pinecone/core-2020-05-10-deduplication huggingface dataset:
Splicing Is Required for Transactivation by the Immediate Early Gene 1 of theLymantria disparMultinucleocapsid Nuclear Polyhedrosis Virus

AbstractA region of theLymantria disparmultinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirusie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPVie-1 and contains a highly conserved splice acceptor consensus sequence. Plasmids containing the LdMNPV ORF141 andie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing theie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of splicedie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of theL. disparcell line Ld652Y. Using a plasmid containing theie-0 spliced form of the gene as a transactivator,hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPVhr5 enhancer did not show increased activity when cotransfected with LdMNPVie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system, LdMNPVie-0 acted as an essential replication gene, but LdMNPVie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line
Sequence-Specific Repression of Cotranslational Translocation of the Hepatitis B Virus Envelope Proteins Coincides with Binding of Heat Shock Protein Hsc70

AbstractThe large L envelope protein of the hepatitis B virus has the peculiar capacity to adopt two transmembrane topologies. The N-terminal preS domain of L initially remains in the cytosol while the S domain is cotranslationally inserted into the endoplasmic reticulum membrane. The preS region of about half of the L molecules is posttranslationally translocated to the lumenal space. We now demonstrate that the repression of cotranslational translocation of preS is conferred by a preS1-specific sequence. By analysis of L deletion mutants, the cytosolic anchorage determinant was mapped to amino acid sequence 70 to 94 of L. The intrinsic potential of this determinant to suppress cotranslational translocation was confirmed by transfer to the HBV middle envelope protein. In searching for cellular factors potentially involved in this novel process, we identified the cytosolic heat shock protein Hsc70 as a specific binding partner of L. The interaction site(s) for the chaperone was mapped to amino acids 63 to 107 of L using coimmunoprecipitation andin vitrobinding analyses. Deletion of the cytosolic anchorage determinant almost completely abolished ATP-dependent Hsc70 binding. Therefore, interaction between Hsc70 and L is likely to be responsible for the suppression of cotranslational translocation of the preS domain
The Immunoreceptor Tyrosine-Based Activation Motif of Epstein–Barr Virus LMP2A Is Essential for Blocking BCR-Mediated Signal Transduction

AbstractThe Epstein–Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B lymphocytesin vitro.The cytoplasmic amino-terminal domain of LMP2A contains an immunoreceptor tyrosine activation motif (ITAM). ITAMs consist of paired tyrosine and leucine residues and play a central role in signal transduction of the BCR and the T-cell receptor (TCR). To investigate the importance of the LMP2A ITAM, two EBV recombinants were constructed, each containing a tyrosine-to-phenylalanine point mutation at amino acid 74 or 85 within the ITAM of LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were no longer blocked in the LMP2A ITAM mutant LCLs following BCR cross-linking. In addition, the Syk protein tyrosine kinase (PTK) was unable to bind LMP2A in unstimulated LCLs infected with either of the LMP2A ITAM mutants. Analysis of Syk phosphorylation before and after BCR cross-linking in the LMP2A mutant ITAM LCLs compared with wild-type EBV LCLs indicates a specific role of the LMP2A ITAM on the LMP2A-mediated negative effect on the Syk PTK. These data indicate the importance of the LMP2A ITAM motif in the LMP2A-mediated block on BCR signal transduction and position the role of the Syk PTK as being central to the function of LMP2A
Field and Vaccine Strains of Fowlpox Virus Carry Integrated Sequences from the Avian Retrovirus, Reticuloendotheliosis Virus

AbstractFor baculoviruses and herpesviruses, integration of transposons or retroviruses into the virus genome has been documented. We report here that field and vaccine strains of fowlpox virus (FPV) carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus (REV). Using PCR and hybridization analysis we observed that vaccine and field strains of FPV carry REV sequences integrated into a previously uncharacterized region of the right 1/3 of the FPV genome. Long-range PCR, hybridization, and nucleotide sequence determination demonstrated that one vaccine strain (FPV S) and recently isolated field strains carry a near-full-length REV provirus. For another vaccine strain (FPV M) a rearranged remnant of the LTR was found at the same insertion site. By Western blotting and reverse transcriptase assays we were unable to demonstrate free REV in supernatants of FPV S cultures. The near-full-length REV provirus integrated into the FPV genome is infectious since FPV S DNA gave rise to REV upon transfection into chicken embryo fibroblasts. Upon infection of chickens with FPV S, all chickens developed high-titered antibodies to REV, and REV was isolated from the blood of half of the inoculated chickens. Our observations add to the list of targets for retrovirus integration into DNA virus genomes. The integration of a near-full-length, and apparently infectious, REV provirus into FPV provides additional transmission routes for the retrovirus by way of the infectious cycle of FPV, including the possibility of mechanical transmission by biting insects since FPV is believed to be transmitted by this route. For large DNA viruses, including the poxviruses, retrovirus integration with attendant possibilities of gene transduction may be an important mechanism for virus evolution, including the acquisition of cellular genes with the potential to modify virus virulence and pathogenicity
Persistent Echovirus Infection of Mouse Cells Expressing the Viral Receptor VLA-2

AbstractMouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (α2β1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (α2β1 3T3 cells) or the α2 subunit of human VLA-2 (α2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected α2 3T3 cells produce infectious virus. All EV-1-infected α2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected α2 3T3 or α2β1 L cells. Since α2β1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing
A Conserved African Swine Fever Virus IκB Homolog, 5EL, Is Nonessential for Growthin Vitroand Virulence in Domestic Swine

AbstractAn African swine fever virus (ASFV) gene with similarity to the cellular inhibitor of NFκB (IκB) was described in the pathogenic African isolate Malawi Lil-20/1 (ORF 5EL) and a cell-culture-adapted European virus, BA71V (ORF A238L). Recently, this gene was shown to be a functional IκB homolog capable of downregulating NFκB-regulated gene expression. This observation suggests the gene may be of significance to aspects of ASFV pathogenesis and virulence in domestic swine by interfering with a normal antiviral host response. Here we show, using nucleotide sequence analysis, that5ELis highly conserved among various African and European pathogenic field isolates and that in all cases its similarity to IκB genes is limited to the presence of four low complexity ankyrin repeats in the ASFV gene. The5ELgene of Malawi Lil-20/1 encodes a 28-kDa protein which was expressed early in virus-infected macrophage cell cultures with maximum levels observed at 3 to 5 hr postinfection. To study gene function, a Malawi Lil-20/15ELgene deletion mutant (Δ5EL) was constructed. Growth characteristics of Δ5EL in porcine macrophage cell cultures were indistinguishable from those of the parental virus. And, Δ5ELexhibited an unaltered parental Malawi Lil-20/1 disease and virulence phenotype in domestic swine. Thus, although highly conserved among ASFV isolates,5ELis nonessential for growth in porcine macrophagesin vitroand for viral virulence in domestic swine. A possible role for this gene in transmission of ASFV in nature, a setting which involves the cycling of ASFV between two highly adapted hosts,Ornithodorosticks and warthogs or bush pigs, in sub-Saharan Africa is discussed
Methyltransferase Activity of the Insect Orbivirus JKT-7400: Photoaffinity Labeling of a Minor Virion Protein, VP4, withS-Adenosylmethionine

AbstractJKT-7400 virus is an orbivirus originally isolated fromCulexmosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor. The apparentKmof the MTase forS-adenosylmethionine (AdoMet) was 25 μM.Photoaffinity labeling experiments in which3H-[methyl]-AdoMet was incubated with virions or viral cores demonstrated labeling of VP4, a minor protein present in the viral core, suggesting that this protein is the viral MTase. Labeling of VP4 was inhibited by addition of unlabeled AdoMet orS-adenosylhomocysteine (AdoHcy)
Baculovirus Expression of a Human G2-Specific, Neutralizing IgG Monoclonal Antibody to Puumala Virus

AbstractWe amplified by polymerase chain reaction the heavy- and light-chain antibody genes of a human hybridoma secreting a neutralizing, IgG monoclonal antibody to the G2 protein of Puumala virus. The heavy- and light-chain genes were cloned and sequenced and the deduced amino acids were aligned with those of other human antibodies to identify the constant and variable regions. The genes were cloned into the baculovirus plasmid transfer vector pACUW51 such that the heavy-chain and light-chain genes were under control of the baculovirus polyhedrin or p10 promoters, respectively. The transfer vector plasmid was cotransfected into culturedSpodoptera frugiperda(Sf9) cells with linearized DNA of the baculovirusAutographa californicanuclear polyhedrosis virus, and recombinant baculoviruses were selected by plaque formation on monolayers ofSf9 cells. Expression and secretion of an IgG monoclonal antibody was confirmed by assay of recombinant-infectedSf9 cell supernatants for the presence of the heavy and light chains. Specificity of the expressed human antibody was determined by immune-precipitation of radiolabeled Puumala virus proteins and by ELISA with Puumala virus-infected cell lysates. Similar quantities of the expressed IgG and the authentic monoclonal antibody neutralized Puumala virus in plaque-reduction neutralization assays. Neither the authentic nor the recombinant antibody could passively protect hamsters from challenge with Puumala virus; however, our results demonstrate the potential of this methodology for production of biologically active neutralizing antibodies
Membrane Permeability Changes Induced inEscherichia coliby the SH Protein of Human Respiratory Syncytial Virus

AbstractThe small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed inEscherichia coli.In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein. These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the β-galactoside substrateo-nitrophenyl-β-d-galactopyranoside and the exit of preloaded [3H]uridine from bacterial cells. Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes. In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity. This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of α-helix for all residues of the SH transmembrane domain. Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein. However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes. Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product
Recovery of Infectious Human Parainfluenza Virus Type 3 from cDNA

AbstractInfectious HPIV3 was produced by the intracellular coexpression of four plasmid-borne cDNAs. These separately encoded a complete HPIV3 genome (negative-sense), the HPIV3 nucleocapsid protein N, the phosphoprotein P, and the polymerase protein L. The cDNA-encoded HPIV3 genome differed from the JS wildtype (wt) strain of HPIV3 used in its construction by seven point mutations: four of these are silent mutations in the HN or L gene coding regions that serve as markers of a cDNA-derived virus, two were introduced to create an amino acid substitution that ablates an epitope recognized by the HN-specific monoclonal neutralizing antibody 423/6, and the remaining point mutation results in an incidental amino acid substitution in the HN protein at amino acid position 263. The four plasmids were transfected into HEp-2 cell monolayers and their expression was driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The titer of virus present in the harvested transfection supernatant was low (<5 PFU/ml), and the recovered recombinant virus (rJS) retained each of the seven mutations present in the cDNA from which it was derived. Despite the introduced and incidental mutations, rJS retained the wt phenotypes as regards replication at elevated temperaturein vitroand efficient replication in the upper and lower respiratory tract of hamsters. rJS was also recovered from a cDNA encoding a complete antigenome (positive-sense) with slightly greater efficiency than from the negative-sense construct. The ability to generate infectious HPIV3 from cDNA should greatly enhance our ability to develop new live-attenuated parainfluenza virus vaccines, including chimeric PIV1 and PIV2 vaccines, and to understand the genetic basis of attenuation of PIV3 candidate vaccines
How Lysophosphatidylcholine Inhibits Cell–Cell Fusion Mediated by the Envelope Glycoprotein of Human Immunodeficiency Virus

AbstractWe have investigated the effect of lysophosphatidylcholine (LPC) on fusion of cells expressing the envelope glycoprotein of HIV-1, gp120/gp41, with cells expressing the receptor for this virus, CD4. Fusion was inhibited by micromolar concentrations of LPC added from an aqueous stock solution, but not by membrane-associated LPC or LPC produced in the membrane by the action of phospholipase A2. No inhibition was found for LPCs with acyl chain lengths shorter than 12 carbon atoms, and the inhibitory effect of longer molecules increased with their length. gp120-CD4 specific cell–cell binding was inhibited with a chain length dependence corresponding to that of fusion, but with palmitoyl-LPC, fusion was more strongly affected than binding. These data indicate that gp120/gp41-induced fusion is inhibited by LPC because LPC affects viral protein–host cell binding and not because LPC prevented the formation of lipid intermediates required for fusion
Histones as a Target for Influenza Virus Matrix Protein M1

AbstractMatrix protein M1 purified from influenza A and B viruses has been analyzed for its ability to specifically interact with cellular proteins by immune coprecipitation and by anin vitrobinding assay on nitrocellulose on PVDF membranes. When M1 was mixed with lysates of uninfected cells there was selective binding of histones H2A, H2B, H3, and H4. Week binding of H1 was also observed. The binding specificity of M1 was confirmed by using purified histones. The M1–histone complexes were dependent on pH and ionic strength, indicating electrostatic interactions. Chemical cleavage of M1 by formic acid into an N-terminal 9-kDa fragment and a C-terminal 18-kDa fragment did not abolish interaction with histones. However, after treatment with 1Msodium chloride cleaved M1 no longer bound to histones, whereas uncleaved M1 showed an increased binding activity after salt treatment. These findings suggest that both N- and C-terminal domains of M1 are involved in histone binding and that conformation of M is an important factor in this interaction. The data support the notion that there is specific interaction of M1 with nucleosomes during the nuclear phase of influenza virus replication
Phosphorylation of the Sendai Virus M Protein Is Not Essential for Virus Replication Eitherin Vitroorin Vivo

AbstractA large proportion of intracellular Sendai virus (SeV) M proteins is phosphorylated, but in mature virions the M protein is not phosphorylated or dephosphorylated. Phosphorylated M protein in cells is bound to the cytoskeletal components more firmly than unphosphorylated M protein. Thus it has been hypothesized that M protein phosphorylation plays an important role in the virus life cycle, especially in the step of maturation. Here, a transient expression-mutation experiment of the M gene demonstrated that a change of the Ser residue at the 70th position from the N-terminus to Ala (S70A) totally abolished M protein phosphorylation, strongly suggesting that this residue is phosphorylated. The mutated M gene was then placed in the corresponding region in the cDNA plasmid which generates a full-length antigenome SeV RNA, and a mutant SeV M-S70A was successfully recovered from the cDNA. This mutant virus was indeed defective in M protein phosphorylation but did not differ at all from the wild-type SeV recovered from the parental cDNA either in the replication kinetics and plaque morphology in cultured cells or inin vivoreplication and pathogenicity for mice. We thus concluded that no phosphorylation of the M protein was required for SeV replication eitherin vitroorin vivo
The Sendai Virus C Protein Binds the L Polymerase Protein to Inhibit Viral RNA Synthesis

AbstractThe Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathioneS-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcriptionin vitro,showing that the fusion protein retains biological function. Pulse–chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1.2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose–response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis
Characterization of an Overlapping CD8+and CD4+T-Cell Epitope on Rubella Capsid Protein

AbstractA synthetic peptide corresponding to rubella virus capsid protein residues 263 to 275 which contains an epitope recognized by a cloned CD4+cytotoxic T-lymphocyte (CTL) line was used to induce CD8+T-cell lines specific to this peptide. A peptide-specific CD8+CTL clone was derived and characterized. This peptide-specific CD8+CTL clone exhibited cytotoxicity against target cells infected by a vaccinia recombinant virus expressing rubella virus capsid protein, but not by target cells infected by vaccinia recombinant virus expressing rubella virus E1 or E2 envelope proteins. Analysis of HLA class I restriction of the CD8+CTL clone revealed that A11 and A3 were restrictive elements. Fine mapping with truncated and overlapping peptide analogs revealed a nonamer sequence, C(264-272), as the T-cell epitope eliciting stronger cytotoxicity. Two anchor residues for binding to HLA A11 and A3 were identified at position 2 (isoleucine) and at position 9 (histidine) or at position 8 (arginine) of the epitope sequence. The identification of overlapping CD4+and CD8+T-cell epitopes within the capsid protein sequence C(263–275) implicates a strategy for using such epitopes in a candidate peptide-based rubella vaccine