pinecone/core-2020-05-10-deduplication huggingface dataset:
Assembly of SIV Virus-like Particles Containing Envelope Proteins Using a Baculovirus Expression System

AbstractThe requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gagprecursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envtprotein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP
Strain Variability and Localization of Important Epitopes on the Major Structural Protein (VP2) of Infectious Pancreatic Necrosis Virus1

AbstractInfectious pancreatic necrosis virus (IPNV), a birnavirus, is an important pathogen in fish farms. Analyses of viral proteins showed that VP2 is the major structural and immunogenic polypeptide of the virus. All neutralizing monoclonal antibodies (mAbs) against IPNV are specific to VP2 and bind to continuous or discontinuous epitopes. In order to determine which parts of the protein are involved in antigenic variations, five IPNV strains were sequenced over the VP2 coding region. Comparison of the sequences obtained with three previously published strains revealed a central variable domain (positions 183 to 335) which encompasses two hydrophilic hypervariable segments. Viral mutants which escaped neutralization were then selected with anti-VP2 mAbs directed against discontinuous epitopes. Sequencing of three mutants revealed a single amino acid mismatch in each of them. All of these substitutions occurred in the hypervariable segments, suggesting that these regions are involved in the formation of a discontinuous epitope. Finally, expression of different truncated VP2s inEscherichia coliallowed localization of the binding site for neutralizing mAbs which recognize continuous epitopes. One of these mAbs bound to the region adjacent to the C-terminus of the variable domain of VP2, while two others reacted with the central and C-terminal parts of the variable domain. No antibody reacted with the N-terminus of VP2. These results suggest that the variable domain of VP2 and the 20 adjacent amino acids of the conserved C-terminal part are the most important in inducing an immune response for the protection of animals
Differences in the Role of Glycoprotein C of HSV-1 and HSV-2 in Viral Binding May Contribute to Serotype Differences in Cell Tropism

AbstractHeparan sulfate serves as a receptor for several herpesviruses. For herpes simplex virus 1 (HSV-1), pseudorabies virus, and bovine herpesvirus 1, glycoprotein C homologues have been shown to mediate the binding to cell-surface heparan sulfate. It has been assumed that glycoprotein C of HSV-2 (gC-2) plays a similar role in HSV-2 entry, but this has not been established experimentally. We first determined, using heparin-affinity chromatography, that gC-2 is a heparin-binding glycoprotein. To examine the role of gC-2 in HSV-2 infection, we constructed a gC-2 deletion mutant, HSV-2(G)gC−. In contrast to results obtained for the other α herpesviruses, we found that the HSV-2(G)gC−virus showed no loss in specific binding activity (particles bound/cell) or specific infectivity (PFU/particle) compared to the parental wild-type virus. Moreover, while gC-1 mutants show a marked lag in the rate of viral penetration, the gC-2-deletion virus did not. We did find that gC-2, like gC-1, protects virus from complement-mediated neutralization. These results suggest that, in contrast to HSV-1, gC-2 does not play the key role in viral binding. The major role of gC-2 may be to protect virus from complement-mediated neutralization. We speculate that serotype differences in the contribution of gC to viral binding may contribute to serotype differences in cell tropism
Tyrosine Phosphorylation of Phospholipase C-γ1 by Vaccinia Virus Growth Factor

AbstractVaccinia virus growth factor (VGF), a highly glycosylated polypeptide encoded in the genome of vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors, EGF and TGF-α. Although the mitogenic activity of the purified or synthetic VGF suggested that the factor may be beneficial for viral replication by stimulation of host cell growth, neither the role in virus life cycle nor the step next to the EGF receptor activation had been firmly established. We found tyrosine-phosphorylations of PLC-γ1 and a concomitant increase of the phosphoinositides level in the human epidermoidal A431 cells either treated with purified VGF or infected with vaccinia virus. In contrast to the wild-type virus, a VGF−mutant virus did not induce tyrosine phosphorylation of PLC-γ1. Phosphopeptide analysis indicated that the phosphorylation of the PLC-γ1 by VGF includes tyrosine residues identical to those phosphorylated by EGF. These results suggest that tyrosine phosphorylation of PLC-γ1, mediated by VGF, leads to activation of PLC-γ1 and a concomitant increase in phosphatidylinositol turnover
A Feline Herpesvirus-1 Recombinant with a Deletion in the Genes for Glycoproteins gI and gE Is Effective as a Vaccine for Feline Rhinotracheitis

AbstractUsing a site-directed mutagenesis technique we constructed a new feline herpesvirus-1 recombinant strain containing a deletion in two genes encoding glycoproteins gI and gE. These proteins may have a role in virulence, the establishment of latency, and viral recurrence as shown in other herpesviruses of the varicella and simplex types. This recombinant was characterized and used to immunize juvenile cats against virulent virus challenge. Significant protection resulted from vaccination of cats by the subcutaneous route
Inhibition of Viral Aphid Transmission by the N-Terminus of the Maize Dwarf Mosaic Virus Coat Protein

AbstractSince removal of the exposed N-terminus of the coat protein of some potyviruses abolishes aphid transmission, the role of this coat protein region of maize dwarf mosaic potyvirus (MDMV) in aphid transmission was investigated. The viral cDNA encoding this region was cloned and expressed as a fusion protein in bacteria. The resulting purified N-terminus of the coat protein was used in controlled aphid transmission experiments in competition with MDMV. The results show that this region inhibits aphid transmission of MDMV, indicating a direct involvement of the N-terminal region of the coat protein in aphid transmission
In VitroPropagation and Production of Hepatitis E Virus fromin Vivo-Infected Primary Macaque Hepatocytes

AbstractHepatitis E virus (HEV) is responsible for sporadic cases as well as large epidemics of acute viral hepatitis in many developing countries. The nucleotide sequence of HEV appears to be unique among known viruses and thus may represent a prototype human pathogen in a novel class of single-stranded, positive-sense RNA viruses. To facilitate further studies of the biology of HEV, a tissue culture system using a serum-free medium formulation has been developed to propagate the virusin vitro.Hepatocytes were isolated from livers of cynomolgus macaques experimentally infected with a HEV (Burma strain) inoculum and maintained in long-term cultures. Using a highly strand-specific RT–PCR assay, both the positive-sense and the negative replicative strands of HEV RNA were detected in these hepatocytes throughout the course of the experiments. Positive-strand genomic RNA was also detected in the culture medium, suggesting the production and secretion of HEV virus particles. The virus particles were successfully concentrated 200-fold from the medium using ultrafiltration, and they could be observed by immunoelectron microscopy using anti-HEV-positive immune serum. These results demonstrate the capacity of this hepatocyte culture system to replicate HEVin vitro,thus providing an experimental means to study the replicative process of the virus
Influence of ORF2 on Host Cell Tropism of Feline Immunodeficiency Virus

AbstractFeline immunodeficiency virus (FIV) is a lentivirus associated with an immunodeficiency syndrome of the domestic cat. A short open reading frame (ORF2), of unknown function, is present in all FIV isolates. We have investigated the role of ORF2 in determining the cell tropism of two infectious molecular clones of FIV. FIV-PPR is able to productively infect feline peripheral blood leukocytes (PBLs) and a T lymphocyte cell line (MCH5-4), but not a feline astrocyte cell line (G355-5) or Crandell feline kidney cells (CrFK). In contrast, FIV-34TF10 is able to productively infect G355-5 and CrFK cells, but not PBLs or MCH5-4 cells. The major difference in these FIV clones is that ORF2 in FIV-PPR is capable of encoding a 79-amino-acid peptide, whereas there is a stop codon in ORF2 after 43 amino acids in FIV-34TF10. We performed site-directed mutagenesis to change the stop codon (TGA) in FIV-34TF10 to a tryptophan (TGG), the amino acid present at this location in FIV-PPR. FIV-34TF10 with ORF2 repaired (FIV-ORF2rep) productively infected PBLs, MCH5-4 cells, and primary macrophages, as well as CrFK and G355-5 cells, indicating that a protein encoded by ORF2 plays a role in determining the host cell tropism of FIV. ORF2 contains hydrophobic, acidic, and leucine-rich domains similar to those shown to be important for transactivating proteins of other lentiviruses. Coexpression of a plasmid expressing the ORF2 gene product with another construct expressing the chloramphenicol acetyl transferase (CAT) gene driven by the FIV LTR, resulted in transactivation of CAT expression in both feline and human cells
Mechanism of Interferon Action Biochemical and Genetic Evidence for the Intermolecular Association of the RNA-Dependent Protein Kinase PKR from Human Cells

AbstractThe interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation. Structure–function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1–551)K296R display a dominant negative behavior when expressed in transfected cells. The potential for PKR to form protein multimers has therefore been examined. Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA. First, the intermolecular association of PKRin vitrowas demonstrated in the context of an enzyme–substrate interaction. Purified recombinant histidine-tagged PKR(1–551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA. At a fixed RNA concentration, high concentrations of the HIS-PKR(1–551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself. Second, the yeast two-hybrid system was used to probe the intermolecular association of PKRin vivo.Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1–551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes,HIS3 andlacZ. The full-length RNA-binding deficient PKR(1–551)K64E/K296R double mutant also interacted with PKR(1–551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1–280)wt and PKR(281–551)K296R as well as p53, RAS, and BCL2 did not. Third, both PKR(1–551)K296R and PKR(1–551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells. By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1–551)K296R or PKR(1–551)K64E/K296R. These results indicate that PKR interacts with itself in an intermolecular manner bothin vivoandin vitro,and that RNA binding is neither necessary nor sufficient for PKR multimerization
In VitroProduction of Stable Epstein–Barr Virus-Positive Epithelial Cell Clones Which Resemble the Virus:Cell Interaction Observed in Nasopharyngeal Carcinoma

AbstractThe interaction of Epstein–Barr virus (EBV) with epithelial cells and the consequent role of the virus in the aetiology of undifferentiated nasopharyngeal carcinoma (NPC) is poorly understood. One important obstacle to work in this area has been the lack of an epithelial cell culture system in which EBV is stably maintained. Using a previously described approach in which CR2-transfected epithelial cells (SVK-CR2) are rendered susceptible to transient EBV infection (Liet al., Nature356, 347, 1992), we now demonstrate that the pattern of EBV latent gene transcription in these acutely infected epithelial cells differs from that observed in virus-infected primary B cells. In addition, some of these epithelial cells spontaneously entered the EBV lytic cycle. By cloning Akata virus-infected SVK-CR2 cells we generated two stable lines which remained EBV positive for more than 1.5 years at which time further subclones were isolated. These cloned lines carry the EBV genome as an episome and exclusively use the FQp promoter for driving EBNA1 transcription, display no Cp/Wp promoter activity, and express low levels of the LMP mRNAs. Unlike acutely infected SVK-CR2 cells, the cloned lines responded poorly to suspension-induced terminal differentiation and were impaired in their ability to enter the virus lytic cycle. These results, showing similarities between the cloned EBV-positive SVK-CR2 lines and NPC tumour cells, suggest that stable maintenance of EBV in epithelial cells may require an undifferentiated cellular environment
Transmission of HIV-1 from Productively Infected Mature Langerhans Cells to Primary CD4+ T Lymphocytes Results in Altered T Cell Responses with Enhanced Production of IFN-γ and IL-10

AbstractMature Langerhans cells (mLC), theex vivocorrelates of interdigitating dendritic cells (IDC), are susceptible to infection with HIV-1. As IDC are important activators of T helper (Th) cellsin vivo,we examined the interaction of HIV-1-infected mLC with CD4+ T lymphocytes. HIV-1-infected mLC readily formed clusters with the T cells and efficiently transmitted HIV-1 to the CD4+ Th cells. Formation of syncytia between mLC and T cells was initiated by HIV-1-infected mLC. In the clusters of HIV-1-infected mLC and activated T cells a massive HIV-1 production was observed leading to the subsequent elimination of the activated and infected T helper cells. Examination of the cytokine pattern produced during interaction of infected mLC with CD4+ T cells revealed an enhanced production of IFN-γ and IL-10 in the cocultures. These results suggest that during antigen presentation-driven T cell activation by IDC in the lymphoid tissues, HIV-1-infected IDC might efficiently transmit the virus to Th cells, leading to altered Th cell responses
Nucleotide Sequence and Molecular Analysis of the Rhesus Cytomegalovirus Immediate-Early Gene and the UL121–117 Open Reading Frames

AbstractCytomegalovirus (CMV) has been isolated from many nonhuman primates, including rhesus macaques (Macaca mulatta). To better understand the molecular biology of rhesus CMV (RhCMV), a 9.2-kb DNA restriction fragment spanning the immediate-early (IE) gene has been molecularly cloned and sequenced. Open reading frames (ORF) have been identified and transcripts mapped for regions corresponding to exons 1, 2, 3, and 4 of the IE1 protein of human CMV (HCMV) and to exons 1, 2, 3, and 5 of IE2. The predicted RhCMV IE1 protein was 29 and 40% identical with the HCMV and African green monkey (AGM) CMV IE1 proteins, respectively, and the predicted RhCMV IE2 protein was 48 and 65% identical with the HCMV and AGM CMV IE2 proteins, respectively. Five additional ORF 3′ to the RhCMV IE gene were identified which contained significant homologies with the HCMV UL121–UL117 ORF. The predicted translation products ranged from 29 to 47% identical with, and 52 to 66% similarity to, the corresponding ORF of HCMV. Conservation of nucleic and amino acid sequences, and colinearity of genes, between primate CMV genomes contribute to a better understanding of primate CMV evolution, regulation, and pathogenesis
Human Papillomavirus E7 Oncoproteins Bind a Single Form of Cyclin E in a Complex with cdk2 and p107

AbstractThe E6 and E7 proteins of the high-risk human papillomaviruses (HPVs) act coordinately to immortalize human keratinocytes. These viral oncoproteins function by binding and altering the activity of cellular proteins which regulate cell cycle progression. Among the proteins bound by E7 are the retinoblastoma protein, Rb, as well as the related p107 and p130 proteins. In addition, E7 binds cyclin A, which regulates transit through the S and G2/M phases of the cell cycle. In this study, we demonstrate that HPV 18 E7 also associates with cyclin E which controls the G1/S transition. E7/cyclin E complexes were immunoprecipitated from E7-expressing cells as well as from cell extracts using GST–E7 fusion proteins. E7 was found to complex with a single form of cyclin E, and the binding was mediated through p107. Both E7/cyclin E and E7/cyclin A complexes exhibit kinase activity through associated cdk2 proteins which can contribute to phosphorylation of p107. The association of E7 with proteins which regulate transit through the cell cycle may provide an additional mechanism by which infection with human papillomaviruses results in cellular hyperproliferation
Natural Isolates of ECHO Virus Type 25 with Extensive Variations in IRES Sequences and Different Translational Efficiencies

AbstractThe complete nucleotide sequence of the 5′-untranslated region (5′-UTR) of the genome of three ECHO virus type 25 strains (the JV4 reference strain and two wild isolates) was determined. The 5′-UTR of the two isolates shared 85 and 82% nucleotide identity with the reference strain. The overall folding of the predicted secondary structure of the ECHO virus 5′-UTRs showed significant conservation with that of the poliovirus. Most significant differences were observed in specific domains of the predicted IRES of the wild isolates. The efficiency with which ECHO virus type 25 5′-UTRs promoted internal initiation of translation was examined in anin vitrotranslation system. At low doses of input RNA, all three 5′-UTRs were similarly efficient in promoting internal ribosome entry and behaved similarly to the poliovirus IRES in that downstream cistron translation was markedly stimulated by the addition of HeLa cell extracts to standard reticulocyte lysates. At high RNA doses, the JV4 5′-UTR was much more efficient than the other two ECHO virus 5′-UTRs. This difference was much less marked when reticulocyte lysates were supplemented with 20% HeLa S-10 extracts, which suggests that the two less efficient 5′-UTRs were characterized by a reduced binding capacity to at least one factor present in HeLa cells. In MRC5 cell S-10 extract, internal initiation of translation was stimulated from the IRES of all three ECHO strains, the M1262 IRES being the least stimulated
SIVsmmPBj14Induces Expression of a Mucosal Integrin on Macaque Lymphocytes

AbstractThe PBj14 isolate of simian immunodeficiency virus, SIVsmmPBj14, is an acutely pathogenic lentivirus that causes severe gastrointestinal disease in macaque monkeys. The studies reported here examine the basis for the enteropathic phenotype of SIVsmmPBj14, using flow cytometric analysis of cultured macaque lymphocytes and immunohistochemical staining of tissue specimens from virus-infected macaques. The data show that enteropathic molecular clones of SIVsmmPBj14induce expression of the αEβ7integrin, which is believed to mediate mucosal retention of T-cells, whereas molecular clones from nonenteropathic derivatives of SIVsmmPBj14do not do so. Thus, elevated expression of αEβ7may be responsible, at least in part, for the accumulation of abnormally large numbers of T-cells within the intestinal mucosa during acute SIVsmmPBj14infection
Protection of Nude Mice by Passive Immunization with a Type-Common Human Recombinant Monoclonal Antibody against HSV

AbstractHerpes simplex viral disease is an important cause of morbidity and mortality in man. Although the development of very effective nucleoside analogs with a high therapeutic index has greatly improved the clinical management of herpetic infections, the emergence of drug-resistant viral strains has become a cause of serious concern both because of its clinical implications and in terms of viral ecology. The present report is the first demonstration of thein vivoprotective activity of a type-common human recombinant monoclonal antibody derived from a combinatorial antibody library. Athymic nude mice were infected with HSV type 1 either intracutaneously in the flank or by corneal scarification. Beside reducing mortality rates when administered before infection, the antibody dramatically and significantly prolonged survival times (P< 0.0001) when administered up to 24 hr postinfection, a time when the virus had already reached the peripheral nervous system. This suggests that the antibody may act, at least in part, by interfering with axonal transport of the virus and/or with viral expression. These results indicate that human recombinant antibodies isolated by antigen selection from combinatorial libraries can be effectivein vivo.Such antibodies could complement antiviral chemotherapy and represent valuable tools for the prophylaxis of infections by the herpes simplex viruses
Priming of Measles Virus-Specific CTL Responses after Immunization with a CTL Epitope Linked to a Fusogenic Peptide

AbstractIn this study, the potential of the amino-terminal sequence from the F1polypeptide responsible for the fusion of measles virus (MV) with cell membranes as a carrier system for a CTL epitope from the MV nucleoprotein was examined. The addition of the fusion sequence (FP) at either the amino or the carboxyl terminus of the CTL epitope peptide rendered it immunogenic after intraperitoneal immunization in mice. The CTLs induced were able to lyse target cells pulsed with the peptide or persistently infected with MV. After intranasal administration of a FP–CTL chimera with or without cholera toxin B subunit (CTB) as an adjuvant, CTL responses to the peptide pulsed and to MV-infected target cells were detected. Responses in groups of mice where CTB was used as an adjuvant were stronger. However, intranasal administration of the CTL epitope did not induce a protective response against intracranial challenge with a neuroadapted strain of MV. These findings highlight the potential of fusion sequences as a carrier system for CTL epitopes and the potential of the intranasal route for administration of synthetic peptides representing MV sequences