binding protein (LBP) has been reported to associate with metabolic diseases, such as obesity, diabetes, and non-alcoholic fatty liver disease. Since chronic hepatitis C virus (HCV) infection is associated with metabolic derangements, the relationship between LBP and HCV deserves additional studies. This study aimed to determine the serum LBP level in subjects with or without HCV infection and investigate the change of its level after anti-viral treatments with or without interferon. Methods and Findings We recruited 120 non-HCV subjects, 42 and 17 HCV-infected subjects respectively treated with peginterferon α-2a/ribavirin and direct-acting antiviral drugs. Basic information, clinical data, serum LBP level and abdominal ultrasonography were collected. All the subjects provided written informed consent before being enrolled approved by the Research Ethics Committee of the National Taiwan University Hospital. Serum LBP level was significantly higher in HCV-infected subjects than non-HCV subjects (31.0 ± 8.8 versus 20.0 ± 6.4 μg/mL; p-value < 0.001). After multivariate analyses, LBP at baseline was independently associated with body mass index, hemoglobin A1c, alanine aminotransferase (ALT) and HCV infection. Moreover, the baseline LBP was only significantly positively associated with ALT and inversely with fatty liver in HCV-infected subjects. The LBP level significantly decreased at sustained virologic response (27.4 ± 6.6 versus 34.6 ± 7.3 μg/mL, p-value < 0.001; 15.9 ± 4.4 versus 22.2 ± 5.7 μg/mL, p-value = 0.001), regardless of interferon-based or -free therapy. Conclusions LBP, an endotoxemia associated protein might be used as an inflammatory biomarker of both infectious and non-infectious origins in HCV-infected subjects. PMID:28107471

  4. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    NASA Astrophysics Data System (ADS)

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-05-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

  5. Regulation of promyelocytic leukemia (PML) protein levels and cell morphology by bovine herpesvirus 1 infected cell protein 0 (bICP0) and mutant bICP0 proteins that do not localize to the nucleus.

    PubMed

    Gaudreault, Natasha; Jones, Clinton

    2011-03-01

    BHV-1 is an important pathogen of cattle. The infected cell protein 0 (bICP0) encoded by BHV-1 is an important regulatory protein because it is constitutively expressed and can activate all viral promoters. The mechanism by which bICP0 activates viral promoters is not well understood because bICP0 does not appear to be a sequence specific binding protein. A C(3)HC(4) zinc RING (really interesting novel gene) motif at the N-terminus of bICP0 has E3 ubiquitin ligase activity, which is important for activating viral gene expression and inhibiting interferon dependent transcription. Like other alpha-herpesvirinae ICP0 homologues, bICP0 is associated with promyelocytic leukemia (PML) protein-containing nuclear domains. During productive infection of cultured cells, BHV-1 induces degradation of the PML protein, which correlates with efficient productive infection. In this study, we demonstrated that a plasmid expressing bICP0 reduces steady state levels of the PML protein, and the C(3)HC(4) zinc RING finger is important for PML degradation. Surprisingly, bICP0 mutants with an intact C(3)HC(4) zinc RING finger that lack a nuclear localization signal also reduces steady PML protein levels. In addition, mutant bICP0 proteins that primarily localize to the cytoplasm induced morphological changes in transfected cells. During productive infection, bICP0 was detected in the cytoplasm of low-passage bovine kidney, but not established bovine kidney cells. These studies demonstrated that bICP0, even when not able to efficiently localize to the nucleus, was able to induce degradation of the PML protein and alter the morphology of transfected cells.

  6. Artificial Neural Networks Trained to Detect Viral and Phage Structural Proteins

    PubMed Central

    Seguritan, Victor; Raymond, Amy; Lorimer, Don; Burgin, Alex B.; Salamon, Peter; Segall, Anca M.

    2012-01-01

    Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is

  7. Non-human Primate Schlafen11 Inhibits Production of Both Host and Viral Proteins

    PubMed Central

    Stabell, Alex C.; Hawkins, John; Li, Manqing; Gao, Xia; David, Michael; Press, William H.; Sawyer, Sara L.

    2016-01-01

    Schlafen11 (encoded by the SLFN11 gene) has been shown to inhibit the accumulation of HIV-1 proteins. We show that the SLFN11 gene is under positive selection in simian primates and is species-specific in its activity against HIV-1. The activity of human Schlafen11 is relatively weak compared to that of some other primate versions of this protein, with the versions encoded by chimpanzee, orangutan, gibbon, and marmoset being particularly potent inhibitors of HIV-1 protein production. Interestingly, we find that Schlafen11 is functional in the absence of infection and reduces protein production from certain non-viral (GFP) and even host (Vinculin and GAPDH) transcripts. This suggests that Schlafen11 may just generally block protein production from non-codon optimized transcripts. Because Schlafen11 is an interferon-stimulated gene with a broad ability to inhibit protein production from many host and viral transcripts, its role may be to create a general antiviral state in the cell. Interestingly, the strong inhibitors such as marmoset Schlafen11 consistently block protein production better than weak primate Schlafen11 proteins, regardless of the virus or host target being analyzed. Further, we show that the residues to which species-specific differences in Schlafen11 potency map are distinct from residues that have been targeted by positive selection. We speculate that the positive selection of SLFN11 could have been driven by a number of different factors, including interaction with one or more viral antagonists that have yet to be identified. PMID:28027315

  8. Quality by design approach for viral clearance by protein a chromatography.

    PubMed

    Zhang, Min; Miesegaes, George R; Lee, Michael; Coleman, Daniel; Yang, Bin; Trexler-Schmidt, Melody; Norling, Lenore; Lester, Philip; Brorson, Kurt A; Chen, Qi

    2014-01-01

    Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.

  9. The Ubiquitin System and Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Ashizawa, Akira; Higashi, Chizuka; Masuda, Kazuki; Ohga, Rie; Taira, Takahiro; Fujimuro, Masahiro

    2012-01-01

    Ubiquitination is a post-translational modification in which one or more ubiquitin molecules are covalently linked to lysine residues of target proteins. The ubiquitin system plays a key role in the regulation of protein degradation, which contributes to cell signaling, vesicular trafficking, apoptosis, and immune regulation. Bacterial and viral pathogens exploit the cellular ubiquitin system by encoding their own proteins to serve their survival and replication in infected cells. Recent studies have revealed that Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the ubiquitin system of infected cells to facilitate cell proliferation, anti-apoptosis, and evasion from immunity. This review summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cellular ubiquitin machinery. PMID:22375140

  10. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    SciTech Connect

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui; Zhang, Jun; Xia, Ning-Shao; Miao, Ji; Zhao, Qinjian

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  11. T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

    PubMed Central

    Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A.; Edwards, David M.; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K.; Pickup, David J.; Lewinsohn, David M.; Gold, Marielle C.; Wong, Scott W.; Sacha, Jonah B.; Slifka, Mark K.; Früh, Klaus

    2014-01-01

    Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

  12. New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

    PubMed Central

    Tyulkina, L.G.; Skurat, E.V.; Frolova, O.Yu.; Komarova, T.V.; Karger, E.M.; Atabekov, I.G.

    2011-01-01

    The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines. PMID:22649706

  13. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  14. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2005-07-05

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

  15. Intraventricular injection of myxoma virus results in transient expression of viral protein in mouse brain ependymal and subventricular cells.

    PubMed

    France, Megan R; Thomas, Diana L; Liu, Jia; McFadden, Grant; MacNeill, Amy L; Roy, Edward J

    2011-01-01

    Oncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.

  16. Regulation of Wnt/β-catenin signaling by herpesviruses

    PubMed Central

    Zwezdaryk, Kevin J; Combs, Joseph A; Morris, Cindy A; Sullivan, Deborah E

    2016-01-01

    The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda. PMID:27878101

  17. Characterization of the DNA polymerase gene of human herpesvirus 6.

    PubMed Central

    Teo, I A; Griffin, B E; Jones, M D

    1991-01-01

    The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group. Images PMID:1651403

  18. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    PubMed Central

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  19. Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection

    PubMed Central

    Lee, Jennifer S.; Raja, Priya

    2016-01-01

    ABSTRACT Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1) genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3) lysine 9-trimethylation (H3K9me3) and lysine 27-trimethylation (H3K27me3) during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms. PMID:26758183

  20. Identification and characterization of Kaposi's sarcoma-associated herpesvirus open reading frame 11 promotor activation

    SciTech Connect

    Chen, Lei

    2008-01-01

    Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family shared by some members of the gamma- herpesvirus subfamily. Little is known about this ORF11 homologous protein family. We have characterized an unknown open reading frame, ORF11, located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis reveals that ORF11 is expressed during the KSHV lytic cycle with delayed-early transcription kinetics. We have determined the 5{prime} and 3{prime} untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Core promoter region, representing ORF11 promoter activity, was mapped to a 159nt fragment 5{prime} most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. Interestingly, we found that ORF11 transcriptional activation is not responsive to Rta, the KSHV lytic switch protein. We also discovered that part of the ORF11 promoter region, the 209nt fragment upstream of the transcription start site, was repressed by phorbol esters. Our data help to understand transcription regulation of ORF11 and to elucidate roles of ORF11 in KSHV pathogenesis and life cycle.

  1. The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

    PubMed Central

    Kousoulas, Konstantin G.

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761–6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  2. A Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50 deletion mutant is defective for reactivation of latent virus and DNA replication.

    PubMed

    Xu, Yiyang; AuCoin, David P; Huete, Alicia Rodriguez; Cei, Sylvia A; Hanson, Lisa J; Pari, Gregory S

    2005-03-01

    Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Delta50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36Delta50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Delta50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Delta50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Delta50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.

  3. Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

    PubMed Central

    Cohen, Efrat M.

    2016-01-01

    Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells. PMID:27923068

  4. A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

    PubMed Central

    Campeau, Eric; Ruhl, Victoria E.; Rodier, Francis; Smith, Corey L.; Rahmberg, Brittany L.; Fuss, Jill O.; Campisi, Judith; Yaswen, Paul; Cooper, Priscilla K.; Kaufman, Paul D.

    2009-01-01

    The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we

  5. A proteomic study of the differential protein expression in MDBK cells after bovine herpesvirus type 1 infection (BHV-1) strain treatment

    PubMed Central

    Guo, Li; Yang, Yanling; Liu, Linna; Liao, Peng; Wen, Yongjun; Wu, Hua; Cheng, Shipeng

    2015-01-01

    Different BHV-1 strains, such as the virulent IBRV LN01/08 strains and the attenuated vaccine strain IBRV LNM, produces different clinical immune responses; however, the study of the differential protein expression in Madin-Darby bovine kidney (MDBK) cells after BHV-1-infection still remains unclear. Here, we applied a comparative proteomic strategy, based on 2D and MALDI-TOF/MS platforms, to examine the differential expression of proteins in MDBK cells that were treated and not treated with virulent IBRV LN01/08 and attenuated IBRV LNM strains. A total of eight differential proteins, including pyruvate kinase, heat shock protein (HSP) 90 (HSP90AA1 and HSP90AB1), annexin A, albumin (ALB), scinderin (SCIN), tubulin (alpha 1a) and vimentin (VIM), were identified. Among these proteins, pyruvate kinase, and HSP90 (HSP90AB1), tubulin and vimentin were identified in the virulent IBRV LN01/08 strain group, but were not identified in the attenuated IBRV LNM group. These results play an important role in tumor formation and development, cell migration, tumor cell line apoptosis, cell invasion and viral infection. The HSP90 (HSP90AA1) protein was identified in the control group and the attenuated IBRV LNM-infected group. Most studies have shown that HSP90 proteins were more of a cancer gene target, and inhibiting its function would result to oncogene degradation during cancer treatment. On the other hand, ALB is associated to cell differentiation, apoptosis, necrosis, cell death, viral infection, autophagy, interstitial tissue inflammation, and cell survival. These results provide a theoretical basis for the systematic understanding of BHV-1-infection mechanisms and BHV-1-induced immune responses. PMID:26064331

  6. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles.

    PubMed Central

    Furlong, D B; Nibert, M L; Fields, B N

    1988-01-01

    Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the mu 1C or sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state. Images PMID:3275434

  7. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

    SciTech Connect

    Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L. )

    1990-03-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

  8. Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus.

    PubMed

    Deroubaix, Aurélie; Osseman, Quentin; Cassany, Aurélia; Bégu, Dominique; Ragues, Jessica; Kassab, Somar; Lainé, Sébastien; Kann, Michael

    2015-01-01

    Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

  9. Concurrent oral shedding of feline calicivirus and feline herpesvirus 1 in cats with chronic gingivostomatitis.

    PubMed

    Lommer, M J; Verstraete, F J M

    2003-04-01

    Oral mucosal salivary samples were collected from 25 cats with chronic gingivostomatitis and 24 cats with periodontal disease. Viral culture and isolation of feline calicivirus and feline herpesvirus 1 were performed. Eighty-eight per cent of cats with chronic gingivostomatitis were shedding both viruses, compared to 21% of cats without chronic oral inflammatory disease. Cats with chronic gingivostomatitis are significantly more likely to concurrently shed both feline calicivirus and feline herpesvirus 1 than are cats with classical periodontal disease.

  10. Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission.

    PubMed

    Lorizate, Maier; Huarte, Nerea; Sáez-Cirión, Asier; Nieva, José L

    2008-01-01

    Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

  11. Protective effect of post-ischaemic viral delivery of heat shock proteins in vivo

    PubMed Central

    Badin, Romina A; Modo, Michael; Cheetham, Mike; Thomas, David L; Gadian, David G; Latchman, David S; Lythgoe, Mark F

    2009-01-01

    Heat shock proteins (HSPs) function as molecular chaperones involved in protein folding, transport and degradation and, in addition, they can promote cell survival both in vitro and in vivo after a range of stresses. Although some in vivo studies have suggested that HSP27 and HSP70 can be neuroprotective, current evidence is limited, particularly when HSPs have been delivered after an insult. The effect of overexpressing HSPs after transient occlusion of the middle cerebral artery in rats was investigated by delivering an attenuated herpes simplex viral vector (HSV-1) engineered to express HSP27 or HSP70 30 mins after tissue reperfusion. Magnetic resonance imaging scans were used to determine lesion size and cerebral blood flow at six different time points up to 1 month after stroke. Animals underwent two sensorimotor tests at the same time points to assess the relationship between lesion size and function. Results indicate that post-ischaemic viral delivery of HSP27, but not of HSP70, caused a statistically significant reduction in lesion size and induced a significant behavioural improvement compared with controls. This is the first evidence of effective post-ischaemic gene therapy with a viral vector expressing HSP27 in an experimental model of stroke. PMID:18781161

  12. Viral proteins of bovine papillomavirus type 4 during the development of alimentary canal tumours.

    PubMed

    Anderson, R A; Scobie, L; O'Neil, B W; Grindlay, G J; Campo, M S

    1997-07-01

    In cattle infection of the upper alimentary canal mucosa by bovine papillomavirus type 4 (BPV-4) results in the development of papillomas which can progress to cancer in animals fed on bracken fern. This paper describes a study of the cellular and subcellular distribution of a number of different BPV-4 products in experimentally-induced BPV-4 tumours. E8 and E4 proteins were detected solely as cytoplasmic antigens in the undifferentiated and differentiated layers of the papilloma, respectively; L2 was detected solely as a nuclear antigen in the differentiated layers, whereas E7 was present in either the nucleus or the cytoplasm depending on the differentiation stage of the keratinocyte. Replicative forms of viral DNA were detected from the spinous to the squamous layers. Viral antigens were not detected during papilloma regression or in carcinomas. E8 was most prominent in early developmental stages, while E4 and L2 were most abundant in mature papillomas. E7 was present in large amounts in both early and mature stages, declining at later stages. These results suggest a temporal and spatial requirement for the expression and function of the viral proteins.

  13. [HIV-1 p17 matrix protein is transported into the cell nucleus and binds with genomic viral RNA].

    PubMed

    Bukrinskaia, A G; Vorkunova, G K; Tentsov, Iu Iu

    1993-01-01

    We have shown that gag polyprotein p55 is cleaved in cytosol rapidly after its synthesis, during 2 h, and p17 enters the nuclei while p24 resides in cytosol. To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. Monoclonal antibodies against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential part from membranes while monoclonal antibodies against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and rise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

  14. The phiX174 protein J mediates DNA packaging and viral attachment to host cells.

    PubMed

    Bernal, Ricardo A; Hafenstein, Susan; Esmeralda, Raquel; Fane, Bentley A; Rossmann, Michael G

    2004-04-09

    Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational

  15. Regulation of herpesvirus macromolecular synthesis: temporal order of transcription of alpha genes is not dependent on the stringency of inhibition of protein synthesis.

    PubMed Central

    Mackem, S; Roizman, B

    1981-01-01

    Operationally, alpha genes of herpes simplex virus 1 were defined on the basis of the observations that they are the earliest genes expressed in the infected cell and that the transcription, processing, accumulation of the mRNA's in the infected cell cytoplasm can take place in the presence of inhibitors of protein synthesis, such as cycloheximide. In these studies, we translated in vitro the viral mRNA's extracted from cells infected maintained in the presence of cycloheximide, emetine, or anisomycin. Inasmuch as all the major alpha proteins (no. 0, 4, 22, and 27) were translated, we conclude that the transcription of all previously defined alpha genes is independent of the stringency of inhibition of protein synthesis and that pre-alpha genes cannot be detected in such experiments. Images PMID:6270385

  16. Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function.

    PubMed

    Donofrio, Gaetano; Herath, Shan; Sartori, Chiara; Cavirani, Sandro; Flammini, Cesidio Filippo; Sheldon, Iain Martin

    2007-07-01

    Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

  17. Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

    PubMed Central

    Pautasso, Sara; von Einem, Jens; Marschall, Manfred; Plachter, Bodo

    2016-01-01

    ABSTRACT A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the

  18. Myc is required for the maintenance of Kaposi's sarcoma-associated herpesvirus latency.

    PubMed

    Li, Xudong; Chen, Shijia; Feng, Jun; Deng, Hongyu; Sun, Ren

    2010-09-01

    Myc is deregulated by Kaposi's sarcoma-associated herpesvirus (KSHV) latent proteins, but its role in KSHV latency is not clear. We found that Myc knockdown with RNA interference (RNAi) induced KSHV reactivation and increased the protein and mRNA levels of RTA, a key viral regulator of KSHV reactivation. Myc knockdown increased, whereas Myc overexpression inhibited, RTA promoter activity. KSHV reactivation and the activation of the RTA promoter induced by Myc depletion were inhibited by c-Jun N-terminal kinase (JNK) and p38 inhibitors but not by a MEK1 inhibitor. Myc knockdown inhibited primary effusion lymphoma (PEL) cell proliferation through inducing apoptosis and G(1) cell cycle arrest. Thus, Myc may be a key cellular node coupling cellular transformation and KSHV latency.

  19. The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

    PubMed

    Belyavskyi, M; Braunagel, S C; Summers, M D

    1998-09-15

    Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

  20. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  1. Antiviral agents targeted to interact with viral capsid proteins and a possible application to human immunodeficiency virus.

    PubMed Central

    Rossmann, M G

    1988-01-01

    The tertiary structure of most icosahedral viral capsid proteins consists of an eight-stranded antiparallel beta-barrel with a hydrophobic interior. In a group of picornaviruses, this hydrophobic pocket can be filled by suitable organic molecules, which thereby stop viral uncoating after attachment and penetration into the host cell. The antiviral activity of these agents is probably due to increased rigidity of the capsid protein, which inhibits disassembly. The hydrophobic pocket may be an essential functional component of the protein and, therefore, may have been conserved in the evolution of many viruses from a common precursor. Since eight-stranded anti-parallel beta-barrels, with a topology as in viral capsid proteins, are not generally found for other proteins involved in cell metabolism, this class of antiviral agents is likely to be more virus-specific and less cytotoxic. Furthermore, the greatest conservation of viral capsid proteins occurs within this pocket, whereas the least conserved part is the antigenic exterior. Thus, compounds that bind to such a pocket are likely to be effective against a broader group of serologically distinct viruses. Discovery of antiviral agents of this type will, therefore, depend on designing compounds that can enter and fit snugly into the hydrophobic pocket of a particular viral capsid protein. The major capsid protein, p24, of human immunodeficiency virus would be a likely suitable target. PMID:3133655

  2. Regulation and autoregulation of the promoter for the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Jeong, Joseph H; Orvis, Joshua; Kim, Jong Wook; McMurtrey, Curtis P; Renne, Rolf; Dittmer, Dirk P

    2004-04-16

    Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 has been established as the etiological agent of Kaposi's sarcoma and certain AIDS-associated lymphomas. KSHV establishes latent infection in these tumors, invariably expressing high levels of the viral latency-associated nuclear antigen (LANA) protein. LANA is necessary and sufficient to maintain the KSHV episome. It also modulates viral and cellular transcription and has been implicated directly in oncogenesis because of its ability to bind to the p53 and pRb tumor suppressor proteins. Previously, we identified the LANA promoter (LANAp) and showed that it was positively regulated by LANA itself. Here, we present a detailed mutational analysis and define cis-acting elements and trans-acting factors for the core LANAp. We found that a downstream promoter element, TATA box, and GC box/Sp1 site at -29 are all individually required for activity. This architecture places LANAp into the small and unusual group of eukaryotic promoters that contain both the downstream promoter element and TATA element but lack a defined initiation site. Furthermore, we demonstrate that LANA regulates its own promoter via its C-terminal domain and does bind to a defined site within the core promoter.

  3. Application and correlation of nano resolution microscopy techniques to viral protein localization

    NASA Astrophysics Data System (ADS)

    Hodges, Jeffery Allen

    This dissertation is primarily focused on the application of super-resolution microscopy techniques to localization of viral proteins within envelope viruses. Advances in optical super-resolution microscopy techniques have enabled scientists to observe phenomena much smaller than the Abbe diffraction limit by stochastically limiting the number of molecules excited at a given instance and localizing their positions one at a time. Additionally, methods such as Atomic Force Microscopy (AFM) allow scientists to measure the topological features and material properties of samples through contact with a force probe. This dissertation describes the application of these two techniques to virology in order to localize internal viral proteins of enveloped virions, and measure their effect on the elastic properties of the virion. By utilizing super-resolution microscopy techniques such as Fluorescent Photo-Activated Localization Microscopy (fPALM) on virions, which have had their surface glycoproteins labeled with a photo-switchable label, the viral envelope may be accurately recovered. This dissertation describes the development and application of this technique as it applies to envelope recovery of Vesicular Stomatitis Virus (VSV) and Human Immunodeficiency Virus-1 (HIV-1). By fluorescently labeling proteins, which are internal to each of these viruses, I have been able to localize a variety of viral proteins within their recovered envelopes. This is done without significant damage to the virion, making this method a highly effective in vivo technique. In the case of VSV, an asymmetric localization along the central axis towards the blunt 5' end was found to exist for both the polymerase and phosphoproteins. These have been determined to occupy a region in the central cavity of ˜57 +/- 12 nm on the 5' end. This inhomogeneity of the underlying proteins such an asymmetry would predict that the Young's modulus would vary along the central axis of the virion. This dissertation

  4. Human herpesvirus 6.

    PubMed Central

    Braun, D K; Dominguez, G; Pellett, P E

    1997-01-01

    Human herpesvirus 6 variant A (HHV-6A) and human herpesvirus 6 variant B (HHV-6B) are two closely related yet distinct viruses. These visuses belong to the Roseolovirus genus of the betaherpesvirus subfamily; they are most closely related to human herpesvirus 7 and then to human cytomegalovirus. Over 95% of people older than 2 years of age are seropositive for either or both HHV-6 variants, and current serologic methods are incapable of discriminating infection with one variant from infection with the other. HHV-6A has not been etiologically linked to any human disease, but such an association will probably be found soon. HHV-6B is the etiologic agent of the common childhood illness exanthem subitum (roseola infantum or sixth disease) and related febrile illnesses. These viruses are frequently active and associated with illness in immunocompromised patients and may play a role in the etiology of Hodgkin's disease and other malignancies. HHV-6 is a commensal inhabitant of brains; various neurologic manifestations, including convulsions and encephalitis, can occur during primary HHV-6 infection or in immunocompromised patients. HHV-6 and distribution in the central nervous system are altered in patients with multiple sclerosis; the significance of this is under investigation. PMID:9227865

  5. Identification and characterization of the genomic termini and cleavage/packaging signals of gallid herpesvirus 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Herpesvirus replication within host cells produces concatameric genomic DNA which is cleaved into unit-length genomes and packaged into the capsid by a complex of proteins. The sites of cleavage have been identified for many herpesviruses and conserved signaling sequences involved in cleavage and p...

  6. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  7. Kinetics of Kaposi’s Sarcoma-Associated Herpesvirus Gene Expression

    PubMed Central

    Sun, Ren; Lin, Su-Fang; Staskus, Katherine; Gradoville, Lyndle; Grogan, Elizabeth; Haase, Ashley; Miller, George

    1999-01-01

    Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the “immediate-early” gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor

  8. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  9. Singapore grouper iridovirus protein VP088 is essential for viral infectivity

    PubMed Central

    Yuan, Yongming; Wang, Yunzhi; Liu, Qizhi; Zhu, Feng; Hong, Yunhan

    2016-01-01

    Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention. PMID:27498856

  10. Alzheimer's Associated β-Amyloid Protein Inhibits Influenza A Virus and Modulates Viral Interactions with Phagocytes

    PubMed Central

    White, Mitchell R.; Kandel, Ruth; Tripathi, Shweta; Condon, David; Qi, Li; Taubenberger, Jeffrey; Hartshorn, Kevan L.

    2014-01-01

    Accumulation of β-Amyloid (βA) is a key pathogenetic factor in Alzheimer's disease; however, the normal function of βA is unknown. Recent studies have shown that βA can inhibit growth of bacteria and fungi. In this paper we show that βA also inhibits replication of seasonal and pandemic strains of H3N2 and H1N1 influenza A virus (IAV) in vitro. The 42 amino acid fragment of βA (βA42) had greater activity than the 40 amino acid fragment. Direct incubation of the virus with βA42 was needed to achieve optimal inhibition. Using quantitative PCR assays βA42 was shown to reduce viral uptake by epithelial cells after 45 minutes and to reduce supernatant virus at 24 hours post infection. βA42 caused aggregation of IAV particles as detected by light transmission assays and electron and confocal microscopy. βA42 did not stimulate neutrophil H2O2 production or extracellular trap formation on its own, but it increased both responses stimulated by IAV. In addition, βA42 increased uptake of IAV by neutrophils. βA42 reduced viral protein synthesis in monocytes and reduced IAV-induced interleukin-6 production by these cells. Hence, we demonstrate for the first time that βA has antiviral activity and modulates viral interactions with phagocytes. PMID:24988208

  11. Tat acetylation modulates assembly of a viral-host RNA–protein transcription complex

    PubMed Central

    D'Orso, Iván; Frankel, Alan D.

    2009-01-01

    HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat–CycT1–TAR complexes by enhancing an interaction with the CycT1 Tat–TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA–protein complex, thereby tuning the levels of transcription elongation. PMID:19223581

  12. A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3

    PubMed Central

    Origgi, Francesco C.; Tecilla, Marco; Pilo, Paola; Aloisio, Fabio; Otten, Patricia; Aguilar-Bultet, Lisandra; Sattler, Ursula; Roccabianca, Paola; Romero, Carlos H.; Bloom, David C.; Jacobson, Elliott R.

    2015-01-01

    We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel

  13. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  14. Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus.

    PubMed

    Torre, D; Pugliese, A; Ferrario, G; Marietti, G; Forno, B; Zeroli, C

    1994-02-01

    Fibronectin (FN) is present in soluble and matrix forms in various body fluids and tissues, and has been shown to bind to several pathogens, including viruses. The interaction of FN with viral proteins of human immunodeficiency virus (HIV-1) was investigated by immunofluorescence technique using a cell line chronically infected with HIV-1 (H9-V). The results of this study showed that FN binds to HIV-1 infected cells, especially at FN concentration of 5 micrograms/ml. In addition, FN-pentapeptide has shown the ability to bind to HIV-1 infected cells. On the other hand, preincubation with antibodies against FN abolished the binding of FN to HIV-1 infected cells. Finally, FN has shown to bind to HIV-1 glycoproteins, including gp41 and gp120. In contrast, no binding to HIV-1 core proteins, including p15 and p24, was noted. We suggest that FN, in binding HIV-1 particles, may reduce viremia and thus may be involved in the clearance of viral proteins from the cells.

  15. Importance of SARS-CoV spike protein Trp-rich region in viral infectivity

    SciTech Connect

    Lu Yanning; Neo, T.L.; Liu, D.Xi.; Tam, James P.

    2008-07-04

    SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.

  16. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  17. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    PubMed Central

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  18. Protein engineering strategies for the development of viral vaccines and immunotherapeutics.

    PubMed

    Koellhoffer, Jayne F; Higgins, Chelsea D; Lai, Jonathan R

    2014-01-21

    Vaccines that elicit a protective broadly neutralizing antibody (bNAb) response and monoclonal antibody therapies are critical for the treatment and prevention of viral infections. However, isolation of protective neutralizing antibodies has been challenging for some viruses, notably those with high antigenic diversity or those that do not elicit a bNAb response in the course of natural infection. Here, we discuss recent work that employs protein engineering strategies to design immunogens that elicit bNAbs or engineer novel bNAbs. We highlight the use of rational, computational, and combinatorial strategies and assess the potential of these approaches for the development of new vaccines and immunotherapeutics.

  19. A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

    PubMed Central

    Du, Zhiyou; Murphy, Alex M.; Anggoro, Damar Tri; Tungadi, Trisna; Luang-In, Vijitra; Lewsey, Mathew G.; Rossiter, John T.; Powell, Glen; Smith, Alison G.; Carr, John P.

    2013-01-01

    Background Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’). Methodology/Principal Findings Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. Conclusions/Significance Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between

  20. Humanized-BLT mouse model of Kaposi's sarcoma-associated herpesvirus infection.

    PubMed

    Wang, Lin-Xu; Kang, Guobin; Kumar, Pankaj; Lu, Wuxun; Li, Yue; Zhou, You; Li, Qingsheng; Wood, Charles

    2014-02-25

    Lack of an effective small-animal model to study the Kaposi's sarcoma-associated herpesvirus (KSHV) infection in vivo has hampered studies on the pathogenesis and transmission of KSHV. The objective of our study was to determine whether the humanized BLT (bone marrow, liver, and thymus) mouse (hu-BLT) model generated from NOD/SCID/IL2rγ mice can be a useful model for studying KSHV infection. We have tested KSHV infection of hu-BLT mice via various routes of infection, including oral and intravaginal routes, to mimic natural routes of transmission, with recombinant KSHV over a 1- or 3-mo period. Infection was determined by measuring viral DNA, latent and lytic viral transcripts and antigens in various tissues by PCR, in situ hybridization, and immunohistochemical staining. KSHV DNA, as well as both latent and lytic viral transcripts and proteins, were detected in various tissues, via various routes of infection. Using double-labeled immune-fluorescence confocal microscopy, we found that KSHV can establish infection in human B cells and macrophages. Our results demonstrate that KSHV can establish a robust infection in the hu-BLT mice, via different routes of infection, including the oral mucosa which is the most common natural route of infection. This hu-BLT mouse not only will be a useful model for studying the pathogenesis of KSHV in vivo but can potentially be used to study the routes and spread of viral infection in the infected host.

  1. Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence.

    PubMed

    Tanaka, Michiko; Kato, Akihisa; Satoh, Yuko; Ide, Takahiro; Sagou, Ken; Kimura, Kayo; Hasegawa, Hideki; Kawaguchi, Yasushi

    2012-05-01

    Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.

  2. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    PubMed

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  3. Aquareovirus NS80 recruits viral proteins to its inclusions, and its C-terminal domain is the primary driving force for viral inclusion formation.

    PubMed

    Shao, Ling; Guo, Hong; Yan, Li-Ming; Liu, Huan; Fang, Qin

    2013-01-01

    Cytoplasmic inclusion bodies formed in reovirus-infected cells are the sites of viral replication and assembly. Previous studies have suggested that the NS80 protein of aquareovirus may be involved in the formation of viral inclusion bodies. However, it remains unknown whether other viral proteins are involved in the process, and what regions of NS80 may act coordinately in mediating inclusion formation. Here, we observed that globular cytoplasmic inclusions were formed in virus-infected cells and viral proteins NS80 and NS38 colocalized in the inclusions. During transfection, singly expressed NS80 could form cytoplasmic inclusions and recruit NS38 and GFP-tagged VP4 to these structures. Further treatment of cells with nocodazole, a microtubule inhibitor, did not disrupt the inclusion, suggesting that inclusion formation does not rely on microtubule network. Besides, we identified that the region 530-742 of NS80 was likely the minimal region required for inclusion formation, and the C-tail, coiled-coil region as well as the conserved linker region were essential for inclusion phenotype. Moreover, with series deletions from the N-terminus, a stepwise conversion occurred from large condensed cytoplasmic to small nuclear inclusions, then to a diffused intracellular distribution. Notablely, we found that the nuclear inclusions, formed by NS80 truncations (471 to 513-742), colocalized with cellular protein β-catenin. These data indicated that NS80 could be a major mediator in recruiting NS38 and VP4 into inclusion structures, and the C-terminus of NS80 is responsible for inclusion formation.

  4. Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

    PubMed

    Souquere-Besse, Sylvie; Pichard, Evelyne; Filhol, Odile; Legrand, Valerie; Rosa-Calatrava, Manuel; Hovanessian, Ara G; Cochet, Claude; Puvion-Dutilleul, Francine

    2002-03-15

    The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.

  5. Tales from the crypt and coral reef: the successes and challenges of identifying new herpesviruses using metagenomics

    PubMed Central

    Houldcroft, Charlotte J.; Breuer, Judith

    2015-01-01

    Herpesviruses are ubiquitous double-stranded DNA viruses infecting many animals, with the capacity to cause disease in both immunocompetent and immunocompromised hosts. Different herpesviruses have different cell tropisms, and have been detected in a diverse range of tissues and sample types. Metagenomics—encompassing viromics—analyses the nucleic acid of a tissue or other sample in an unbiased manner, making few or no prior assumptions about which viruses may be present in a sample. This approach has successfully discovered a number of novel herpesviruses. Furthermore, metagenomic analysis can identify herpesviruses with high degrees of sequence divergence from known herpesviruses and does not rely upon culturing large quantities of viral material. Metagenomics has had success in two areas of herpesvirus sequencing: firstly, the discovery of novel exogenous and endogenous herpesviruses in primates, bats and cnidarians; and secondly, in characterizing large areas of the genomes of herpesviruses previously only known from small fragments, revealing unexpected diversity. This review will discuss the successes and challenges of using metagenomics to identify novel herpesviruses, and future directions within the field. PMID:25821447

  6. Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets.

    PubMed

    Dansako, Hiromichi; Hiramoto, Hiroki; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2014-08-01

    During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs.

  7. The ORF3 Protein of Porcine Circovirus Type 2 Is Involved in Viral Pathogenesis In Vivo

    PubMed Central

    Liu, Jue; Chen, Isabelle; Du, Qingyun; Chua, Huikheng; Kwang, Jimmy

    2006-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a novel identified protein, ORF3, was not essential for PCV2 replication in cultured PK15 cells and played a major role in virus-induced apoptosis. To evaluate the role of the ORF3 protein in viral pathogenesis in vivo, we inoculated 8-week-old BALB/c mice that have been developed for PCV2 replication with ORF3-deficient mutant PCV2 (mPCV2). By 42 days postinoculation, all of the mice inoculated with the mPCV2, as well as with wild-type PCV2 (wPCV2), had seroconverted to PCV2 capsid antibody, and the mutant induced levels of PCV2 antibodies that were higher than those of the wPCV2. The PCV2 genomic copy numbers in serum were significantly higher (P < 0.05) in the wPCV2-inoculated mice than in mice inoculated with the mPCV2. Also, the wPCV2 caused microscopic lesions characterized by lymphocyte depletion with histiocytic infiltration of lymphoid organs, but the mutant virus failed to induce any obvious pathological lesions. In situ hybridization and immunohistochemical analyses also showed that larger amounts of viral DNA and antigens were detected in the lymph nodes of the wPCV2-inoculated than mPCV2-inoculated mice. Furthermore, animals of the wPCV2-inoculated group showed significant downshifts of CD8+ T-cell subsets of peripheral blood lymphocytes compared to the control mice (P < 0.05) at various time points postinoculation. Also, the proportions of the CD4+ and CD4+ CD8+ cells were significantly reduced in wPCV2-inoculated mice at some time points postinoculation. In contrast, there are some reductions in the proportions of these subsets in the mutant virus-inoculated mice, but the proportions do not decrease significantly. Taken together, these results demonstrate that the ORF3 protein is also dispensable for viral replication in vivo and that it plays an important role in viral pathogenesis. PMID

  8. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  9. Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

    PubMed Central

    Gutiérrez, C; Freire, R; Salas, M; Hermoso, J M

    1994-01-01

    The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex. Images PMID:8306969

  10. Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions.

    PubMed Central

    Lu, Y L; Spearman, P; Ratner, L

    1993-01-01

    The subcellular localization of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) was examined by subcellular fractionation. In HIV-1-infected peripheral blood mononuclear cells, Vpr was found in the nuclear and membrane fractions as well as the conditioned medium. Expression of Vpr without other HIV-1 proteins, in two different eukaryotic expression systems, demonstrated a predominant localization of Vpr in the nuclear matrix and chromatin extract fractions. Deletion of the carboxyl-terminal 19-amino-acid arginine-rich sequence impaired Vpr nuclear localization. Indirect immunofluorescence confirmed the nuclear localization of Vpr and also indicated a perinuclear location. Expression of Vpr alone did not result in export of the protein from the cell, but when coexpressed with the Gag protein, Vpr was exported and found in virus-like particles. A truncated Gag protein, missing the p6 sequence and a portion of the p9 sequence, was incapable of exporting Vpr from the cell. Regulation of Vpr localization may be important in the influence of this protein on virus replication. Images PMID:8411357

  11. Diagnosis and Control of Viral Diseases of Reproductive Importance: Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea.

    PubMed

    Newcomer, Benjamin W; Givens, Daniel

    2016-07-01

    Both bovine viral diarrhea virus and bovine herpesvirus 1 can have significant negative reproductive impacts on cattle health. Vaccination is the primary control method for the viral pathogens in US cattle herds. Polyvalent, modified-live vaccines are recommended to provide optimal protection against various viral field strains. Of particular importance to bovine viral diarrhea control is the limitation of contact of pregnant cattle with potential viral reservoirs during the critical first 125 days of gestation.

  12. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    PubMed Central

    Esparo, Nicolle M.; Child, Stephanie J.; Geballe, Adam P.

    2016-01-01

    During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α. PMID:27780231

  13. Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression.

    PubMed

    Chiang, Ya Wen; Yeh, Chia Feng; Yen, Ming Hong; Lu, Chi Yu; Chiang, Lien Chai; Shieh, Den En; Chang, Jung San

    2017-02-23

    Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

  14. Integrated Computational Approach for Virtual Hit Identification against Ebola Viral Proteins VP35 and VP40

    PubMed Central

    Mirza, Muhammad Usman; Ikram, Nazia

    2016-01-01

    The Ebola virus (EBOV) has been recognised for nearly 40 years, with the most recent EBOV outbreak being in West Africa, where it created a humanitarian crisis. Mortalities reported up to 30 March 2016 totalled 11,307. However, up until now, EBOV drugs have been far from achieving regulatory (FDA) approval. It is therefore essential to identify parent compounds that have the potential to be developed into effective drugs. Studies on Ebola viral proteins have shown that some can elicit an immunological response in mice, and these are now considered essential components of a vaccine designed to protect against Ebola haemorrhagic fever. The current study focuses on chemoinformatic approaches to identify virtual hits against Ebola viral proteins (VP35 and VP40), including protein binding site prediction, drug-likeness, pharmacokinetic and pharmacodynamic properties, metabolic site prediction, and molecular docking. Retrospective validation was performed using a database of non-active compounds, and early enrichment of EBOV actives at different false positive rates was calculated. Homology modelling and subsequent superimposition of binding site residues on other strains of EBOV were carried out to check residual conformations, and hence to confirm the efficacy of potential compounds. As a mechanism for artefactual inhibition of proteins through non-specific compounds, virtual hits were assessed for their aggregator potential compared with previously reported aggregators. These systematic studies have indicated that a few compounds may be effective inhibitors of EBOV replication and therefore might have the potential to be developed as anti-EBOV drugs after subsequent testing and validation in experiments in vivo. PMID:27792169

  15. Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

    PubMed

    Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

    2017-04-01

    Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD(+)-dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV.

  16. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    PubMed

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  17. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  18. The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids

    PubMed Central

    Cowan, Graham H.; Roberts, Alison G.; Chapman, Sean N.; Ziegler, Angelika; Savenkov, Eugene I.; Torrance, Lesley

    2012-01-01

    The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1–2 μm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts. PMID:23269927

  19. Human herpesvirus 8 glycoprotein B binds the entry receptor DC-SIGN.

    PubMed

    Hensler, Heather R; Tomaszewski, Monica J; Rappocciolo, Giovanna; Rinaldo, Charles R; Jenkins, Frank J

    2014-09-22

    We have previously shown that human herpesvirus 8 (HHV-8) uses DC-SIGN as an entry receptor for dendritic cells, macrophages and B cells. The viral attachment protein for DC-SIGN is unknown. HHV-8 virions contain five conserved herpesvirus glycoproteins, a single unique glycoprotein, and two predicted glycoproteins. Previous studies have shown that DC-SIGN binds highly mannosylated glycoproteins. The HHV-8 glycoprotein B (gB) has been reported to be highly mannosylated, and therefore we hypothesized that gB will bind to DC-SIGN. In this report we confirm that gB has a high mannose carbohydrate structure and demonstrate for the first time that it binds DC-SIGN in a dose-dependent manner. We also identify key amino acids in the DC-SIGN carbohydrate recognition domain that are required for HHV-8 infection and compare these results with published binding regions for ICAM-2/3 and HIV-1 gp120. These results clarify some of the initial events in HHV-8 entry and can be used for the design of targeted preventive therapies.

  20. Equine herpesvirus-1 infection disrupts interferon regulatory factor-3 (IRF-3) signaling pathways in equine endothelial cells.

    PubMed

    Sarkar, Sanjay; Balasuriya, Udeni B R; Horohov, David W; Chambers, Thomas M

    2016-05-01

    Equine herpesvirus-1 (EHV-1) is a major respiratory viral pathogen of horses, causing upper respiratory tract disease, abortion, neonatal death, and neurological disease that may lead to paralysis and death. EHV-1 replicates initially in the respiratory epithelium and then spreads systemically to endothelial cells lining the small blood vessels in the uterus and spinal cord leading to abortion and EHM in horses. Like other herpesviruses, EHV-1 employs a variety of mechanisms for immune evasion including suppression of type-I interferon (IFN) production in equine endothelial cells (EECs). Previously we have shown that the neuropathogenic T953 strain of EHV-1 inhibits type-I IFN production in EECs and this is mediated by a viral late gene product. But the mechanism of inhibition was not known. Here we show that T953 strain infection of EECs induced degradation of endogenous IRF-3 protein. This in turn interfered with the activation of IRF-3 signaling pathways. EHV-1 infection caused the activation of the NF-κB signaling pathways, suggesting that inhibition of type-I IFN production is probably due to interference in IRF-3 and not NF-κB signal transduction.

  1. Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus.

    PubMed

    Chen, Yuming; Lu, Zhen; Zhang, Lizhou; Gao, Li; Wang, Nian; Gao, Xiang; Wang, Yongqiang; Li, Kai; Gao, Yulong; Cui, Hongyu; Gao, Honglei; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2016-01-04

    VP3 protein is a structural protein which plays important roles in the virus assembly and the inhibition of antiviral innate immunity of infectious bursal disease virus (IBDV). To explore the potential roles of VP3 in the interplay of IBDV with the host cell, an immunoprecipitation (IP)-coupled mass spectra (MS) screening was performed and the host cellular ribosomal protein L4 (RPL4) was identified as a putative interacting partner of VP3 protein. The interaction of RPL4 with VP3 was further confirmed by co-immunoprecipitation (co-IP) and their colocalization in DF1 cells were observed by confocal microscopy. In addition, knockdown of RPL4 in DF1 cells resulted in reductions of the viral protein pVP2 expression and the virus titers, which reveals a significant role of RPL4 in IBDV replication. Taken together, we indicated for the first time that ribosomal protein L4 (RPL4) was an interacting partner of VP3 and involved in the modulation of IBDV replication. The present study contributes to further understanding the pathogenic mechanism of IBDV.

  2. Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids

    PubMed Central

    Glasgow, Jeff E.; Capehart, Stacy L.; Francis, Matthew B.; Tullman-Ercek, Danielle

    2012-01-01

    The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments. PMID:22953696

  3. Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

    PubMed

    Smales, C Mark; Pepper, Duncan S; James, David C

    2002-01-05

    To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

  4. Bell's palsy and herpesviruses.

    PubMed

    Gilbert, Stan C

    2002-12-01

    A growing body of evidence links reactivation of herpesviruses (primarily varicella zoster virus and herpes simplex virus type 1) with the development of a large proportion of cases of acute peripheral facial palsy, a syndrome commonly known by its eponym, Bell's palsy. This article reviews the definition and natural history of the disease, its underlying anatomy and pathophysiology, the data linking herpetic reactivation with development of signs and symptoms, and therapeutic trials utilizing antiviral therapy. In addition, it poses the question, would earlier intervention with antivirals make a larger impact on outcomes?

  5. Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

    PubMed

    Mondal, Arindam; Victor, Ken G; Pudupakam, R S; Lyons, Charles E; Wertz, Gail W

    2014-02-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

  6. Emydid herpesvirus 1 infection in northern map turtles (Graptemys geographica) and painted turtles (Chrysemys picta).

    PubMed

    Ossiboff, Robert J; Newton, Alisa L; Seimon, Tracie A; Moore, Robert P; McAloose, Denise

    2015-05-01

    A captive, juvenile, female northern map turtle (Graptemys geographica) was found dead following a brief period of weakness and nasal discharge. Postmortem examination identified pneumonia with necrosis and numerous epithelial, intranuclear viral inclusion bodies, consistent with herpesviral pneumonia. Similar intranuclear inclusions were also associated with foci of hepatocellular and splenic necrosis. Polymerase chain reaction (PCR) screening of fresh, frozen liver for the herpesviral DNA-dependent DNA polymerase gene yielded an amplicon with 99.2% similarity to recently described emydid herpesvirus 1 (EmyHV-1). Molecular screening of turtles housed in enclosures that shared a common circulation system with the affected map turtle identified 4 asymptomatic, EmyHV-1 PCR-positive painted turtles (Chrysemys picta) and 1 asymptomatic northern map turtle. Herpesvirus transmission between painted and map turtles has been previously suggested, and our report provides the molecular characterization of a herpesvirus in asymptomatic painted turtles that can cause fatal herpesvirus-associated disease in northern map turtles.

  7. Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

    PubMed

    Cervantes-Salazar, Margot; Angel-Ambrocio, Antonio H; Soto-Acosta, Ruben; Bautista-Carbajal, Patricia; Hurtado-Monzon, Arianna M; Alcaraz-Estrada, Sofia L; Ludert, Juan E; Del Angel, Rosa M

    2015-10-01

    Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

  8. Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity.

    PubMed

    Yang, Su-Lin; Chou, Ying-Ting; Wu, Cheng-Nan; Ho, Mei-Shang

    2011-11-01

    Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose.

  9. The Chromatin Landscape of Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Toth, Zsolt; Brulois, Kevin; Jung, Jae U.

    2013-01-01

    Kaposi’s sarcoma-associated herpesvirus is an oncogenic γ-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis. PMID:23698402

  10. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    SciTech Connect

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

  11. Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas).

    PubMed

    Barbosa-Solomieu, V; Dégremont, L; Vázquez-Juárez, R; Ascencio-Valle, F; Boudry, P; Renault, T

    2005-01-01

    Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.

  12. Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell recepto...

  13. Isolation and immunizations with hepatitis A viral structural proteins: induction of antiprotein, antiviral, and neutralizing responses.

    PubMed

    Hughes, J V; Stanton, L W

    1985-08-01

    An immune affinity purification procedure for hepatitis A virus (HAV) was designed which yielded milligram quantities of the virus with greater than 95% purity. The major structural proteins VP-1, VP-2, and VP-3 were isolated from the purified virus by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to immunize Lewis rats (three to four doses, 10 to 15 micrograms per dose). The two Lewis rats immunized with VP-1 developed a strong antibody response to VP-1, as determined by Western blot analysis and immune precipitation of the denatured protein. These animals also developed a good antibody response to the whole virus, as demonstrated by a positive response in a competitive radioimmunoassay (HAV antibody test) and by precipitation of the whole virus. In addition, both animals developed a low titer neutralizing antibody to HAV, as demonstrated by an in vitro cell culture assay. While the two rats receiving VP-2 developed only minimal responses to the protein and to the virus by the same assays described above, one of the two developed a significant neutralizing antibody to HAV. The immunization of one Lewis rat with VP-3 induced a good antibody response to both denatured protein and the whole virus. This serum sample was also demonstrated to neutralize the viral infectivity. Finally, two rabbits that had received inoculations of sodium dodecyl sulfate and heat-disrupted HAV (containing 20 to 30 micrograms of each protein per dose) developed good antiprotein responses to all of the proteins and good antiviral responses, including a consistently significant neutralizing activity. The neutralizing antibody responses suggest that the structural proteins of HAV, or a portion of them, could provide the basis for a subunit vaccine for HAV.

  14. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    PubMed

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.

  15. PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription

    PubMed Central

    Faust, Tyler B.; Li, Yang; Jang, Gwendolyn M.; Johnson, Jeffrey R.; Yang, Shumin; Weiss, Amit; Krogan, Nevan J.; Frankel, Alan D.

    2017-01-01

    Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire. PMID:28345603

  16. Toll-interacting protein inhibits HIV-1 infection and regulates viral latency.

    PubMed

    Li, Chuan; Kuang, Wen-Dong; Qu, Di; Wang, Jian-Hua

    2016-06-24

    HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4(+) T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4(+) T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4(+) T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency.

  17. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release

    PubMed Central

    Raghava, Smita; Giorda, Kristina M.; Romano, Fabian B.; Heuck, Alejandro P.; Hebert, Daniel N.

    2014-01-01

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers. PMID:23651212

  18. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    PubMed

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment.

  19. Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens.

    PubMed

    Valansi, Clari; Moi, David; Leikina, Evgenia; Matveev, Elena; Graña, Martín; Chernomordik, Leonid V; Romero, Héctor; Aguilar, Pablo S; Podbilewicz, Benjamin

    2017-03-06

    Cell-cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell-cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus-cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion.

  20. Interplay of viral miRNAs and host mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-10-01

    Recent experiments indicate that several viruses may encode microRNAs (miRNAs) in cells. Such RNAs may interfere with the host mRNAs and proteins. We present a kinetic analysis of this interplay. In our treatment, the viral miRNA is considered to be able to associate with the host mRNA with subsequent degradation. This process may result in a decline of the mRNA population and also in a decline of the population of the protein encoded by this mRNA. With these ingredients, we first show the types of the corresponding steady-state kinetics in the cases of positive and negative regulation of the miRNA synthesis by the protein. In addition, we scrutinize the situation when the protein regulates the virion replication or, in other words, provides a feedback for the replication. For the negative feedback, the replication rate is found to increase with increasing the intracellular virion population. For the positive feedback, the replication rate first increases and then drops. These features may determine the stability of steady states.

  1. 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.

  2. Discovery of herpesviruses in Canadian wildlife.

    PubMed

    Dalton, Chimoné S; van de Rakt, Karen; Fahlman, Åsa; Ruckstuhl, Kathreen; Neuhaus, Peter; Popko, Richard; Kutz, Susan; van der Meer, Frank

    2017-02-01

    Herpesviruses (HVs) have a wide range of hosts in the animal kingdom. The result of infection with HVs can vary from asymptomatic to fatal diseases depending on subtype, strain, and host. To date, little is known about HVs naturally circulating in wildlife species and the impact of these viruses on other species. In our study, we used genetic and comparative approaches to increase our understanding of circulating HVs in Canadian wildlife. Using nested polymerase chain reaction targeting a conserved region of the HV DNA polymerase gene, we analyzed material derived from wildlife of western and northern Canada collected between February 2009 and Sept 2014. For classification of new virus sequences, we compared our viral sequences with published sequences in GenBank to identify conserved residues and motifs that are unique to each subfamily, alongside phylogenetic analysis. All alphaherpesviruses shared a conserved tryptophan (W856) and tyrosine (Y880), betaherpesviruses all shared a serine (S836), and gammaherpesviruses had a conserved glutamic acid (E835). Most of our wildlife HV sequences grouped together with HVs from taxonomically related host species. From Martes americana, we detected previously uncharacterized alpha- and beta-herpesviruses.

  3. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

    PubMed Central

    Deb, S; Jackson, C T; Subler, M A; Martin, D W

    1992-01-01

    Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA. Images PMID:1356162

  4. The immunological relationship between canine herpesvirus and four other herpesviruses.

    PubMed

    Manning, A; Buchan, A; Skinner, G R; Durham, J; Thompson, H

    1988-07-01

    Canine herpesvirus (CHV) was compared with four other herpesviruses by several serological techniques. Cross-neutralization was demonstrated between CHV and herpes simplex virus types 1 and 2 and pseudorabies virus. Non-neutralizing cross-reactions were found with these viruses and also with equine abortion virus and bovine mammillitis virus. The data suggest that CHV is immunologically more closely related to herpes simplex virus than to the other viruses used in this study.

  5. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

    SciTech Connect

    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.

    2009-12-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

  6. Canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins.

    PubMed

    Licitra, Beth N; Duhamel, Gerald E; Whittaker, Gary R

    2014-08-22

    Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.

  7. Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells.

    PubMed

    Yeates, T O; Tsai, Y; Tanaka, S; Sawaya, M R; Kerfeld, C A

    2007-06-01

    Many proteins self-assemble to form large supramolecular complexes. Numerous examples of these structures have been characterized, ranging from spherical viruses to tubular protein assemblies. Some new kinds of supramolecular structures are just coming to light, while it is likely there are others that have not yet been discovered. The carboxysome is a subcellular structure that has been known for more than 40 years, but whose structural and functional details are just now emerging. This giant polyhedral body is constructed as a closed shell assembled from several thousand protein subunits. Within this protein shell, the carboxysome encapsulates the CO(2)-fixing enzymes, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and carbonic anhydrase; this arrangement enhances the efficiency of cellular CO(2) fixation. The carboxysome is present in many photosynthetic and chemoautotrophic bacteria, and so plays an important role in the global carbon cycle. It also serves as the prototypical member of what appears to be a large class of primitive protein-based organelles in bacteria. A series of crystal structures is beginning to reveal the secrets of how the carboxysome is assembled and how it enhances the efficiency of CO(2) fixation. Some of the assembly principles revealed in the carboxysome are reminiscent of those seen in icosahedral viral capsids. In addition, the shell appears to be perforated by pores for metabolite transport into and out of the carboxysome, suggesting comparisons to the pores through oligomeric transmembrane proteins, which serve to transport small molecules across the membrane bilayers of cells and eukaryotic organelles.

  8. Chromosomally integrated human herpesvirus 6: questions and answers.

    PubMed

    Pellett, Philip E; Ablashi, Dharam V; Ambros, Peter F; Agut, Henri; Caserta, Mary T; Descamps, Vincent; Flamand, Louis; Gautheret-Dejean, Agnès; Hall, Caroline B; Kamble, Rammurti T; Kuehl, Uwe; Lassner, Dirk; Lautenschlager, Irmeli; Loomis, Kristin S; Luppi, Mario; Lusso, Paolo; Medveczky, Peter G; Montoya, Jose G; Mori, Yasuko; Ogata, Masao; Pritchett, Joshua C; Rogez, Sylvie; Seto, Edward; Ward, Katherine N; Yoshikawa, Tetsushi; Razonable, Raymund R

    2012-05-01

    Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease.

  9. Chromosomally integrated human herpesvirus 6: questions and answers

    PubMed Central

    Pellett, Philip E; Ablashi, Dharam V; Ambros, Peter F; Agut, Henri; Caserta, Mary T; Descamps, Vincent; Flamand, Louis; Gautheret-Dejean, Agnès; Hall, Caroline B; Kamble, Rammurti T; Kuehl, Uwe; Lassner, Dirk; Lautenschlager, Irmeli; Loomis, Kristin S; Luppi, Mario; Lusso, Paolo; Medveczky, Peter G; Montoya, Jose G; Mori, Yasuko; Ogata, Masao; Pritchett, Joshua C; Rogez, Sylvie; Seto, Edward; Ward, Katherine N; Yoshikawa, Tetsushi; Razonable, Raymund R

    2012-01-01

    SUMMARY Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease. Copyright © 2011 John Wiley & Sons, Ltd. PMID:22052666

  10. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

    PubMed

    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  11. Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

    PubMed

    Lin, Pei-Wen; Huang, Yi-Jen; John, Joseph Abraham Christopher; Chang, Ya-Nan; Yuan, Chung-Hsiang; Chen, Wen-Ya; Yeh, Chiao-Hwa; Shen, San-Tai; Lin, Fu-Pang; Tsui, Wen-Huei; Chang, Chi-Yao

    2008-01-01

    The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.

  12. HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses--a review.

    PubMed

    Becker, Y

    1994-07-01

    The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8+ cytotoxic T cells (CTLs), may be needed. Antiviral CD8+ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8+ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen

  13. Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication

    PubMed Central

    Zengel, James; Pickar, Adrian; Xu, Pei; Lin, Alita

    2015-01-01

    ABSTRACT Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. IMPORTANCE Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development. PMID:25948749

  14. A longitudinal study of poor performance and subclinical respiratory viral activity in Standardbred trotters

    PubMed Central

    Back, Helena; Penell, Johanna; Pringle, John; Isaksson, Mats; Ronéus, Nils; Treiberg Berndtsson, Louise; Ståhl, Karl

    2015-01-01

    Introduction While clinical respiratory disease is considered a main cause of poor performance in horses, the role of subclinical respiratory virus infections is less clear and needs further investigation. Aims and objectives In this descriptive longitudinal study the relationship of markers of subclinical respiratory viral activity to occurrence of poor performance in racing Standardbred trotters was investigated. Material and methods 66 elite Standardbred trotters were followed for 13 months by nasal swabs analysed with qPCR for equine influenza virus, equine arteritis virus, equine rhinitis B virus (ERBV), equine herpesvirus type 1(EHV-1) and equine herpesvirus type 4 (EHV-4) and serology to equine rhinitis A virus (ERAV), ERBV, EHV-1 and EHV-4, as well as the acute phase protein serum amyloid A (SAA). Findings on lab analyses were subsequently assessed for possible correlations to workload performance and trainer opinion measures of poor performance. Results Despite occurrence of poor performance and subclinical viral activity the authors were unable to detect association neither between subclinical viral activity and poor performance, nor between SAA elevations and either viral activity or poor performance. Conclusions Consistent with earlier study results, antibody titres to ERBV remained high for at least a year and few horses two years or older were seronegative to either ERAV or ERBV. In absence of clinical signs, serology to common respiratory viruses appears to have little diagnostic benefit in evaluation of poor performance in young athletic horses. PMID:26392904

  15. Human herpesvirus-6 enhances natural killer cell cytotoxicity via IL-15.

    PubMed Central

    Flamand, L; Stefanescu, I; Menezes, J

    1996-01-01

    The marked tropism of human herpesvirus-6 (HHV-6) for natural killer (NK) cells and T lymphocytes has led us to investigate the effect of HHV-6 on cellular cytotoxicity. We describe here how HHV-6 infection of peripheral blood mononuclear cells (PBMC) leads to upregulation of their NK cell cytotoxicity. The induction of NK cell activity by HHV-6 was abrogated by monoclonal antibodies (mAbs) to IL-15 but not by mAbs to other cytokines (IFN-alpha, IFN-gamma, TNF-alpha, TNF-beta, IL-2, IL-12) suggesting that IL-15 secreted in response to viral infection was responsible for the observed effect. Furthermore, NK activation by HHV-6 was blocked with mAb to CD122, as well as by human anti-HHV-6 neutralizing antibodies. Using RT-PCR, we were able to detect IL-15 mRNA upregulation in purified monocyte and NK cell preparations. IL-15 protein synthesis was increased in response to HHV-6. Finally, addition of IL-15 to PBMC cultures was found to severely curtail HHV-6 expression. Taken together, our data suggest that enhanced NK activity in response to viral infection represent a natural anti-viral defense mechanism aimed at rapidly eliminating virus-infected cells. PMID:8617868

  16. Molecular evolution of viral multifunctional proteins: the case of potyvirus HC-Pro.

    PubMed

    Hasiów-Jaroszewska, Beata; Fares, Mario A; Elena, Santiago F

    2014-01-01

    Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlapping.

  17. Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes

    PubMed Central

    Liu, Jianying; Liu, Yang; Nie, Kaixiao; Du, Senyan; Qiu, Jingjun; Pang, Xiaojing; Wang, Penghua; Cheng, Gong

    2016-01-01

    Summary The arbovirus life cycle involves viral transfer between a vertebrate host and an arthropod vector, and acquisition of virus from an infected mammalian host by a vector is an essential step in this process. Here, we report that flavivirus nonstructural protein-1 (NS1), which is abundantly secreted into the serum of an infected host, plays a critical role in flavivirus acquisition by mosquitoes. The presence of dengue virus (DENV) and Japanese encephalitis virus (JEV) NS1s in the blood of infected interferon alpha and gamma receptor-deficient mice (AG6) facilitated virus acquisition by their native mosquito vectors because the protein enabled the virus to overcome the immune barrier of the mosquito midgut. Active immunization of AG6 mice with a modified DENV NS1 reduced DENV acquisition by mosquitoes and protected mice against a lethal DENV challenge, suggesting that immunization with NS1 could reduce the number of virus-carrying mosquitoes as well as the incidence of flaviviral diseases. Our study demonstrates that flaviviruses utilize NS1 proteins produced during their vertebrate phases to enhance their acquisition by vectors, which might be a result of flavivirus evolution to adapt to multiple host environments. PMID:27562253

  18. The Vpr protein from HIV-1: distinct roles along the viral life cycle

    PubMed Central

    Le Rouzic, Erwann; Benichou, Serge

    2005-01-01

    The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle. PMID:15725353

  19. A Low Protein Binding Cationic Poly(2-oxazoline) as Non-Viral Vector

    PubMed Central

    He, Zhijian; Miao, Lei; Jordan, Rainer; S-Manickam, Devika; Luxenhofer, Robert; Kabanov, Alexander V

    2015-01-01

    Developing safe and efficient non-viral gene delivery systems remains a major challenge. We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA), CPOx forms small (diameter ≈ 80 nm) and narrowly dispersed polyplexes (PDI < 0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(l-lysine) (PEG-b-PLL). CPOx/pDNA polyplexes at N/P = 5 bound considerably less plasma protein compared to polyplexes of PEG-b-PLL at the same N/P ratio. This is a unique aspect of the developed polyplexes emphasizing their potential for systemic delivery in vivo. The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery. PMID:25846127

  20. Protein Trans-Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

    PubMed Central

    Li, Juan; Sun, Wenchang; Wang, Bing; Xiao, Xiao

    2008-01-01

    Abstract Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Split-inteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly functional 6.3-kb Becker-form dystrophin cDNA was broken into two pieces and modified by adding appropriate split-intein coding sequences, resulting in split-genes sufficiently small for packaging in AAV vectors. The two split-genes, after codelivery into target cells, produced two polypeptides that spontaneously trans-spliced to form the expected Becker-form dystrophin protein in cell culture in vitro. Delivering the split-genes by AAV1 vectors into the muscle of a mouse model of Duchenne muscular dystrophy rendered therapeutic gene expression and benefits. PMID:18788906

  1. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    PubMed

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  2. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.

  3. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  4. The conundrum of a unique protein encoded by citrus tristeza virus that is dispensable for infection of most hosts yet shows characteristics of a viral movement protein.

    PubMed

    Bak, Aurélie; Folimonova, Svetlana Y

    2015-11-01

    Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts.

  5. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

    PubMed

    Sun, Yingjie; Dong, Luna; Yu, Shengqing; Wang, Xiaoxu; Zheng, Hang; Zhang, Pin; Meng, Chunchun; Zhan, Yuan; Tan, Lei; Song, Cuiping; Qiu, Xusheng; Wang, Guijun; Liao, Ying; Ding, Chan

    2017-04-01

    Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

  6. Kaposi’s Sarcoma Herpesvirus Genome Persistence

    PubMed Central

    Juillard, Franceline; Tan, Min; Li, Shijun; Kaye, Kenneth M.

    2016-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA’s role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease. PMID:27570517

  7. Purification in an active form of the phage phi 29 protein p4 that controls the viral late transcription.

    PubMed Central

    Barthelemy, I; Lázaro, J M; Méndez, E; Mellado, R P; Salas, M

    1987-01-01

    The phage phi 29 protein p4, that controls viral late transcription, was highly purified from Escherichia coli cells harbouring a gene 4-containing plasmid. This protein, representing about 6% of the total cellular protein, was obtained in a highly purified form. The protein was characterized as p4 by amino acid analysis and NH2-terminal sequence determination. The purified protein was active in an in vitro transcription assay, allowing specific initiation of transcription at the phi 29 A3 late promoter in the presence of Bacillus subtilis sigma 43-RNA polymerase holoenzyme. Images PMID:3671066

  8. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins.

    PubMed

    Suryawanshi, Gajendra W; Hoffmann, Alexander

    2015-12-07

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target

  9. The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication

    PubMed Central

    Gu, Baohua; Liu, Changbao; Lin-Goerke, Juili; Maley, Derrick R.; Gutshall, Lester L.; Feltenberger, Cynthia A.; Del Vecchio, Alfred M.

    2000-01-01

    Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis. PMID:10644352

  10. CD147 and downstream ADAMTSs promote the tumorigenicity of Kaposi's sarcoma-associated herpesvirus infected endothelial cells.

    PubMed

    Dai, Lu; Trillo-Tinoco, Jimena; Chen, Yihan; Bonstaff, Karlie; Del Valle, Luis; Parsons, Chris; Ochoa, Augusto C; Zabaleta, Jovanny; Toole, Bryan P; Qin, Zhiqiang

    2016-01-26

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS), which preferentially arise in immunocompromised patients and lack effective therapeutic options. We have previously shown that KSHV or viral protein LANA up-regulates the glycoprotein CD147, thereby inducing primary endothelial cell invasiveness. In the current study, we identify the global network controlled by CD147 in KSHV-infected endothelial cells using Illumina microarray analysis. Among downstream genes, two specific metalloproteases, ADAMTS1 and 9, are strongly expressed in AIDS-KS tissues and contribute to KSHV-infected endothelial cell invasiveness through up-regulation of IL-6 and VEGF. By using a KS-like nude mouse model, we found that targeting CD147 and downstream ADAMTSs significantly suppressed KSHV-induced tumorigenesis in vivo. Taken together, targeting CD147 and associated proteins may represent a promising therapeutic strategy against these KSHV-related malignancies.

  11. The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

    PubMed

    Luan, Hexiang; Shine, M B; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

    2016-09-01

    The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

  12. Proteomics computational analyses suggest that the carboxyl terminal glycoproteins of Bunyaviruses are class II viral fusion protein (beta-penetrenes)

    PubMed Central

    Garry, Courtney E; Garry, Robert F

    2004-01-01

    The Bunyaviridae family of enveloped RNA viruses includes five genuses, orthobunyaviruses, hantaviruses, phleboviruses, nairoviruses and tospoviruses. It has not been determined which Bunyavirus protein mediates virion:cell membrane fusion. Class II viral fusion proteins (beta-penetrenes), encoded by members of the Alphaviridae and Flaviviridae, are comprised of three antiparallel beta sheet domains with an internal fusion peptide located at the end of domain II. Proteomics computational analyses indicate that the carboxyl terminal glycoprotein (Gc) encoded by Sandfly fever virus (SAN), a phlebovirus, has a significant amino acid sequence similarity with envelope protein 1 (E1), the class II fusion protein of Sindbis virus (SIN), an Alphavirus. Similar sequences and common structural/functional motifs, including domains with a high propensity to interface with bilayer membranes, are located collinearly in SAN Gc and SIN E1. Gc encoded by members of each Bunyavirus genus share several sequence and structural motifs. These results suggest that Gc of Bunyaviridae, and similar proteins of Tenuiviruses and a group of Caenorhabditis elegans retroviruses, are class II viral fusion proteins. Comparisons of divergent viral fusion proteins can reveal features essential for virion:cell fusion, and suggest drug and vaccine strategies. PMID:15544707

  13. Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

    PubMed

    Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

    2012-10-12

    The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  14. The Leeuwenhoek Lecture, 1997. Marek's disease herpesvirus: oncogenesis and prevention.

    PubMed

    Biggs, P M

    1997-12-29

    There are a number of neoplasias for which a herpesvirus is an essential part of the aetiology. Of these, Marek's disease is the most common and provides excellent opportunities for the study of a herpesvirus-induced tumour both experimentally and under natural conditions in the field. Marek's disease is caused by an alpha herpesvirus; it differs from the other oncogenic herpesviruses which are gamma herpesviruses. It is a ubiquitous virus in poultry populations of the world and is highly cell-associated and contagious, yet only a proportion of infected fowl develop tumours. Evidence is presented to suggest that at least one of the reasons for a wide variation in the incidence of the disease is a temporal interplay between virulent viruses and viruses of low or no virulence. The viral genes associated with the oncogenicity of Marek's disease virus (MDV) are discussed and it is concluded that it is likely that several genes are involved. Finally, a brief history of vaccination to control Marek's disease is given and mode of action discussed. It is concluded that the mechanism of protection is mainly through an antiviral cell mediated immune response, resulting in a lowered challenge virus burden. Marek's disease viruses over the past 40 years have been evolving greater oncogenicity, some of which are not adequately controlled by the vaccines that are currently available. It is suggested that for MDV to produce tumours, there is a need for the cytolytic infection phase and that infection must be with an MDV which possesses a functional gC, ICP4 for maintaining latency which allows the expression of at least the 1.8 kb family, pp38, meq, and possibly pp14 genes, for maintaining the tumour state and possibly initiating this state. Intervention in this process reduces the chance of tumour formation and incidence in a population which can occur through natural or man-mediated infection with non-pathogenic MDVs.

  15. Bovine herpesvirus type 1 marker vaccine induces cross-protection against bubaline herpesvirus type 1 in water buffalo.

    PubMed

    Montagnaro, Serena; De Martinis, Claudio; Iovane, Valentina; Ciarcia, Roberto; Damiano, Sara; Nizza, Sandra; De Martino, Luisa; Iovane, Giuseppe; Pagnini, Ugo

    2014-09-01

    Water buffalo (Bubalus bubalis) are susceptible to bovine herpesvirus type 1 (BoHV-1) and a species-specific herpesvirus, bubaline herpesvirus type 1 (BuHV-1). In this study, an attenuated marker BoHV-1 based vaccine against BuHV-1 challenge was evaluated to determine whether it induces protection from viral replication. One group of water buffalo calves was immunized with an attenuated BoHV-1 marker vaccine. A second group was not vaccinated and used as the control. During the post-vaccination period, we monitored the humoral immune response. The efficacy of the vaccine was tested after intranasal challenge of the calves with a BuHV-1 strain. The experiment showed that after vaccination, BuHV-1 replication was significantly reduced by approximately three titer points compared to the controls. The control animals showed high levels of viral shedding and mild signs associated with BuHV-1 infection. Therefore, our study provides evidence for the existence of cross-protection between BoHV-1 and BuHV-1 in buffalo calves.

  16. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    PubMed

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  17. Efficient HIV-1 replication can occur in the absence of the viral matrix protein.

    PubMed Central

    Reil, H; Bukovsky, A A; Gelderblom, H R; Göttlinger, H G

    1998-01-01

    Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells. PMID:9564051

  18. Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)

    PubMed Central

    Milutinovic, Snezana; Heynen-Genel, Susanne; Chao, Elizabeth; Dewing, Antimone; Solano, Ricardo; Milan, Loribelle; Barron, Nikki; He, Min; Diaz, Paul W.; Matsuzawa, Shu-ichi; Reed, John C.; Hassig, Christian A.

    2016-01-01

    Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli. PMID:27031987

  19. Argonaute proteins in cardiac tissue contribute to the heart injury during viral myocarditis.