pinecone/core-2020-05-10-deduplication huggingface dataset:
Activity of Vesicular Stomatitis Virus M Protein Mutants in Cell Rounding Is Correlated with the Ability to Inhibit Host Gene Expression and Is Not Correlated with Virus Assembly Function

AbstractIn addition to its role in virus assembly, the matrix (M) protein of vesicular stomatitis virus (VSV) is involved in virus-induced cell rounding and inhibition of host-directed gene expression. Previous experiments have shown that two M protein mutants genetically dissociate the ability of M protein to inhibit host-directed gene expression from its function in virus assembly: M protein from tsO82 virus is fully functional in virus assembly but defective in the inhibition of host-directed gene expression, while the MN1 deletion mutant, which lacks amino acids 4–21, inhibits host-directed gene expression but cannot function in virus assembly. Experiments presented here compared cell rounding induced by these two mutant M proteins to that of wt M protein. BHK cells were transfected with M protein mRNA transcribedin vitro,and the extent of cell rounding was evaluated at 24 hr posttransfection. The MN1 protein was nearly as effective as wt M protein in the induction of cell rounding, while tsO82 M protein expressed from transfected RNA was not able to induce cell rounding above that observed in negative controls without M protein, although it did cause BHK cells to have a less elongated shape. These results indicate that the ability of MN1 and tsO82 M proteins to induce cell rounding is not correlated with their virus assembly function. Instead the cell rounding activity of these mutants is correlated with their ability to inhibit host-directed gene expression. Previous data suggesting that these two cytopathic activities could be dissociated can be readily accounted for by quantitative differences in M protein expression required. Infection of either BHK cells or L cells with tsO82 virus induced cell rounding, although cell rounding was delayed relative to that following infection with wt VSV, suggesting that tsO82 M protein retains some cytopathic activity. The distribution of actin, vimentin, and tubulin in transfected cells was determined by fluorescence microscopy. In cells transfected with tsO82 M mRNA, these cytoskeletal elements were indistinguishable from those of negative control transfected cells. In cells rounded as a result of transfection with wt M or MN1 mRNA, actin-containing filaments were reorganized into a thick perinuclear ring but were not depolymerized. In contrast, tubulin and vimentin appeared to be diffusely distributed throughout the cytoplasm of rounded cells. These results support the idea that cell rounding induced by M protein results from the depolymerization of microtubules and/or intermediate filaments
Translation and Replication Properties of the Human Rhinovirus Genomein Vivoandin Vitro

AbstractThe poor translation efficiency of genome-length human rhinovirus RNAin vitrousing HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficientin vitrotranslation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particlesin vitro.A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesisin vivo.In order to test this hypothesis and extendin vitrotranslation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed thein vitrosynthesis of T7 transcripts containing the intact poliovirus type 1 (PV1) 5′ noncoding region (5′ NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14) polyprotein-coding region and 3′-terminal sequences. These chimeric RNAs translated efficientlyin vitroand were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimerain vitro.The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirusin vivoare determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5′ NCR may be a limitation in rabbit reticulocyte lysate-basedin vitrotranslation extracts, it does not appear to be a major limiting determinant for growth of rhinovirusin vivoor replication in the HeLa cell-free extract
Human Immunodeficiency Virus Type I Nef Independently Affects Virion Incorporation of Major Histocompatibility Complex Class I Molecules and Virus Infectivity

AbstractWe have recently reported that HIV-1 Nef down-regulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules. MHC-I molecules are one of the predominant cellular proteins associated with HIV-1 virions. Wild-type or nef-mutated HIV-1 virions were analyzed by immunoelectronic microscopy and Western blot for particle-associated MHC-I molecules. The number of MHC-I molecules was significantly higher in HIV-1 virions produced in the absence of Nef than in wild-type virions, indicating that Nef affects the incorporation of MHC-I molecules into virions. Wild-type HIV particles have been shown to be more infectious than Nef−viruses. This difference was maintained when Nef+and Nef−virions devoid of MHC-I molecules were produced in Daudi-CD4 cells. Therefore, the enhancement of virion infectivity and the down-regulation of MHC-I represent independent biological properties of Nef
Isolation and Characterization of Vesicular Stomatitis Virus PolR Revertants: Polymerase Readthrough of the Leader–N Gene Junction Is Linked to an ATP-Dependent Function

AbstractThe switch from transcription to replication of the VSV genome is coupled to assembly of nascent chains and involves an unspecified change in the P–L polymerase complex when it reaches the leader–N gene junction. PolR VSV mutants, in contrast to wild-type virus, read through this first gene junction at high frequency without concurrent assembly, and they show altered ATP requirements for transcriptionin vitro.The mutation(s) responsible for the polR phenotype segregates to the N-RNA template fraction. We report here that both polR1 and polR2 mutants display severe growth restriction in mouse L cells but not in BHK cells. Four of six polR1 revertant viruses, originating from rare plaques on L cells, showed wild-type characteristics for growth, readthrough of leader–N gene junction, and ATP utilization, while two showed partial and quantitatively parallel coreversion of all properties. Sequence analysis of N and P genes of polR mutants and revertants provided proof that a single mutation in the N protein, Arg179 to His, is responsible for the polR phenotype. PolR1, but not polR2, also displayed a phenotypically silent GA-to-GG change in the N–P intergenic dinucleotide sequence. Five of six revertants retained the polR1 N protein mutation and showed no change in their P gene. We conclude that the L protein likely contains second-site suppressors of the polR phenotype, and we propose that the switch from transcription to replication is modulated by an ATP-dependent interaction between the template-associated N protein and the L subunit of the P–L polymerase complex
Sensitization to Fas-Mediated Apoptosis by Hepatitis C Virus Core Protein

AbstractWe have characterized viral–cell interactions of hepatitis C virus (HCV) and liver cells to study the pathogenesis of HCV infection. HepG2 cells constitutively expressing HCV core protein showed apoptotic changes in response to stimulation with anti-Fas monoclonal antibody. Cells treated with the antibody showed extensive cell rounding, shrinkage, and cytoplasmic blebbing and finally detached from plates. Fragmentation of the chromatin was observed in the nucleus and DNA ladders were detected. In contrast, cells expressing HCV envelope, nonstructural proteins or normal HepG2 cells did not exhibit such Fas-mediated apoptosis. However, expression of Fas receptor was not upregulated on the surface of the cells expressing HCV core protein. Apoptotic cell death was prevented by pretreatment with a specific inhibitor of the cysteine protease CPP32, while the specific inhibitor of interleukin-1β-converting enzyme did not show the preventive effect. The results suggest (i) that intracellular expression of HCV core protein makes cells prone to apoptotic death without upregulation of surface Fas expression and (ii) that the CPP32 protease plays a part in the apoptosis effector pathway of HCV core-expressing cells. HCV core protein may have a role in immune-mediated liver cell injury
Major Differences between WHV and HBV in the Regulation of Transcription

AbstractStudies were carried out to further characterize enhancer and promoter elements on the woodchuck hepatitis virus (WHV) genome. We were able to confirm the existence of WHV promoters analogous to the major promoters of the related human hepatitis B virus (HBV) and of an enhancer analogous to the recently described WHV E2 element (Ueda, K., Wei, Y., and Ganem, D.,Virology217, 413, 1996). However, we were unable to identify an enhancer analogous to the E1 element of (HBV), despite the fact that these two viruses share a high degree of sequence homology and genetic organization. Some factor binding sites in the E1 region appeared to be conserved between the two viruses and may be required for the activity of the overlapping X gene promoter of WHV. Others did not appear to be essential for WHV X gene promoter activity, and their functional activity, if any, was not revealed. Our failure to detect a functional enhancer element in the region of WHV homologous to the HBV E1 enhancer may indicate that (i) fundamental differences exist in transcriptional regulation of the small circular genomes of WHV and HBV; (ii) WHV contains an E1 element which is functional in the context of the intact viral genome, but which is unable to function in the context of the various expression constructs used in our experiments; or (iii) correct regulation of WHV transcription via an E1 element is dependent upon transcription factors which are not expressed in the liver-specific cell lines used in our experiments
The T1/35kDa Family of Poxvirus-Secreted Proteins Bind Chemokines and Modulate Leukocyte Influx into Virus-Infected Tissues

AbstractImmunomodulatory proteins encoded by the larger DNA viruses interact with a wide spectrum of immune effector molecules that regulate the antiviral response in the infected host. Here we show that certain poxviruses, including myxoma virus, Shope fibroma virus, rabbitpox virus, vaccinia virus (strain Lister), cowpox virus, and raccoonpox virus, express a new family of secreted proteins which interact with members of both the CC and CXC superfamilies of chemokines. However, swinepox virus and vaccinia virus (strain WR) do not express this activity. Using a recombinant poxviruses, the myxoma M-T1 and rabbitpox virus 35kDa secreted proteins were identified as prototypic members of this family of chemokine binding proteins. Members of this T1/35kDa family of poxvirus-secreted proteins share multiple stretches of identical sequence motifs, including eight conserved cysteine residues, but are otherwise unrelated to any cellular genes in the database. The affinity of the CC chemokine RANTES interaction with M-T1 was assessed by Scatchard analysis and yielded aKdof approximately 73 nM.In rabbits infected with a mutant rabbitpox virus, in which the 35kDa gene is deleted, there was an increased number of extravasating leukocytes in the deep dermis during the early phases of infection. These observations suggest that members of the T1/35kDa class of secreted viral proteins bind multiple members of the chemokine superfamilyin vitroand modulate the influx of inflammatory cells into virus-infected tissuesin vivo
Construction and Characterization of Herpes Simplex Virus Type 1 Mutants with Conditional Defects in Immediate Early Gene Expression

AbstractThe herpes simplex virus type 1 (HSV-1) mutantin1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICP0, to yield mutantin1820. In almost all cell types tested,in1820 initiated infection less efficiently thanin1814, behaving as if lacking both VP16 and ICP0 functions, but in BHK cellsin1820 was less impaired thanin1814. A rescuant ofin1820 at the VP16 locus,in1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types.In1825 was unable to complement an ICP0 null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICP0 protein in functionally significant amounts. The novel host range properties ofin1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, intoin1820 to producein1820K. This mutant could be propagated effectively in BHK cells at 31° but was effectively devoid of the major regulators ICP0, ICP4, and VP16 in other cells types at 38.5°. Cultures could withstand infection with 5 PFU ofin1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative ofin1820K containing theEscherichia colilacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of β-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5°. Cultures infected with 5 PFU per cell of anin1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based onin1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors
Differentiation of the Shutoff of Protein Synthesis by Virion Host Shutoff and Mutant γ134.5 Genes of Herpes Simplex Virus 1

Abstractvhsprotein is the product of the UL41 open reading frame of herpes simplex virus 1. The protein, made late in infection, is packaged into virions and, in newly infected cells, shuts off host protein synthesis by degrading mRNA. γ134.5 gene encodes a protein which precludes total shutoff of protein synthesis after the onset of viral DNA synthesis in infected cells of human derivation. The experiments reported here were designed to test the hypothesis that in cells infected with γ134.5−mutant the total shutoff of protein synthesis reflects the failure to alter the function ofvhsmade late in infection. Hence, double mutants,vhs−and γ134.5−should not cause total shutoff of protein synthesis. The mutants constructed to test the hypothesis were (i) viruses lacking 1 kbp from the coding domain of γ134.5 and carryinglacZ inserted into the coding domain of UL41, (ii) viruses with deletions in γ134.5 genes, (iii) viruses withlacZ inserted into UL41, and (iv) viruses in which the sequences of the deleted or interrupted genes were restored. We report that viruses with wild-type UL41 gene shut off the synthesis of actin, whereas viruses with interrupted genes made amounts of actin comparable to those of mock-infected cells. However, late in infection, protein synthesis in human neuroblastoma cells infected with the γ134.5−mutants was shut off irrespective of the status of the UL41 gene. Conversely, the phenotype of UL41−viruses with wild-type γ134.5 gene could not be differentiated from those of wild-type virus in the same assays. These studies indicate that the functions of the UL41 and γ134.5 genes and their products are independent of each other
Evolutionary Dynamics of Cucumber Mosaic Virus Satellite RNA during Natural Epidemics in Italy

AbstractThe evolutionary dynamics of 22 variants of cucumber mosaic virus satellite RNA (CMV satRNA) isolated in Italy during virus epidemics from 1988 to 1993 were investigated on the basis of their primary structure and biological properties. Most of the variants were amplified from total nucleic acid preparations extracted from field-infected plants, thus representing wild isolates of CMV satRNA. Eleven variants were associated with subgroup II CMV strains, 10 with subgroup I and 1 with a mixed infection by both strains. When inoculated onto tomato seedlings, the variants induced the phenotype (necrogenic or ameliorative) predicted by their nucleotide sequence. Phylogenetic relationships between the satRNA variants were determined using the stationary Markov model, a stochastic model for evolution. For each satRNA, the Markov analysis gave a good correlation between position in the phylogenetic tree and biological properties. The variants with ameliorative and necrogenic phenotypes in tomato followed two different evolutionary dynamics in nature. Tfn-satRNA, a 390-nt-long molecule, followed a third type of evolutionary dynamic far apart from that of the shorter satRNA molecules (i.e., those in the 334- to 340-nt-length class). Average values of the mean constant rate of nucleotide substitutions/site (Ksubs/site) indicated that in nature the variants tend to keep their heterogeneity unchanged from one epidemic episode to the other, even if the outbreaks occur in places very far from each other. This seems to be in agreement with the proposed maintenance of a functional molecular structure as a constraint to CMV satRNA evolution
Growth Transformation of Antigen-Specific T Cell Lines from Rhesus Monkeys byHerpesvirus saimiri

AbstractThis study aims in establishing thein vitrobasis for a primate model to evaluate potential applications ofH. saimiri-transformed T cells. T cell lines specific for myelin basic protein and streptolysin O were derived from rhesus monkeys and transformed to stable antigen-independent growth with strain C488 ofH. saimiri.The transformed T cells from rhesus monkeys did not produce infectious virus and harbored theH. saimirigenome exclusively in an episomal form, whereas transformed T cells from the New World monkeyCalltithrix jacchusreleased infectious virus. Transformed T cells from rhesus monkeys showed an unaltered surface expression of CD2 and CD3, of the activation markers CD25 and CD69, and of the costimulatory molecule CD80 (B7.1). Remarkably, both transformed and nontransformed T cell lines were largely double-positive for CD4 and CD8. In contrast to the parental cell lines, the transformed cells constitutively expressed major histocompatibility complex-DR antigens and were able to present antigen to each other. The transformed T cells from rhesus monkeys continued to express a functionally intact T cell receptor and responded to recognition of their antigen with enhanced proliferation and production of Th1-type cytokines. In conclusion,H. saimiri-transformed rhesus monkey T cells may open a way to primate models for adoptive immunotherapy and studies on the pathogenesis of autoaggressive T cells
In Vivoandin VitroInfection with Two Different Molecular Clones of Visna Virus

AbstractThe behavior of two genetically different molecular clones of visna virus KV1772-kv72/67 and LV1-1KS1 was comparedin vivoandin vitro.On intracerebral inoculation, clone KV1772-kv72/67 induced a similar response in five sheep as has already been reported with neurovirulent derivates of visna virus. Virus was frequently isolated from blood, cerebrospinal fluid (CSF), and lymphoid organs and induced characteristic central nervous system (CNS) lesions. A strong humoral immune response was detected by ELISA, immunoblotting, and neutralization. Six sheep infected with clone LV1-1KS1 showed a completely different picture. No virus could be isolated from blood or CSF during 6 months of infection. At sacrifice all organs were virus-negative except the CNS of one sheep. None of the six sheep developed significant neutralizing antibodies and only low titer antibodies were detected by ELISA and immunoblotting. Minimal CNS lesions were present in one sheep. The molecular clones were also tested in sheep choroid plexus cells (SCP) and macrophages. In macrophages LV1-1KS1 replicated to a significantly lower titer but induced much more cell fusion than KV1772-kv72/67. The clones replicated equally well in SCP cells. Thus, these molecular clones of visna virus, which differ only by 1% in nucleotide sequence, showed a profound difference in replication and pathogenicity bothin vitroandin vivo.These results can be used to map viral genetic determinants important for host–lentivirus interactions
A Highly Conserved Region of the Sendai Virus Nucleocapsid Protein Contributes to the NP–NP Binding Domain

AbstractThe nucleocapsid protein (NP) of Sendai virus is an essential component of both the nucleocapsid template and the NP–NP and NP0–P protein complexes required for viral RNA replication. When expressed alone in mammalian cells NP self-assembles into nucleocapsidlike particles which appear to contain cellular RNA. To identify putative NP–NP binding domains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258–357, MBP–NP1) and the N-terminal 255 amino acids (MBP-NP2) of NP both oligomerized, suggesting that these regions contain sequences important for NP–NP self-assembly. In addition, the MBP–NP1 fusion protein can function as an inhibitor of viral RNA replication. Complementary studies involving site-directed mutagenesis of the full-length NP protein have identified specific residues in the CCR which are essential for viral RNA replicationin vitro.Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP–NP interaction. A third mutant, NP260-1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, appear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific residues in the CCR of the native NP protein which appear to be important for NP–NP or NP–RNA interactions and for genome replication