pinecone/core-2020-05-10-deduplication huggingface dataset:
Expression of a Foreign Epitope by Porcine Reproductive and Respiratory Syndrome Virus

AbstractThe potential of porcine reproductive and respiratory syndrome virus (PRRSV) as a viral vector was explored by the insertion of a sequence encoding a foreign antigen into the infectious cDNA clone of the Lelystad virus isolate. An epitope of the hemagglutinin (HA) protein of human influenza A virus was introduced at the 5′ end and at the 3′ end of ORF7, in each case resulting in a fusion protein between the HA epitope and the nucleocapsid (N) protein. Furthermore, in the construct carrying the HA sequences at the 5′ end of ORF7, additional in-frame insertions encoding the autoprotease 2A of foot-and-mouth disease virus were made between the HA and ORF7 sequences to ensure the generation of a functional N protein from its hybrid precursor. When RNA transcripts from these full-length cDNA clones were transfected into BHK-21 cells, they were each found to replicate, to express the HA epitope, and to produce progeny virus. However, fusion of the HA epitope directly to the nucleocapsid protein either at the N terminus or at the C terminus adversely affected both the viability and the genetic stability of the recombinant PRRS viruses. Serial passage of the recombinant viruses on porcine alveolar macrophages demonstrated that these viruses had lost (part of) the HA epitope at passage four. In contrast, in the PRRS viruses expressing the HA epitope from a precursor cleavable by the autoprotease 2A peptide, the HA epitope was still intact after four passages, and no effect on the viability of these viruses was observed. Immunoprecipitation and pulse chase experiments revealed the efficient and presumably cotranslational cleavage of the HA epitope from the N protein by the 2A protease. Our results demonstrate the feasibility of using PRRSV as a viral vector that might be suitable for the delivery of antigens from other pathogens to the immune system of the pig
Fitness of Human Immunodeficiency Virus Type 1 Protease Inhibitor-Selected Single Mutants

AbstractHuman immunodeficiency virus type 1 (HIV-1) evolution under chemotherapeutic selection pressure in vivo involves a complex interplay between an increasing magnitude of drug resistance and changes in viral replicative capacity. To examine the replicative fitness of HIV-1 mutants with single, drug-selected substitutions in protease (PR), we constructed virus that contained the most common mutations in indinavir-selected clinical isolates, PR M46I and V82T, and the most common polymorphic change in drug-naı&#x0308;ve patients, PR L63P. These mutants were competed in vitro in the absence of drug against the otherwise isogenic WT virus (NL4-3). Phenotypic drug susceptibility was determined with a recombinant virus assay using a single cycle of virus growth. PR M46I and L63P were as fit as WT. However, PR V82T was out-competed by WT. None of these mutants had appreciable phenotypic resistance to any of the protease inhibitors, including indinavir. The PRV82T mutant was hypersusceptible to saquinavir. Thus, the impaired fitness of the V82T single mutant is consistent with its low frequency in protease inhibitor-naı&#x0308;ve patients. The similar fitness of WT (NL4-3), L63P, and M46I is consistent with the common occurrence of L63P in the absence of protease inhibitor-selection pressure, but not with the rare detection of M46I in drug-naı&#x0308;ve patients
Replication of Simian Immunodeficiency Virus (SIV) in ex Vivo Lymph Nodes as a Means to Assess Susceptibility of Macaques in Vivo

AbstractSix macaques, apparently uninfected, following low-dose exposure to the pathogenic SIVmac251 and SIVSME660 by the mucosal route, were used in a pilot study to investigate whether infectability of ex vivo lymph nodes could predict resistance and/or susceptibility to SIV infection in vivo. Of six macaques exposed to the less-pathogenic virus SIVMNE, four resisted viral infection. Analysis of the susceptibility of the PBMC of these four animals before SIVMNE challenge indicated that all of them were resistant to infection by the SIVBK28 isolate and, in three of them, this resistance was dependent on CD8+ T cells. Blocks of lymph nodes of these four macaques were resistant to SIVMNE infection ex vivo following SIVMNE viral challenge exposure. However, the same blocks from the same animals were permissive to the more virulent SIV251(32H). Accordingly, three of these macaques were readily infected following challenge exposure with SIV251(32H). Lymphoproliferative responses in blood or lymph nodes, local C-C chemokine production in the lymph-node explants, and cytotoxic T-cell activity measured throughout the study did not correlate with ex vivo resistance or susceptibility to in vivo infection. In conclusion, PBMC and lymph-node resistance or susceptibility to infection ex vivo appeared to correlate with in vivo infectivity and, thus, these approaches should be further tested for their predictive value for in vivo infection
Sequence Analysis of the Plutella xylostella Granulovirus Genome

AbstractThe Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open reading frames (ORFs) were compared to genes of the first-sequenced GV, Xestia c-nigrum GV (XcGV), and those from other baculoviruses and organisms. PxGV DNA has a size of 100,999 bp with a G + C content of 40.7%. The analysis predicted 120 ORFs with a size of 150 nucleotides or larger that showed minimal overlap. Blast searches followed by a comparison of ORF arrangement with those of completely sequenced baculovirus genomes showed the presence of 102 homologs to other genes in the database. Among them, 74 and 100 were homologous to genes of Autographa californica NPV (AcMNPV) and XcGV, respectively. A striking feature of the relationship between the genomes of PxGV and XcGV was the conservation of the order and orientation of homologous genes. Even though the XcGV genome is much larger than that of PxGV (178 vs 101 kb) and had many more predicted ORFs (181 vs 120) with an average amino acid sequence relatedness of 42%, the order and orientation of almost all homologous genes was conserved. The PxGV genome contained four homologous regions (hrs), each with 10 to 23 repeated sequences of 101 to 105 nucleotides containing a 15-bp imperfect palindrome in the center of the repeats
Epstein–Barr Virus Lacking Glycoprotein gp85 Cannot Infect B Cells and Epithelial Cells

AbstractTo evaluate whether the BXLF2 gene of Epstein–Barr virus (EBV), which codes gp85 protein, is essential for infection of B cells and epithelial cells, we analyzed the infectivity of an EBV recombinant lacking gp85. The cells that were infected with the BXLF2-disrupted virus were unable to express gp85 proteins that could be detected by mouse monoclonal antibody E1D1, specific for gp85/gp25 complexes. The BXLF2-disrupted EBV had the ability to attach to, but not infect, B cells. On the other hand, the same virus failed to bind to and infect NU-GC-3, a human gastric adenocarcinoma cell line that is susceptible to EBV infection. The results indicate that the gp85 is used for infection of not only B cells but also epithelial cells and suggest that the gp85 is necessary for attaching the virus to epithelial cells
Packaging of Tobacco Mosaic Virus Subgenomic RNAs by Brome Mosaic Virus Coat Protein Exhibits RNA Controlled Polymorphism

AbstractThe coat protein (CP) of icosahedral Brome mosaic virus (BMV) was expressed from a genetically engineered rod-shape Tobacco mosaic virus. Molecular characterization of the progeny recovered from symptomatic plants revealed that BMV CP selectively packaged the three subgenomic RNAs of the hybrid virus into two differently sized icosahedral virus-like particles (VLPs). The smaller VLPs packaged only the two smaller subgenomic RNAs. Additional in vitro reassembly assays with BMV CP subunits and transcripts of hybrid subgenomic RNAs further demonstrated that the ability of BMV capsids to display polymorphism is not dependent on the RNA size alone and appears to be controlled by some other feature(s) of the genetically engineered RNA
In Vitro Strand Transfer from Broken RNAs Results in Mismatch but Not Frameshift Mutations

AbstractAn in vitro system to compare the fidelity of strand transfers from truncated vs full-length RNAs was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. All RNAs were derived from the N-terminal portion of the α-lac gene. On full-length donors, transfers occurred when DNAs migrated to the acceptor prior to being completed on the donor. On donors that were truncated, most transfers occurred after DNAs reached the end of the donor. Transfer products were amplified by PCR and used to replace the corresponding region in a vector containing the α-lac gene. Transformed Escherichia coli were screened for α-complementation by blue–white phenotype analysis, with white colonies scored as those with errors in α-lac. These errors were derived from RT synthesis and strand transfer. The mutant colony frequency approximately doubled for transfer products derived from truncated donors (0.026 ± 0.005 vs 0.053 ± 0.011 (three experiments ± SD), for full-length vs truncated, respectively). The increases resulted from additional non-template-directed bases (mostly thymidines) added to the DNAs before transfer. Sequence analysis of DNAs synthesized on truncated donors showed that about 60% had additions (20/34); however, those without additions transferred at a much higher rate than those with. Transfer of the DNAs with additions always resulted in substitutions; no frameshifts were observed. Results are consistent with RT adding nontemplated nucleotides at template termini. Transfer and subsequent extension of these products is severely inhibited relative to products without additions. The potential relevance of these findings to retrovirus replication is discussed
Induction of Apoptosis by the Vpr Protein of Human Immunodeficiency Virus Type 1 Occurs Independently of G2 Arrest of the Cell Cycle

AbstractThe HIV-1 accessory gene product Vpr can inhibit cell proliferation via cell cycle arrest at the G2 phase, and it can induce apoptosis after G2 arrest. We found recently that C81, a carboxy-terminally truncated form of Vpr, induced apoptosis via G1 arrest but did not induce G2 arrest of the cell cycle. Thus, it seemed possible that expression of Vpr in cells might cause apoptosis independently of the ability of Vpr to induce G2 arrest. We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that does not necessarily require induction of G2 arrest. First, it was found that the extent of apoptosis reached a maximum even when few cells were arrested at the G2 phase of the cell cycle and was reduced in inverse proportion to the increased induction of G2 arrest. Thus, the extent of induction of G2 arrest was not correlated with the extent of Vpr-induced apoptosis. Furthermore, we replaced the Ile/Leu residues in the leucine zipper-like domain of Vpr with Ala or Pro and used cells that expressed the mutant protein to demonstrate that Vpr caused apoptosis in a manner that was independent of G2 arrest. Finally, replacement of Ile/Leu by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of wild-type Vpr and C81 revealed that the Ile/Leu residues at positions 60, 67, and 74 in the leucine zipper-like domain were indispensable for induction of apoptosis induced by Vpr and by C81 and confirmed, in addition, that both processes might be regulated by the same pathway. C81 appears to be a useful tool for elucidation of the mechanism of apoptosis induced by expression of Vpr protein
Comparison of Identical Temperature-Sensitive Mutations in the L Polymerase Proteins of Sendai and Parainfluenza3 Viruses

AbstractThe L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal
Inhibition of CCR5-Dependent HIV-1 Infection by Hairpin Ribozyme Gene Therapy against CC-Chemokine Receptor 5

AbstractCCR-5 is a major cellular coreceptor for R5 strains of HIV-1. Individuals carrying a homozygous 32-base-pair deletion in this gene are apparently healthy and are relatively resistant to HIV-1 infection. Since CCR5 appears to be dispensable for the host, but important for initial HIV-1 infection, CCR5 mRNA is an excellent therapeutic target for inhibiting HIV-1 replication via ribozyme knockout. We report here that hairpin ribozymes are able to reduce cellular CCR5 mRNA and cell surface CCR5 when stably introduced into PM1 cells by transduction with recombinant adenoassociated viral vector. The ribozymes effectively protect the cells from infection by R5 HIV-1 strains or non-syncytium-inducing clinical isolates commensurate with a reduction in CCR5 mRNA. These results suggest a novel gene therapy approach to preventing or slowing the disease progression of HIV-1 infection
Interaction of CD82 Tetraspanin Proteins with HTLV-1 Envelope Glycoproteins Inhibits Cell-to-Cell Fusion and Virus Transmission

AbstractThe entry of retroviruses into their target cell involves interactions between the virus envelope glycoproteins and their cellular receptors, as well as accessory ligand–receptor interactions involving adhesion molecules that can also participate in fusion. We have studied the contribution of CD82 proteins to the transmission of the human T-cell leukemia virus type 1 (HTLV-1), which is greatly dependent on cell-to-cell contacts. CD82 proteins belong to a class of cell surface molecules, the tetraspanins, that can act as molecular facilitators in cellular adhesion processes. The coexpression of CD82 proteins with HTLV-1 envelope glycoproteins resulted in marked inhibition of syncytium formation, whereas CD82 proteins had no effect on syncytium formation induced by human immunodeficiency virus type 1 (HIV-1) envelope proteins. The presence of CD82 proteins also inhibited cell-to-cell transmission of HTLV-1. Coimmunoprecipitation and cocapping experiments showed that CD82 associates with HTLV-1 envelope glycoproteins, both within the cell and at the cell surface. Finally, whereas the intracellular maturation of HTLV-1 glycoproteins was not modified by the presence of CD82 proteins, HTLV-1 protein coproduction delayed the intracellular maturation of CD82 proteins. There thus seems to be a reciprocal interaction between virus and cell proteins, and the cellular proteins involved in adhesion modulate retrovirus transmission both positively, as shown in other systems, and negatively, as shown here
Identification and Characterization of Recombinant Subgroup J Avian Leukosis Viruses (ALV) Expressing Subgroup A ALV Envelope

AbstractThree recombinant avian leukosis subgroup J viruses, ADOL 5701A, ADOL 5701AΔ, and ADOL 6803A, carrying a subgroup A envelope have been isolated and characterized. These viruses were identified by their ability to replicate in DF-1/J, a recombinant chicken embryo fibroblast (CEF) cell line expressing the subgroup J envelope that is resistant to subgroup J replication. Flow cytometric analysis of DF-1/J cells infected with ADOL 5701 and ADOL 6803, two subgroup J isolates, indicated cross-reactivity with subgroup A chicken polyclonal serum. Based on published sequences of subgroups A and J isolates, we designed a series of primers to PCR amplify the envelope and LTR of these viruses. PCR products were obtained when the forward primer was specific for subgroup A gp85 envelope protein gene and the reverse primer was specific for subgroup J LTR. Sequence analysis of the PCR products indicated that these viruses had a subgroup A gp85, a subgroup E gp37, and a subgroup J LTR. Interestingly, these viruses had previously been propagated in CEF from the alv6 chicken line, a line that carries a replication defective recombinant endogenous virus expressing a subgroup A envelope (RAV 0-A1). Sequence analysis of RAV 0-A1 gp85 and gp37 envelope proteins indicated that they were almost identical to those of the recombinants ADOL 5701A and ADOL 6803A. These results indicate that these three recombinant viruses arose by recombination between exogenous subgroup J isolates and a recombinant defective endogenous virus with subgroup A envelope
Puumala (PUU) Hantavirus Strain Differences and Insertion Positions in the Hepatitis B Virus Core Antigen Influence B-cell Immunogenicity and Protective Potential of Core-Derived Particles

AbstractHepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1–45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1–45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40–50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18–20 N (aa 1–45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75–119 of PUU (CG18–20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231–240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge
The HCV Core Protein Acts as a Positive Regulator of Fas-Mediated Apoptosis in a Human Lymphoblastoid T Cell Line

AbstractHepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide and is remarkably efficient at establishing persistent infections. Previously, we have shown that the core protein has an immunomodulatory function including the suppression of T lymphocyte responses to viral infection. To investigate the underlying mechanism for the role of core protein in immune modulation, we examined the effect of core on the sensitivity of the human T cell line, Jurkat, to Fas-mediated apoptosis. The transient and stable expression of core protein in Jurkat cells increased the sensitivity of cells to Fas-mediated apoptosis when compared to control cells expressing vector DNA alone. In addition, we demonstrated that the core protein binds to the cytoplasmic domain of Fas which may enhance the downstream signaling event of Fas-mediated apoptosis. The expression of core protein did not alter the cell surface expression of Fas, indicating that the increased sensitivity of core-expressing cells to Fas ligand was not due to upregulation of Fas. Furthermore, we observed the augmentation of caspase-3 activity in core-expressing cells. These results suggest that the core protein may promote the apoptosis of immune cells during HCV infection via the Fas signaling pathway, thus facilitating HCV persistence
Influenza A and Sendai Viruses Induce Differential Chemokine Gene Expression and Transcription Factor Activation in Human Macrophages

AbstractChemokines regulate leukocyte traffic and extravasation into the site of inflammation. Here we show that influenza A- or Sendai virus-infected human macrophages produce MIP-1α, MIP-1β, RANTES, MCP-1, MCP-3, MIP-3α, IP-10, and IL-8, whereas no upregulation of MIP-3β, eotaxin, or MDC production was detected. Influenza A virus was a better inducer of MCP-1 and MCP-3 production than Sendai virus, whereas MIP-1α, MIP-1β, RANTES, MIP-3α, and IL-8 were induced preferentially by Sendai virus. Infection in the presence of protein synthesis inhibitor indicated that ongoing protein synthesis was required for influenza A virus-induced expression of MCP-1, MCP-3, and IP-10 genes, whereas Sendai virus-induced chemokine mRNA expression took place in the absence of de novo protein synthesis. Neutralization of virus-induced IFN-α/β resulted in downregulation of virus-induced IP-10, MCP-1, and MCP-3 mRNA expression. IFN-α or IFN-γ were found to directly enhance MCP-1, MCP-3, and IP-10 mRNA expression. Both influenza A and Sendai viruses similarly activated transcription factor NF-κB. In contrast to NF-κB, IRFs and STATs, the other transcription factors involved in the regulation of chemokine gene expression, were differentially activated by these viruses. Influenza A virus more efficiently activated ISGF3 complex formation and Stat1 DNA-binding compared to Sendai virus, which in turn was a more potent activator of IRF-1. Our results show that during viral infections macrophages predominantly produce monocyte and Th1 cell attracting chemokines. Furthermore, virus-induced IFN-α/β enhanced chemokine gene expression in macrophages emphasizing the role of IFN-α/β in the development of Th1 immune responses