pinecone/core-2020-05-10-deduplication huggingface dataset:
Assembly of Herpes Simplex Virus Replication Proteins at Two Distinct Intranuclear Sites

AbstractHerpes simplex virus DNA replication proteins amplify the viral genome in large globular replication compartments within infected cell nuclei. In the absence of viral DNA synthesis, the replication proteins accumulate at punctate foci throughout the nucleus referred to as prereplicative sites. To more thoroughly understand the nature of this nuclear assembly process, we have examined the viral and cellular factors involved. First, we demonstrate that six viral replication proteins are sufficient for formation of functional replication compartments in transfected cells in the absence of viral origin-containing DNA. Second, we show that the viral replication proteins form two distinct types of prereplicative sites within infected cells. One type of punctate structure assembles in S-phase cells, colocalizes with cellular DNA synthesis, and contains components of the host-cell replication apparatus as indicated by the presence of Replication Protein A. However, the other class of prereplicative sites is independent of host-cell DNA synthesis as evidenced by their formation in cells arrested in G1 byn-butyrate. These complexes are significantly less abundant and closely correspond with cellular Nuclear Domain 10 structures to which viral DNA has recently been demonstrated to be targeted early in infection (G. G. Maul, A. M. Ishov, and R. D. Everett, 1996,Virology217, 67–75). Hence, this second type appears to represent the subset of prereplicative sites destined to become replication compartments
Characterization of the Rep78/Adeno-Associated Virus Complex

AbstractAdeno-associated virus (AAV) replication proteins Rep78 and Rep68 play major roles in the life cycle of AAV. We have recently providedin vivoevidence for the existence of a covalent association between Rep78 and virion single-stranded (ss) AAV DNA (K. M.R. Prasad and J.P. Trempe(1995)Virology214, 360–370). In this work we have further characterized the Rep78 protein–AAV DNA covalent linkage. Exonuclease and primer extension analyses revealed that in the majority of isolated ssDNA, Rep78 protein is covalently linked to one of the 5′ terminal thymidines. Pulse–chase experiments with radiolabeled methionine suggest that Rep protein remains associated with the virus particle for up to 8 hr after labeling in infected cells. Quantitative immunoprecipitation indicated that ∼30% of the ssDNA remains associated with the Rep protein after cell fractionation and partial purification. When cells are infected with Rep-associated AAV particles, a significant proportion of viral DNA remains attached to Rep after entry into the nucleus. However, the linkage does not persist after nuclear entry. These observations suggest that covalently linked Rep78 protein may play a key role during wild-type AAV infections and AAV vector transductions
Regulation of the Long Terminal Repeat in Visna Virus by a Transcription Factor Related to the AML/PEBP2/CBF Superfamily

AbstractThe long terminal repeats of maedi visna virus strain 1514 contain a consensus AP-1 binding site which has been shown to be important in controlling virus transcription. However, this consensus site is absent in strain EV-1. Here, we have compared the ability of oligonucleotides corresponding to LTR sequences from EV-1 with those from 1514 to bind transcription factors in competitive gel retardation assays and activate reporter gene expression. The experiments demonstrated no observable binding of AP-1 to the EV-1-derived sequences and significant differences in the abilities of the 1514 and EV-1 sequences to activate transcription. However, both viral sequences interacted with a second, previously undetected, transcription factor. This factor gave specific gel shifts which were competed by an oligonucleotide containing the consensus sequence for the AML/PEBP2/CBF family of transcriptional factors, but not by control AP-1 or OCT-1 oligonucleotides. The factor was therefore denoted AML (vis). A second AML (vis) site, noted upstream of the TATA box proximal AP-1 site, gave single shifts which were competed by the downstream AML (vis) oligonucleotide. Both sites were functional in transfection assays. In gel shift retardation assays, polyclonal antisera directed against known runt domain proteins were able to supershift part of the AML (vis) binding activity in nuclear extracts from physiologically relevant cell types. The results thus suggest that the AML (vis) binding factor belongs to the AML/PEBP2/CBF family of transcription factors and may be important in controlling virus replication in these and other strains of ruminant lentiviruses
A Novel Strategy for Determining Protective Antigens of the Parapoxvirus, Orf Virus

AbstractWe investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy
Molecular Characterization, Sequence Analysis, and Taxonomic Position of Newly Isolated Fish Iridoviruses

AbstractWithin the past decade, iridoviruses have been identified as the causative agents of systemic disease in a variety of commercially and recreationally important fish. Here we examine nine iridoviruses from fish, reptiles, and amphibians and demonstrate that all isolates were more similar to frog virus 3, the type species of the genusRanavirus,than to lymphocystis disease virus, the type species of the genusLymphocystivirus.Comparison of viral protein synthesis profiles, restriction endonuclease digestion patterns, and the amino acid sequence of the major capsid protein indicated that iridoviruses isolated from the same geographic region were similar, if not identical, whereas viruses from different areas were distinct. Moreover, using primers complementary to the conserved major capsid protein, we found that both PCR and RT-PCR successfully amplified virus-specific nucleic acid from all nine isolates. These studies demonstrate that the piscine iridoviruses examined here were members of the genusRanavirus,and suggest that surveys of pathogenic “fish viruses” may need to include neighboring amphibian and reptilian populations. In addition, the results indicate that PCR readily identified vertebrate iridoviruses and suggest that PCR will be useful in the diagnosis of fish disease
Generation ofHerpes Virus Saimiri-Transformed T-Cell Lines from Macaques Is Restricted by Reactivation ofSimian Spuma Viruses

AbstractHerpes virus saimiri(HVS) transforms human T-cellsin vitroto stable growth. These T-cell lines retain their immunological characteristics of the parent cells and do not release infectious virus. Recently, lymphocytes of Old World monkeys were efficiently transformed by HVS. In parallel to these studies we initiated transformation experiments by infecting peripheral blood cell cultures of 45 monkeys, 35 rhesus and 10 cynomolgus macaques. In only three cases, we obtained transformed T-cell lines. The transformed T-cells were largely double-positive for CD4 and CD8. They responded with increased proliferation to mitogenic or IL-2 stimulation and transcribed mRNA for IL-2, IL-4, and IL-10. However, most initiated T-cell cultures from macaques developed giant cells. The cytopathic agent was identified assimian foamy virus(SFV) as confirmed by PCR, immunofluorescence, and coculture experiments. Treatment of the T-cell cultures with AZT- and SFV-specific sera did only shortly prolong the life-span of the cultures. Therefore, the reactivation of SFV caused remarkable difficulties in the establishment of macaque T-cell lines by HVS. This seems to be a general problem since most animals from several breeding colonies are SFV-positive
Identification of Sites That Act Together to Direct Dimerization of Human Foamy Virus RNAin Vitro

AbstractRetroviral particles contain two molecules of genomic RNA, which are noncovalently linked near their 5′ ends in a region called the dimer linkage structure (DLS). By using complementary DNA oligonucleotides and deletion mutants to impair RNA dimerization of the human foamy virus (HFV), three sites, designated SI, SII, and SIII, were found within a 159-nucleotide RNA fragment of HFV that are involved in dimerizationin vitro.SI overlaps the primer-binding site; SII contains the palindromic sequence, UCCCUAGGGA, the disruption of which impairs dimer formation; and SIII extends into thegaggene. The first two sites are highly conserved in the other primate foamy viruses, SFV-1, SFV-3, and SFVcpz, whereas the third appears to be shared only by HFV and SFVcpz. RNA of HFV and SFV-3 could form heterodimers, indicating that both viruses dimerize by similar mechanisms. On testing thermal stability, dimers of the 159-nucleotide fragment dissociated between 40 and 70°, with half of the dimers dissociating at 55°. Since the splice donor site of HFV is located at position 51 of viral RNA, the DLS is part of the genomic RNA exclusively
Interferon-γ Enhances Resolution of Herpes Simplex Virus Type 2 Infection of the Murine Genital Tract

AbstractThe requirement for interferon-γ (IFNγ) in resolution of an HSV-2 vaginal infection and the cellular sources of this cytokine in the vaginal mucosa were assessed. IFNγ levels in vaginal secretions peaked on Days 2 and 5 following HSV-2 inoculation. Natural killer (NK) cell depletion greatly diminished the early production of IFNγ but had no significant effect on the rate of virus clearance. CD4+T cells were primarily responsible for the second peak of IFNγ levels and neutralization of this IFNγ beginning 3 days after virus inoculation delayed, but did not prevent, virus clearance from the vagina. HSV-2 persisted in mice depleted of both CD4+and CD8+T cells while clearance was delayed in CD4+, but not CD8+T cell-depleted mice, demonstrating the T cell dependence and predominant role of CD4+T cells in resolution of the infection. Together, these data suggest that IFNγ is not essential for virus clearance but plays an important role in enhancing T cell-mediated clearance mechanisms. The implication of these results is that IFNγ produced locally in the genital tract enhances virus clearance and may ultimately be important for reducing the amount of virus available to infect sensory ganglia
Low-Affinity Cytotoxic T-Lymphocytes Require IFN-γ to Clear an Acute Viral Infection

AbstractThe majority of the response of cytotoxic T-lymphocytes (CTL) to lymphocytic choriomeningitis virus (LCMV) in H-2dmice is directed toward one epitope located on the nucleoprotein (NP, aa 118–126), and usually no primary responses to other epitopes are detectable. Previous studies have shown that thymic expression of lymphocytic choriomeningitis virus-nucleoprotein (LCMV-NP) in H-2dtransgenic mice (Thy-NP mice) leads to deletion of high-affinity anti-LCMV-NP CTL by negative selection. Selection is incomplete, so that low-affinity NP-specific CTL pass through the thymus and are detectable in the periphery. To analyze the importance of interferon-γ (IFN-γ) in the ability of low-affinity antiviral CTL to clear an acute viral infection, double transgenic mice were generated that are IFN-γ deficient and express the NP of LCMV in the thymus (Thy-NP × IFN-γ −/− mice). When infected with LCMV, these bigenic mice were unable to clear the infection despite generating low-affinity primary antiviral CTL, and they became persistently infected. In contrast, IFN-γ competent Thy-NP mice cleared LCMV within 7–8 days and IFN-γ deficient mice that did not express NP in their thymus generated high-affinity CTL that terminated an acute LCMV infection within 10–12 days post-viral challenge. Persistently infected IFN-γ deficient mice selectively depleted LCMV-specific CTL and displayed reduced levels of antigen-presenting cells in the spleen, and 60% of these mice died at 2–3 months postinfection. Thus, IFN-γ is required for clearing an acute viral infection in the absence of a high-affinity CTL response. In the absence of IFN-γ persistent viral infection results despite the presence of low-affinity CTL
Binding of Antibodies to Virion-Associated gp120 Molecules of Primary-like Human Immunodeficiency Virus Type 1 (HIV-1) Isolates: Effect on HIV-1 Infection of Macrophages and Peripheral Blood Mononuclear Cells

AbstractUsing immunobiochemical approaches we previously studied the conformation and surface exposure of different immunodominant regions within the oligomeric, virion-associated form of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) (L. Stamatatos and C. Cheng-Mayer (1995)J. Virol.69, 6191–6198). These studies allowed us to determine to what extent epitopes within these immunodominant regions of the oligomeric gp120 are occluded or accessible to antibody binding on the virion surface of two primary-like (HIV-1SF162and HIV-1SF128A) and one T-cell-line-adapted (HIV-1SF2) isolates. Here, we investigate whether binding of anti-gp120 monoclonal antibodies (MAbs) to exposed epitopes of the immunodominant regions of oligomeric gp120 results in neutralization of HIV-1 infection and whether certain exposed sites are better targets for neutralization than others. We also test whether proposed mechanisms for antibody-mediated neutralization of T-cell-line-adapted HIV-1 isolates, e.g., antibody-mediated gp120-virion dissociation, are applicable to primary-like HIV-1 isolates. We assess the neutralization potential of anti-V2 loop, anti-V3 loop, and anti-CD4 binding site MAbs using human primary macrophages or peripheral blood mononuclear cells (PBMC) as target cells and HIV-1SF162and HIV-1SF128Aas infecting isolates. Our data indicate that: (i) not every exposed epitope of the immunodominant regions of gp120 oligomers on the virion surface is a target for neutralization; (ii) during virus–cell fusion specific exposure of previously occluded V3 loop epitopes within gp120 oligomers occurs, which may become targets for neutralization; (iii) antibody-mediated gp120-virion dissociation does not appear to be a major mechanism of neutralization for the primary-like HIV-1 isolates tested here; and (iv) infection of macrophages is more sensitive to neutralization by anti-gp120 monoclonal antibodies than PBMC. We also report that binding of one of the two anti-CD4 binding site MAbs tested mediated enhancement of macrophage cell infection in a concentration-dependent fashion, while binding of the other anti-CD4 binding site MAb resulted in efficient neutralization of infection of both macrophages and PBMC
Evidence That Marek's Disease Virus Exists in a Latent State in a Sustainable Fibroblast Cell Line

AbstractPreviously, we reported the development of two fibroblastic cell lines (MDV OU2.1 and OU2.2) infected with Marek's disease virus (MDV). The two cell lines, in nonconfluent continuous cultures, displayed characteristics consistent with MDV existing in a latent state. However, presence of distinct plaques in confluent cell monolayers and the ability to transfer cytolytic infection to susceptible birds and primary chick embryo fibroblasts, suggest that, if latent, the virus is easily reactivated from MDV OU2 cell lines. In this report, we present evidence which supports the hypothesis that MDV genomes in MDV OU2 cells are latent. PCR analyses andin vivoexperiments demonstrate that CHCC-OU2 cells stabilize MDV so that serialin vitropassage does not attenuate the virus. Following two years of active culture, MDV genomes in MDV OU2 cells are still oncogenic, similar to that seen in MDV-lymphoblastoid cell lines. Expansion of the 132-bp repeat within MDVBamHI fragments H and D, typical of highly passaged serotype 1 MDV, has not been observed beyond two copies in MDV OU2 cells. Indirect immunofluorescence assays clearly demonstrate that MDV OU2 cells do not express glycoproteins B and I when subconfluent. However, upon reaching confluence these proteins are expressed in readily detectable amounts. Using RT-PCR we demonstrate that glycoproteins E and D are highly expressed in confluent MDV OU2 cells but absent from subconfluent cells, and MDV latency-associated transcripts (LATs), which are antisense to ICP4 transcripts and have been associated with latent MDV infection, are expressed in subconfluent MDV OU2 cells. Coincident with an increase in ICP4 expression, MDV LAT expression is down regulated when MDV OU2 cells become confluent
HIV-1 Env Glycoproteins from Two Series of Primary Isolates: Replication Phenotype and Immunogenicity

AbstractSeven envelope regions from two series of patient isolates have been molecularly cloned and analyzed for replication phenotypes and immunogenicity. Growth potential was analyzed forenvsequences substituted into an HIV-1-NL4-3 backbone (NL4-3/env recombinants). Immunogenicity studies were conducted on secreted monomeric (gp120) and oligomeric (gp140) forms of the Envs using Env-expressing plasmid DNAs for immunizations. Theenvregions of the patient isolates conferred a spectrum of replication kinetics and cytotropisms on the NL4-3/env recombinants. Both patient series included non-syncytium-inducing viruses with no ability to grow on T-cell lines, and highly syncytium inducing viruses which grew well on T-cell lines. These differences in growth potential did not correlate with the ability of the DNA-expressed Envs to raise antibody in rabbits. Rather, the relative immunogenicity of the Envs was patient and form specific. The Envs from patient 5 raised higher titers of antibody than the Envs from patient 6. For each primary Env, the gp120 form of the Env raised higher titers of antibody than the gp140 form. Thus, structural features of Env that affect replication do not necessarily affect the ability to raise antibody
Identification of a Contiguous 6-Residue Determinant in the MHV Receptor That Controls the Level of Virion Binding to Cells

AbstractMurine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (MHV) into cells. Numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (NTD) to which MHV binds via its protruding spike proteins. Some of these receptor alterations may affect the ability to bind viral spikes. To identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of MHV receptor differing only in their NTD. The two receptors, designated biliary glycoproteins (Bgp) 1aand 1bNTD, varied by 29 residues in the 107 amino acid NTD. When expressed from cDNAs in receptor-negative HeLa cells, these two Bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. Infectious center assays revealed that the 1aisoform was 10 to 100 times more effective than 1bNTDin its ability to confer sensitivity to MHV (strain A59) infection. Bgp1awas also more effective than Bgp1bNTDin comparative virus adsorption assays, binding 6 times more MHV (strain A59) and 2.5 times more MHV (strain JHMX). Bgp1awas similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. To identify residues influencing these differences, we inserted varying numbers of 1bresidues into the Bgp1abackground via restriction fragment exchange and site-directed mutagenesis. Analysis of the resulting chimeric receptors showed that residues 38 to 43 of the NTD were key determinants of the binding and fusion differences between the two receptors. These residues map to an exposed loop (C-C′ loop) in a structural model of the closely related human carcinoembryonic antigen
Concatemeric Intermediates of Equine Herpesvirus Type 1 DNA Replication Contain Frequent Inversions of Adjacent Long Segments of the Viral Genome

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

AbstractThe nucleotide sequence of theOrgyia pseudotsugatamultinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and theAutographa californicaMNPV were found. These include the presence in OpMNPV of three completeiapgene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses