bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

During 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

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

Antibodies (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

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

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

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

Antiviral 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

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

Regulatory 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

The 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.
Foamy Virus Integration
Authors: Juretzek Thomas , Holm Teresa , Gärtner Kathleen , Kanzler Sylvia , Lindemann Dirk , Herchenröder Ottmar , Picard-Maureau Marcus , Rammling Matthias , Heinkelein Martin , Rethwilm Axel
Published: February 2004

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5′ end of the U3 region in the 3′ LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.
Influence of Long Terminal Repeat and Env on the Virulence Phenotype of Equine Infectious Anemia Virus
Authors: Payne Susan L. , Pei Xiao-fang , Jia Bin , Fagerness Angela , Fuller Frederick J.
Published: February 2004

The molecular clones pSPeiav19 and p19/wenv17 of equine infectious anemia virus (EIAV) differ in
env
and long terminal repeats (LTRs) and produce viruses (EIAV
19
and EIAV
17
, respectively) of dramatically different virulence phenotypes. These constructs were used to generate a series of chimeric clones to test the individual contributions of LTR, surface (SU), and transmembrane (TM)/Rev regions to the disease potential of the highly virulent EIAV
17
. The LTRs of EIAV
19
and EIAV
17
differ by 16 nucleotides in the transcriptional enhancer region. The two viruses differ by 30 amino acids in SU, by 17 amino acids in TM, and by 8 amino acids in Rev. Results from in vivo infections with chimeric clones indicate that both LTR and
env
of EIAV
17
are required for the development of severe acute disease. In the context of the EIAV
17
LTR, SU appears to have a greater impact on virulence than does TM. EIAV
17SU
, containing only the TM/Rev region from the avirulent parent, induced acute disease in two animals, while a similar infectious dose of EIAV
17TM
(which derives SU from the avirulent parent) did not. Neither EIAV
17SU
nor EIAV
17TM
produced lethal disease when administered at infectious doses that were 6- to 30-fold higher than a lethal dose of the parental EIAV
17
. All chimeric clones replicated in primary equine monocyte-derived macrophages, and there was no apparent correlation between macrophage tropism and virulence phenotype.
Vaccinia Virus A36R Membrane Protein Provides a Direct Link between Intracellular Enveloped Virions and the Microtubule Motor Kinesin
Authors: Ward Brian M. , Moss Bernard
Published: February 2004

Previous work demonstrated that intracellular enveloped vaccinia virus virions use microtubules to move from the site of membrane wrapping to the cell periphery. The mechanism and direction of intracellular virion movement predicted that viral proteins directly or indirectly interact with the microtubule motor protein kinesin. The yeast two-hybrid assay was used to test for interactions between the light chain of kinesin and the cytoplasmic tails from five viral envelope proteins. We found that the N-terminal tetratricopeptide repeat region of the kinesin light chain (KLC-TPR) interacted with the cytoplasmic tail of the viral A36R protein. A series of C- and N-terminal truncations of A36R further defined a region from residues 81 to 111 that was sufficient for interaction with KLC-TPR. Interactions were confirmed by using pull-down assays with purified glutathione
S
-transferase (GST)-A36R and
35
S-labeled KLC-TPR. The defined region on A36R for interaction with kinesin overlaps the recently defined region (residues 91 to 111) for interaction with the A33R envelope protein. The yeast three-hybrid system was used to demonstrate that expression of A33R interrupted the interaction between A36R and KLC-TPR, indicating that the binding of A36R is mutually exclusive to either A33R or kinesin. Pull-down assays with purified GST-A36R and
35
S-labeled KLC-TPR in the presence of competing A33R corroborated these findings. Collectively, these results demonstrated that the viral A36R protein interacts directly with the microtubule motor protein kinesin and that the viral protein A33R may regulate this interaction.
Identification of Exogenous Forms of Human-Tropic Porcine Endogenous Retrovirus in Miniature Swine
Authors: Wood James C. , Quinn Gary , Suling Kristen M. , Oldmixon Beth A. , Van Tine Brian A. , Cina Robert , Arn Scott , Huang Christine A. , Scobie Linda , Onions David E. , Sachs David H. , Schuurman Henk-Jan , Fishman Jay A. , Patience Clive
Published: February 2004

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.
Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine
Authors: Scobie Linda , Taylor Samantha , Wood James C. , Suling Kristen M. , Quinn Gary , Meikle Sharon , Patience Clive , Schuurman Henk-Jan , Onions David E.
Published: February 2004

The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol.
76:
3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long
env
open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.
A Virulence Factor of Myxoma Virus Colocalizes with NF-κB in the Nucleus and Interferes with Inflammation
Authors: Camus-Bouclainville Christelle , Fiette Laurence , Bouchiha Sophie , Pignolet Béatrice , Counor Dorian , Filipe Cédric , Gelfi Jacqueline , Messud-Petit Frédérique
Published: February 2004

NF-κB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses.
Myxoma virus
, a member of the
Poxviridae
family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-κBα, which regulates NF-κB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-κB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-κB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.
Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference Directed against Human Transcription Elongation Factor P-TEFb (CDK9/CyclinT1)
Authors: Chiu Ya-Lin , Cao Hong , Jacque Jean-Marc , Stevenson Mario , Rana Tariq M.
Published: February 2004

The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.
In Vivo Role of Nectin-1 in Entry of Herpes Simplex Virus Type 1 (HSV-1) and HSV-2 through the Vaginal Mucosa
Authors: Linehan Melissa M. , Richman Susan , Krummenacher Claude , Eisenberg Roselyn J. , Cohen Gary H. , Iwasaki Akiko
Published: February 2004

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.
Molecular Phylogenetic Analyses Indicate a Wide and Ancient Radiation of African Hepatitis Delta Virus, Suggesting a
Deltavirus
Genus of at Least Seven Major Clades
Authors: Radjef Nadjia , Gordien Emmanuel , Ivaniushina Valeria , Gault Elyanne , Anaïs Patricia , Drugan Tudor , Trinchet Jean-Claude , Roulot Dominique , Tamby Mathieu , Milinkovitch Michel C. , Dény Paul
Published: February 2004

Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and propagation and infects nearly 20 million people worldwide. The HDV genome is a compact circular single-stranded RNA genome with extensive intramolecular complementarity. Despite its different epidemiological and pathological patterns, the variability and geographical distribution of HDV are limited to three genotypes and two subtypes that have been characterized to date. Phylogenetic reconstructions based on the delta antigen gene and full-length genome sequence data show an extensive and probably ancient radiation of African lineages, suggesting that the genetic variability of HDV is much more complex than was previously thought, with evidence of additional clades. These results relate the geographic distribution of HDV more closely to the genetic variability of its helper HBV.
Immunization Strategies To Block the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor
Authors: Lin Xiaoqing , Lubinski John M. , Friedman Harvey M.
Published: February 2004

Herpes simplex virus type 1 (HSV-1) glycoprotein gE functions as an immunoglobulin G (IgG) Fc receptor (FcγR) that promotes immune evasion. When an IgG antibody binds by the F(ab′)2domain to an HSV antigen, the Fc domain of some of the same antibody molecules binds to the FcγR, which blocks Fc-mediated functions. gE is a type 1 membrane glycoprotein with a large ectodomain that is expressed on the virion envelope and infected-cell surface. Our goal was to determine if immunizing with gE protein fragments could produce antibodies that bind by the F(ab′)2domain to gE and block the FcγR, as measured by competitively inhibiting nonimmune human IgG binding to the FcγR. Three gE peptides were constructed in baculovirus spanning almost the entire ectodomain and used to immunize mice and rabbits. Two fragments were highly effective at producing antibodies that bind by the F(ab′)2domain and block the FcγR. The most potent of these two antibodies was far more effective at blocking the FcγR than antibodies that are only capable of binding by the Fc domains to the FcγR, including anti-gC, anti-gD, and nonimmune IgG. These results suggest that immunizing with gE fragments has potential for preventing immune evasion by blocking activities mediated by the HSV-1 FcγR.
Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses
Authors: Worgall Stefan , Busch Annette , Rivara Michael , Bonnyay David , Leopold Philip L. , Merritt Robert , Hackett Neil R. , Rovelink Peter W. , Bruder Joseph T. , Wickham Thomas J. , Kovesdi Imi , Crystal Ronald G.
Published: February 2004

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+as well as the CD8+β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P< 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines.
Extraepitopic Compensatory Substitutions Partially Restore Fitness to Simian Immunodeficiency Virus Variants That Escape from an Immunodominant Cytotoxic-T-Lymphocyte Response
Authors: Friedrich Thomas C. , Frye Christopher A. , Yant Levi J. , O'Connor David H. , Kriewaldt Nancy A. , Benson Meghan , Vojnov Lara , Dodds Elizabeth J. , Cullen Candice , Rudersdorf Richard , Hughes Austin L. , Wilson Nancy , Watkins David I.
Published: February 2004

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in
gag
in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.
Inflammatory Cytokines Inhibit Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Transcription in In Vitro-Infected Endothelial Cells
Authors: Milligan Steven , Robinson Mairi , O'Donnell Elizabeth , Blackbourn David J.
Published: February 2004

The response of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) to inflammatory cytokine treatment of experimentally infected endothelial cells was investigated. The cytokines inhibited spontaneous KSHV lytic gene expression but not the level of infection. The data suggest that if inflammatory cytokines present in KS lesions contribute to KSHV pathogenesis, they do so in part by promoting latent KSHV infection of the endothelial cells.
Specific Interaction of Feline Immunodeficiency Virus Surface Glycoprotein with Human DC-SIGN
Authors: de Parseval Aymeric , Su Stephen V. , Elder John H. , Lee Benhur
Published: February 2004

DC-SIGN, a specific C-type lectin expressed on dendritic cells, binds and transmits multiple strains of primate immunodeficiency viruses to susceptible cells. Here, we report that human DC-SIGN also captures feline immunodeficiency virus via high-affinity (1 nM), Ca2+-dependent,d-mannose-inhibited binding to the major envelope glycoprotein, gp95.
Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition
Authors: Das Atze T. , Brummelkamp Thijn R. , Westerhout Ellen M. , Vink Monique , Madiredjo Mandy , Bernards René , Berkhout Ben
Published: February 2004

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.
Cryoelectron Microscopy of Mouse Mammary Tumor Virus
Authors: Briggs John A. G. , Watson Barbara E. , Gowen Brent E. , Fuller Stephen D.
Published: February 2004

Cryoelectron microscopy of
Mouse mammary tumor virus
, a
Betaretrovirus
, provided information about glycoprotein structure and core formation. The virions showed the broad range of diameters typical of retroviruses. Betaretroviruses assemble cytoplasmically, so the broad size range cannot reflect the use of the plasma membrane as a platform for assembly.
Requirement of a 12-Base-Pair TATT-Containing Sequence and Viral Lytic DNA Replication in Activation of the Kaposi's Sarcoma-Associated Herpesvirus K8.1 Late Promoter
Authors: Tang Shuang , Yamanegi Koji , Zheng Zhi-Ming
Published: February 2004

Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by
trans
.
Bovine Papillomavirus Replicative Helicase E1 Is a Target of the Ubiquitin Ligase APC
Authors: Mechali Francisca , Hsu Chiung-Yueh , Castro Anna , Lorca Thierry , Bonne-Andrea Catherine
Published: February 2004

The papillomavirus E1 replicative helicase is essential for replication and maintenance of extrachromosomal viral genomes in infected cells. We previously found that the bovine papillomavirus E1 protein is a substrate of the ubiquitin-dependent proteolytic pathway. Here we show that E1 is targeted for degradation by the anaphase-promoting complex (APC). Inhibition of APC activity by the specific inhibitor Emi1 or point mutations in the D-box and KEN-box motifs of E1 stabilize the protein and increase viral DNA replication in both a cell-free system and in living cells. These findings involve APC as the ubiquitin ligase that controls E1 levels to maintain a constant low copy number of the viral genome during latent infection.
Roles of the E6 and E7 Proteins in the Life Cycle of Low-Risk Human Papillomavirus Type 11
Authors: Oh Stephen T. , Longworth Michelle S. , Laimins Laimonis A.
Published: February 2004

Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.
Binding of the 2F5 Monoclonal Antibody to Native and Fusion-Intermediate Forms of Human Immunodeficiency Virus Type 1 gp41: Implications for Fusion-Inducing Conformational Changes
Authors: de Rosny Eve , Vassell Russell , Jiang Shibo , Kunert Renate , Weiss Carol D.
Published: February 2004

We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.
Role of Indoleamine-2,3-Dioxygenase in Alpha/Beta and Gamma Interferon-Mediated Antiviral Effects against Herpes Simplex Virus Infections
Authors: Adams O. , Besken K. , Oberdörfer C. , MacKenzie C. R. , Takikawa O. , Däubener W.
Published: February 2004

Gamma interferon (IFN-γ)-mediated indoleamine-2,3-dioxygenase (IDO) activity in human astrocytoma cells and in native astrocytes was found to be responsible for the inhibition of herpes simplex virus replication. The effect is abolished in the presence of excess amounts ofl-tryptophan. Both IFN-α and IFN-β restricted herpes simplex virus replication in both cell types, but (in contrast to the results seen with IFN-γ) the addition of an excess amount ofl-tryptophan did not inhibit the induced antiviral effect.