bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis
Authors: Gillet L. , Minner F. , Detry B. , Farnir F. , Willems L. , Lambot M. , Thiry E. , Pastoret P.-P. , Schynts F. , Vanderplasschen A. 
Published: February 2004

ABSTRACT
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.
Vaccinia Virus A28L Gene Encodes an Essential Protein Component of the Virion Membrane with Intramolecular Disulfide Bonds Formed by the Viral Cytoplasmic Redox Pathway
Authors: Senkevich Tatiana G. , Ward Brian M. , Moss Bernard 
Published: February 2004

ABSTRACT
We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.
Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene
Authors: Senkevich Tatiana G. , Ward Brian M. , Moss Bernard 
Published: February 2004

ABSTRACT
The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.
Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses
Authors: Keelapang Poonsook , Sriburi Roongtawan , Supasa Sanpaechuda , Panyadee Nantaya , Songjaeng Adisak , Jairungsri Aroonroong , Puttikhunt Chunya , Kasinrerk Watchara , Malasit Prida , Sittisombut Nopporn 
Published: February 2004

ABSTRACTDuring the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.
The Matrix Protein of Marburg Virus Is Transported to the Plasma Membrane along Cellular Membranes: Exploiting the Retrograde Late Endosomal Pathway
Authors: Kolesnikova Larissa , Bamberg Sandra , Berghöfer Beate , Becker Stephan 
Published: February 2004

ABSTRACT

VP40, the matrix protein of Marburg virus, is a peripheral membrane protein that has been shown to associate with membranes of multivesicular bodies (MVBs) (L. Kolesnikova, H. Bugany, H.-D. Klenk, and S. Becker, J. Virol.
76:
1825-1838, 2002). The present study revealed that VP40 is bound to cellular membranes rapidly after synthesis. Time course studies were performed to trace the distribution of VP40 during the course of expression. First, VP40 was homogenously distributed throughout the cytoplasm, although the majority of protein (70%) was already membrane associated. Next, VP40 accumulated in MVBs and in tubular protrusions emerging from MVBs. Finally, VP40 appeared in a patch-like pattern beneath the plasma membrane. These morphological results were supported by iodixanol density gradient analyses. The majority of VP40-positive membranes were first detected comigrating with small vesicles. VP40 was then shifted to fractions containing endosomal marker proteins, and later, to fractions containing plasma membrane marker proteins. Blocking of protein synthesis by use of cycloheximide at the time when VP40 was mainly associated with the small vesicles did not prevent the redistribution of VP40 to the late endosomes and further to the plasma membrane. The inhibition of intracellular vesicular trafficking by monensin significantly reduced the appearance of VP40 at the plasma membrane. In conclusion, we suggest that the transport of the Marburg virus matrix protein VP40 involves its accumulation in MVBs followed by the redistribution of VP40-enriched membrane clusters to the plasma membrane.
The V3 Loop Is Accessible on the Surface of Most Human Immunodeficiency Virus Type 1 Primary Isolates and Serves as a Neutralization Epitope
Authors: Gorny Miroslaw K. , Revesz Kathy , Williams Constance , Volsky Barbara , Louder Mark K. , Anyangwe Christopher A. , Krachmarov Chavdar , Kayman Samuel C. , Pinter Abraham , Nadas Arthur , Nyambi Phillipe N. , Mascola John R. , Zolla-Pazner Susan 
Published: February 2004

ABSTRACTAntibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoclonal Abs (MAbs). The neutralizing activities of anti-V3 polyclonal Abs and MAbs may, however, be limited due to antigenic variations of the V3 region, a lack of V3 exposure on the surface of intact virions, or Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for neutralization of an SF162-pseudotyped virus in a luciferase assay. MAbs selected with a V3 fusion protein whose V3 region mimics the conformation of the native virus were significantly more potent than MAbs selected with V3 peptides. Seven MAbs were further tested for neutralizing activity against 13 clade B viruses in a single-round peripheral blood mononuclear cell assay. While there was a spectrum of virus sensitivities to the anti-V3 MAbs observed, 12 of the 13 viruses were neutralized by one or more of the anti-V3 MAbs. MAb binding to intact virions correlated significantly with binding to solubilized gp120s and with the potency of neutralization. These results demonstrate that the V3 loop is accessible on the native virus envelope, that the strength of binding of anti-V3 Abs correlates with the potency of neutralization, that V3 epitopes may be shared rather than type specific, and that Abs against the V3 loop, particularly those targeting conformational epitopes, can mediate the neutralization of primary isolates.
The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells
Authors: Raghavan Vineetha , Malik Punjab S. , Choudhury Nirupam Roy , Mukherjee Sunil K. 
Published: February 2004

ABSTRACT

Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain,
Indian mung bean yellow mosaic virus
. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.
Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus
Authors: Agapov Eugene V. , Murray Catherine L. , Frolov Ilya , Qu Lin , Myers Tina M. , Rice Charles M. 
Published: February 2004

ABSTRACT

Despite increasing characterization of pestivirus-encoded proteins, functions for nonstructural (NS) proteins NS2, NS2-3, NS4B, and NS5A have not yet been reported. Here we investigated the function of bovine viral diarrhea virus (BVDV) uncleaved NS2-3. To test whether NS2-3 has a discrete function, the uncleaved protein was specifically abolished in two ways: first by inserting a ubiquitin monomer between NS2 and NS3, and second by placing an internal ribosome entry site between the two proteins (a bicistronic genome). In both cases, complete processing of NS2-3 prevented infectious virion formation without affecting RNA replication. We tested the hypothesis that uncleaved NS2-3 was involved in morphogenesis by creating a bicistronic genome in which NS2-3 was restored in the second cistron. With this genome, both uncleaved NS2-3 expression and particle production returned. We then investigated the minimal regions of the polyprotein that could rescue an NS2-3 defect by developing a
trans
-complementation assay. We determined that the expression of NS4A in
cis
with NS2-3 markedly increased its activity, while p7 could be supplied in
trans
. Based on these data, we propose a model for NS2-3 action in virion morphogenesis.
Unselected Mutations in the Human Immunodeficiency Virus Type 1 Genome Are Mostly Nonsynonymous and Often Deleterious
Authors: Gao Feng , Chen Yalu , Levy David N. , Conway Joan A. , Kepler Thomas B. , Hui Huxiong 
Published: February 2004

ABSTRACT

Mutation rates of human immunodeficiency virus type 1 (HIV-1) genomes have been estimated using purified reverse transcriptase or single-round infection system. Since small sequences were used as templates, the overall mutation rates could only be extrapolated and the biological significance of mutations is unknown. For direct estimation of HIV-1 mutation rates and understanding of the potential biological influences of mutations, we obtained 19 complete or nearly full-length proviral genomes from single-round-infected adherent cells of lymphocytes by using a lambda phage library method and a long-range PCR technique. Analysis of 160,000 bp of sequences showed that the overall mutation rate of HIV-1 genomes was 5.4 × 10
−5
per base per replication cycle. On average, 1.1 mutations (range, 0 to 3) were generated in each viral genome during one infection cycle. Inspection of the mutations in the HIV-1 genome revealed that all site mutations within protein-coding regions were nonsynonymous mutations. Among all mutations, half were deleterious (premature stop codon and deletions) and would result in defective genomes. By applying the same system to an HIV-1 genome with a G262A mutation in the thumb region of the reverse transcriptase, a significant increase was observed in deletion and insertion mutation rates but no increase in the overall mutation rate in viral genomes was found.
Highly Uneven Distribution of Tenofovir-Selected Simian Immunodeficiency Virus in Different Anatomical Sites of Rhesus Macaques
Authors: Magierowska Magdalena , Bernardin Flavien , Garg Seema , Staprans Silvija , Miller Michael D. , Van Rompay Koen K. A. , Delwart Eric L. 
Published: February 2004

ABSTRACTAntiviral tenofovir monotherapy was used to determine whether drug-selected simian immunodeficiency virus (SIV) variants replaced their wild-type progenitors at the same rate in different tissues of six rhesus macaques. The relative frequencies of drug-resistant and wild-type genotypes were measured longitudinally in blood and in 23 lymphoid and nonlymphoid tissues collected at necropsy. The mutant/wild-type genotype ratio was measured using a heteroduplex tracking assay targeting tenofovir-selected SIV reverse transcriptase codons. After the initiation of tenofovir treatment in animals with high steady-state viremia levels, resistant genotypes emerged in the plasma within 1 to 8 weeks and in five of six cases reached frequencies of nearly 100% within 4 to 25 weeks. The appearance of tenofovir-resistant genotypes in peripheral blood mononuclear cell (PBMC) DNA was generally delayed by 1 to 2 weeks and in one case was completely absent. Necropsies performed 8 to 55 weeks after the initiation of tenofovir treatment showed the frequency of resistant SIV genotypes to be generally higher in tissue RNA than DNA fractions. The frequency of drug-resistant genotypes varied widely between anatomical sites, including different lymph nodes of the same animal. Except for the epidydimis, the tissues with the lowest rates of proviral replacement by tenofovir-resistant genotypes differed between animals. The highly uneven distribution of tenofovir-resistant genotypes in different tissues seen shortly after the initiation of tenofovir monotherapy may reflect differences in local antiviral drug selection pressures and/or the stochastic effect of small effective populations of drug-resistant variants randomly seeding different anatomical sites early in therapy.
African Swine Fever Virus Multigene Family 360 Genes Affect Virus Replication and Generalization of Infection in
Ornithodoros porcinus
Ticks
Authors: Burrage T. G. , Lu Z. , Neilan J. G. , Rock D. L. , Zsak L. 
Published: February 2004

ABSTRACT

Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host,
Ornithodoros porcinus porcinus
, an MGF360/530 gene deletion mutant (Pr4Δ35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Δ35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Δ3-C2, which lacked three MGF360 genes (
3HL
,
3Il
, and
3LL
), exhibited reduced viral growth in ticks. Pr4Δ3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Δ3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Δ3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.
Human CD4+CD25+Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens
Authors: Aandahl Einar M. , Michaëlsson Jakob , Moretto Walter J. , Hecht Frederick M. , Nixon Douglas F. 
Published: February 2004

ABSTRACTRegulatory T (TR) cells maintain tolerance to self-antigens and control immune responses to alloantigens after organ transplantation. Here, we show that CD4+CD25+human TRcells suppress virus-specific T-cell responses. Depletion of TRcells from peripheral blood mononuclear cells enhances T-cell responses to cytomegalovirus and human immunodeficiency virus antigens. We propose that chronic viral infections lead to induction of suppressive TRcells that inhibit the antiviral immune response.
Gα Protein Selectivity Determinant Specified by a Viral Chemokine Receptor-Conserved Region in the C Tail of the Human Herpesvirus 8 G Protein-Coupled Receptor
Authors: Liu Chaoqi , Sandford Gordon , Fei Guo , Nicholas John 
Published: February 2004

ABSTRACTThe viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-κB, mitogen-activated protein kinase, and Ca2+signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Gα protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Gαi(pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [35S]GTPγS incorporation assays revealed preferential coupling of vGPCR.15 to Gαqand an inability of vGPCR.8 to couple functionally to Gαq. However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Gαq. Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Gα interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Gα protein coupling specificity.