bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion
Authors: Senkevich Tatiana G. , Moss Bernard 
Published: March 2005

ABSTRACT
The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.
Visualization and Characterization of the Intracellular Movement of Vaccinia Virus Intracellular Mature Virions
Authors: Ward Brian M. 
Published: March 2005

ABSTRACT
Previous work indicated that vaccinia intracellular mature virus (IMV) utilizes microtubules to move from the viral factory to the site of intracellular envelopment and that expression of the viral A27 protein is required for this transport. To investigate further the role of A27 in IMV intracellular transport, a recombinant vaccinia virus was constructed that had the A27L gene deleted and expressed a yellow fluorescent protein (YFP)-A4 chimera in place of the normal A4 protein. The resulting recombinant, vYFP-A4/ΔA27, produced relatively normal quantities of virus in a one-step growth curve but had a small plaque phenotype. Subsequent experiments demonstrated that vYFP-A4/ΔA27 was severely defective in envelope virus production. Despite the absence of A27, live digital video fluorescent microscopy visualized YFP-labeled IMV movement in cells infected with the recombinant. Virion movement approached 3 μm/s and was sensitive to the microtubule depolymerizing drug nocodazole. In addition, IMV could be discerned transiting away from and back towards viral factories. Immunofluorescent staining determined that the distance traveled by A27-deficient virions was sufficient for transport to the site of envelopment. These results indicate that IMVs are capable of bidirectional movement on microtubules, suggesting that they are able to interact with both kinesin and dynein microtubule motors in the absence of A27 and that the distance traveled is sufficient to deliver IMV to the site of wrapping.
Expression of Matrix Metalloproteinases and Their Tissue Inhibitor during Viral Encephalitis
Authors: Zhou Jiehao , Marten Norman W. , Bergmann Cornelia C. , Macklin Wendy B. , Hinton David R. , Stohlman Stephen A. 
Published: March 2005

ABSTRACTMatrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.
Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120
Authors: Desmezieres Emmanuel , Gupta Nidhi , Vassell Russell , He Yong , Peden Keith , Sirota Lev , Yang Zhongning , Wingfield Paul , Weiss Carol D. 
Published: March 2005

ABSTRACTHuman immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.
Inhibition of Murine Leukemia Virus Envelope Protein (Env) Processing by Intracellular Expression of the Env N-Terminal Heptad Repeat Region
Authors: Ou Wu , Silver Jonathan 
Published: March 2005

ABSTRACT
A conserved structural motif in the envelope proteins of several viruses consists of an N-terminal, alpha-helical, trimerization domain and a C-terminal region that refolds during fusion to bind the N-helix trimer. Interaction between the N and C regions is believed to pull viral and target membranes together in a crucial step during membrane fusion. For several viruses with type I fusion proteins, C regions pack as alpha-helices in the grooves between N-helix monomers, and exogenously added N- and C-region peptides block fusion by inhibiting the formation of the six-helix bundle. For other viruses, including influenza virus and murine leukemia virus (MLV), there is no evidence for comparably extended C-region alpha-helices, although a short, non-alpha-helical interaction structure has been reported for influenza virus. We tested candidate N-helix and C-region peptides from MLV for their ability to inhibit cell fusion but found no inhibitory activity. In contrast, intracellular expression of the MLV N-helix inhibited fusion by efficiently blocking proteolytic processing and intracellular transport of the envelope protein. The results highlight another mechanism by which the N-helix peptides can inhibit fusion.
Comprehensive Analysis of Ebola Virus GP1 in Viral Entry
Authors: Manicassamy Balaji , Wang Jizhen , Jiang Haiqing , Rong Lijun 
Published: March 2005

ABSTRACT
Ebola virus infection is initiated by interactions between the viral glycoprotein GP1 and its cognate receptor(s), but little is known about the structure and function of GP1 in viral entry, partly due to the concern about safety when working with the live Ebola virus and the difficulty of manipulating the RNA genome of Ebola virus. In this study, we have used a human immunodeficiency virus-based pseudotyped virus as a surrogate system to dissect the role of Ebola virus GP1 in viral entry. Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with respect to protein expression, processing, viral incorporation, and viral entry has allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues. Furthermore, six amino acids in this region have been identified as critical residues for early events in Ebola virus entry, and among these, three are clustered and are implicated as part of a potential receptor-binding pocket. In addition, substitutions of some 30 residues in GP1 are shown to adversely affect GP1 expression, processing, and viral incorporation, suggesting that these residues are involved in the proper folding and/or overall conformation of GP. Sequence comparison of the GP1 proteins suggests that the majority of the critical residues for GP folding and viral entry identified in Ebola virus GP1 are conserved in Marburg virus. These results provide information for elucidating the structural and functional roles of the filoviral glycoproteins and for developing potential therapeutics to block viral entry.
The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4
Authors: Baxter Michael K. , McPhillips Maria G. , Ozato Keiko , McBride Alison A. 
Published: March 2005

ABSTRACT
The papillomavirus transcriptional activator, E2, is involved in key functions of the viral life cycle. These include transcriptional regulation, viral DNA replication, and viral genome segregation. The transactivation domain of E2 is required for each of these functions. To identify the regions of the domain that mediate binding to mitotic chromosomes, a panel of mutations has been generated and their effect on various E2 functions has been analyzed. A structural model of the bovine papillomavirus type 1 (BPV1) E2 transactivation domain was generated based on its homology with the solved structure of the human papillomavirus type 16 (HPV16) domain. This model was used to identify distinct surfaces of the domain to be targeted by point mutation to further delineate the functional region of the transactivation domain responsible for mitotic chromosome association. The mutated E2 proteins were assessed for mitotic chromosome binding and, in addition, transcriptional activation and transcriptional repression activities. Mutation of amino acids R37 and I73, which are located on a surface of the domain that in HPV16 E2 is reported to mediate self-interaction, completely eliminated mitotic chromosome binding. Mitotic chromosome binding activity was found to correlate well with the ability to interact with the cellular chromosomal associated factor Brd4, which has recently been proposed to mediate the association between BPV1 E2 and mitotic chromosomes.
Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo
Authors: Ito Hideki , Sommer Marvin H. , Zerboni Leigh , Baiker Armin , Sato Bunji , Liang Ruibin , Hay John , Ruyechan William , Arvin Ann M. 
Published: March 2005

ABSTRACT
Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaΔ35(#1) and rOkaΔ35(#2), and replaced ORF35 at a nonnative site, generating two rOkaΔ35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaΔ35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaΔ35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaΔ35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaΔ35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaΔ35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaΔ35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.
Role of Low CD4 Levels in the Influence of Human Immunodeficiency Virus Type 1 Envelope V1 and V2 Regions on Entry and Spread in Macrophages
Authors: Walter Brandon L. , Wehrly Kathy , Swanstrom Ronald , Platt Emily , Kabat David , Chesebro Bruce 
Published: March 2005

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo.
Constitutive Expression of Alpha Interferon by Skin Dendritic Cells Confers Resistance to Infection by Foot-and-Mouth Disease Virus
Authors: Bautista Elida M. , Ferman Geoffrey S. , Gregg Douglas , Brum Mario C. S. , Grubman Marvin J. , Golde William T. 
Published: March 2005

ABSTRACTThe role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-β) mRNA but not IFN-α mRNA. However, these DC secreted both IFN-α and IFN-β proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-α/β, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-α protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-α in uninfected animals. This is the first demonstration of the constitutive expression of IFN-α in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.
The p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication
Authors: Monkewich Sandra , Lin Han-Xin , Fabian Marc R. , Xu Wei , Na Hong , Ray Debashish , Chernysheva Olena A. , Nagy Peter D. , White K. Andrew 
Published: March 2005

ABSTRACT

The replication of positive-strand RNA viral genomes involves various
cis
-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of
Tomato bushy stunt virus
. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.
Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication
Authors: Pogany Judit , White K. Andrew , Nagy Peter D. 
Published: March 2005

ABSTRACT

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus,
Tomato bushy stunt virus
(TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C · C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related
Turnip crinkle virus
and distantly related
Hepatitis C virus
to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in
Saccharomyces cerevisiae
cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in
Tombusviridae
encode comparable replicase proteins, these results may be relevant to other members of this large virus family.
Escape Mutations Alter Proteasome Processing of Major Histocompatibility Complex Class I-Restricted Epitopes in Persistent Hepatitis C Virus Infection
Authors: Kimura Yoichi , Gushima Toshifumi , Rawale Sharad , Kaumaya Pravin , Walker Christopher M. 
Published: March 2005

ABSTRACTMutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.
Interleukin-15 Increases Effector Memory CD8+T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques
Authors: Mueller Yvonne M. , Petrovas Constantinos , Bojczuk Paul M. , Dimitriou Ioannis D. , Beer Brigitte , Silvera Peter , Villinger Francois , Cairns J. Scott , Gracely Edward J. , Lewis Mark G. , Katsikis Peter D. 
Published: March 2005

ABSTRACTInterleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 μg/kg of body weight,n= 3; high dose of IL-15, 100 μg/kg,n= 3; control [saline],n= 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3−NK cells. Furthermore, CD8+T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA−CD62L−and CD45RA+CD62L−effector memory CD8+T cells. Expression of Ki-67 in the CD8+T cells indicated expansion of CD8+T cells and not redistribution. IL-15 did not affect CD4+T-cell, B-cell, and CD14+macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.