pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression

AbstractIn addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression in cells persistently infected with VSV. Viruses isolated from L cells persistently infected with VSV inhibited host RNA synthesis more slowly than wild-type (wt) VSV. M genes of the persistent viral population were cloned and sequenced. One mutation, an N to D change at position 163 of the protein sequence (N163D), was common to all the molecular clones. The N163D M protein was synthesized from transfected mRNA at a rate that was 30% of that of wt M protein, but was turned over at a rate that was similar to that of wt M protein. Transfection of mRNA encoding N163D M protein inhibited expression of a cotransfected target gene encoding chloramphenicol acetyl transferase (CAT), but the inhibition was 6 to 10 times less effective than transfection of equivalent amounts of wt M mRNA. This difference could not be accounted for by differences in translation of CAT mRNA. Thus, when the differences in M protein expression were taken into account, N163D M protein was 2 to 3 times less effective than wt M protein in the inhibition of host-directed gene expression, similar to the differences in host transcription observed in virus-infected cells. Point mutations in addition to the N163D mutation were found in about half of the M gene molecular clones. The M gene of an independently isolated molecular clone, N163D.2, contained two additional point mutations in its carboxy terminal region. N163D.2 M protein was highly defective in inhibition of host gene expression and was turned over more rapidly than wt M protein. These results support the idea that M gene mutations contribute to a reduced cytopathic effect in cells persistently infected with VSV
The Cysteine Residues of the M2 Protein Are Not Required for Influenza A Virus Replication

AbstractThe M2 protein of influenza A virus functions as an ion channel. It contains three cysteine residues: cysteines 17 and 19, which form disulfide bonds in the ectodomain, and cysteine 50, which is acylated. To understand the role of these cysteine residues in virus replication, we used reverse genetics to create influenza viruses in which the individual cysteines were mutated and a virus in which all three cysteines were changed to serine. The M2 cysteine mutants that lacked either of the cysteine residues in the ectodomain and the mutant that lacked all three residues had appreciably lower amounts of M2 oligomers than did the wild-type virus when examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. None of the mutants, however, were defective in replication, eitherin vitroor in ferrets and mice. These findings demonstrate that noncovalent interactions are sufficient for the M2 protein to form functional oligomers for virus replication and that its cysteine residues are dispensable for influenza virus replicationin vitroandin vivo
Mutations Affecting the Sensitivity of the Influenza Virus Neuraminidase to 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic Acid

Abstract4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en) specifically inhibits the influenza virus neuraminidase (NA) through interaction of the guanidino group with conserved Glu 119 and Glu 227 residues in the substrate binding pocket of the enzyme. To understand the mechanism by which influenza viruses become resistant to 4-guanidino-Neu5Ac2en, we investigated mutations at amino acid residues 119 and 227 in the influenza virus NA for their effects on this compound and on NA activity. The NA gene was cloned from the NWS-G70c virus, and mutations were introduced at the codon for amino acid residue 119 or 227. All of the 13 mutants containing a change at residue 119 were transported to the cell surface, although their expression levels ranged from 68.2 to 91.3% of wild type. Mutant NAs that retained at least 20% of the wild-type enzymatic activity were tested for their sensitivity to 4-guanidino-Neu5Ac2en and found to be sevenfold less sensitive to this compound than was the wild-type NA. By contrast, only 6 of 13 mutants defined by modifications at residue 227 were transported to the cell surface, and those NAs lacked substantial enzymatic activity (9% of wild type, at most). These results suggest that only a limited number of resistant viruses arise through mutations at Glu 119 and Glu 227 under selective pressure from 4-guanidino-Neu5Ac2en and that the development of compounds which interact with 227 Glu more strongly than does 4-guanidino-Neu5Ac2en may reduce the likelihood of drug-resistant viruses still further
Passage of HIV-1 Molecular Clones into Different Cell Lines Confers Differential Sensitivity to Neutralization

AbstractIn this study, progeny viruses of four HIV-1 molecular clones were tested for sensitivity to neutralization following prolonged passage in peripheral blood mononuclear cells (PBMC) and MT-2, H9, and CEM T-lymphoid cell lines. Two of the viruses were able to establish persistent infection with no cytopathic effect in H9 and CEM cells. Such adaptation conferred increased sensitivity to neutralization by a panel of human sera obtained from HIV-1-infected asymptomatic individuals, by soluble CD4 and by monoclonal antibodies directed to a linear epitope in the V3 region (268-D) and a conformational epitope in the CD4 binding site of the envelope gp120 (1.5e). Increased sensitivity to neutralizatiom was paralleled by increased binding of these mAbs to native envelope glycoproteins and by increased binding capacity to CD4 expressed on the cell surface. Our results show that virus–host cell interactions are important in influencing sensitivity to neutralization of HIV-1. In primary PBMC or in cytopathic interactions in cell lines, like in MT-2 cells, envelope epitopes important for neutralization remain masked. In contrast, noncytopathic but productive virus–host cell interactions may lead to an increased exposure of neutralizing epitopes and more efficient binding capacity to CD4 resulting in an increased sensitivity to neutralization
Envelope Glycoprotein Nucleotide Sequence and Genetic Characterization of North American Ovine Lentiviruses

AbstractOvine lentiviruses (OvLV) resemble human immunodeficiency viruses in genomic organization, viral heterogeneity, and spectrum of cytophenotypic expression. To gain a better understanding of the relationship of North American OvLV isolates with other characterized OvLV strains, the complete DNA nucleotide sequence of theenvregion of a highly lytic (rapid/high) OvLV strain (85/34) was determined and compared with the sequence of amplicons withinenvof three other OvLV strains of varying cytophenotype and isolated from the same flock of sheep. LTR andpolregions also were compared among these strains. Theenvregion of 85/34 was 986 codons in length and the reported nucleotide sequence showed features shared by other OvLV including heavy glycosylation and conserved and hypervariable regions within the surface membrane protein region. Phylogenetic analyses of regions within LTR, reverse transcriptase, andenvgrouped the four virus strains together and similar to the maedi-visna OvLV strains, including visna virus, South African ovine maedi visna virus, and EV1 (British OvLV isolate), but they were distinct from caprine arthritis encephalitis virus
Phylogenetic Relationship of the Complete Rauscher Murine Leukemia Virus Genome with Other Murine Leukemia Virus Genomes

AbstractWe report the complete nucleotide sequence of the genome of Rauscher murine leukemia virus (R-MuLV), the replication-competent helper virus present in the Rauscher virus complex, and its phylogenetic relationship with other murine leukemia virus genomes. An overall sequence identity of 97.6% was found between R-MuLV and the Friend helper virus (F-MuLV), and the two viruses were closely related on the phylogenetic trees constructed from eithergag, pol,orenvsequences. Moloney murine leukemia virus (Mo-MuLV) was the next closest relative to R-MuLV and F-MuLV on all trees, followed by Akv and radiation leukemia virus (RadLV). The most distantly related helper virus was Hortulanus murine leukemia virus (Ho-MuLV). Interestingly, Cas-Br-E branched with Mo-MuLV on thegagandpoltrees, whereas on theenvtree, it revealed the highest degree of relatedness to Ho-MuLV, possibly due to an ancient recombination with an Ho-MuLV ancestor. In summary, a phylogenetic analysis involving various MuLVs has been performed, in which the postulated close relationship between R-MuLV and F-MuLV has been confirmed, consistent with the pathobiology of the two viruses
The Complete Nucleotide Sequence of the Egg Drop Syndrome Virus: An Intermediate between Mastadenoviruses and Aviadenoviruses

AbstractThe complete nucleotide sequence of an avian adenovirus, the egg drop syndrome (EDS) virus, was determined. The total genome length is 33,213 nucleotides, resulting in a molecular weight of 21.9 × 106. The GC content is only 42.5%. Between map units 3.5 and 76.9, the distribution of open reading frames with homology to known genes is similar to that reported for other mammalian and avian adenoviruses. However, no homologies to adenovirus genes such as E1A, pIX, pV, and E3 could be found. Outside this region, several open reading frames were identified without any obvious homology to known adenovirus proteins. In the region organized similarily as other adenoviral genomes, most homologies were found to an ovine adenovirus (OAV strain 287). The highest level of amino acid identity was found for the hexon proteins of EDS and OAV. The virus-associated RNA (VA RNA) was identified thanks to the homology with the VA RNA of fowl adenovirus serotype 1 (FAV1). Similarities with FAV1 were also found in the fiber protein. Our results demonstrate that the avian EDS virus represents an intermediate between mammalian and avian adenoviruses. The nucleotide sequence and genomic organization of the EDS virus reflect the heterogeneity of the aviadenovirus genus and theAdenoviridaefamily
Liquefaction ofAutographa californicaNucleopolyhedrovirus-Infected Insects Is Dependent on the Integrity of Virus-Encoded Chitinase and Cathepsin Genes

AbstractWe examined the role of theAutographa californicanucleopolyhedrovirus (AcMNPV)-encoded chitinase in virus pathogenesis inTrichoplusia nilarvae. In conjunction with the AcMNPV-encoded cathepsin, it promotes liquefaction of the host in the latter stages of infection. Insects infected with virus mutants lacking either the chitinase A gene (chiA) or cathepsin gene (cath) remained intact several days after death. However, if both viruses were used to infect insects, liquefaction of the host was restored. Chitinase was readily detected in AcMNPV-infected insects using a chitinase-specific antibody, but it was absent from insects infected with achiAdeletion mutant (AcchiA−). The chitinase was also detected in polyhedra purified from AcMNPV-infected insects but not in those from AcchiA−. However, polyhedra derived from a virus lacking an intactchiAwere no less effective in initiating an infection in second instarT. nilarvae than those of the unmodified AcMNPV. It was also demonstrated that the virus chitinase retained high levels of activity between pH 3.0 and 10.0. In contrast, chitinases isolated fromSerratia marcescens,although active under acidic conditions, rapidly lost activity above pH 7.0 illustrating that despite 57% sequence identity, the two proteins have distinct enzymic activities
In VitroInfection and Replication of Hepatitis E Virus in Primary Cynomolgus Macaque Hepatocytes

AbstractAnin vitromodel was developed to replicate hepatitis E virus (HEV) in normal primary cynomolgus macaque hepatocytes using a hormonally defined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liver wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PCR) assay, which could detect the positive- and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectious HEV (Burma strain) inocula were titered by this RT-PCR assay, and a minimum effective infectious dose was determined. Appearance of newly replicated virus was demonstrated by detection of both strands of HEV RNA in experimentally infected hepatocytes as well as the genomic positive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and replication of the virus in culture. Using thisin vitroinfection system, a neutralization assay was developed to assess the ability of anti-HEV antibodies to block virus infection of liver cells. Results presented in this report represent the firstin vitrodemonstration of a neutralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein
Identification of the Functional Regions Required for Hepatitis D Virus Replication and Transcription by Linker-Scanning Mutagenesis of Viral Genome

AbstractTo define the importantcis-elements in hepatitis delta virus (HDV) RNA, the viral genome was mutated by a linker-scanning mutagenesis strategy that maintained the native rod-like structure of HDV RNA. Mutant HDV cDNAs or their corresponding RNA transcripts were transfected into a Huh-7-derived cell line which continuously expressed small hepatitis delta antigen to study the viral replication and transcription. Here we report the following findings. (i) Although most of the mutant RNAs could self-process to generate the 1.7-kb genomic RNA and all their stabilities were similar, positions which surround the genomic ribozyme domain were found to be important for the self-processing of the dimeric RNA. (ii) The replication of viral RNA was greatly diminished in many mutants, suggesting that multiple regions in HDV RNA were required for replication. (iii) In certain mutants, replication of the HDV antigenomic RNA was selectively abolished but that of the genomic RNA was not. Therefore, this was the first report to show that thecis-elements needed for the replication of genomic or antigenomic HDV RNA could be different. (iv) A continuous region (nt 1625 to nt 431), spanning the HDAg mRNA initiation site and containing thein vitroidentified RNA promoter, was found to be important for mRNA productionin vivo.(v) The HDV RNA replication and transcription was previously proposed to be governed by a single “double-acting promoter.” However, two mutants which were deficient in mRNA synthesis still retained active viral RNA replication. It suggested that the HDV replication could initiate from sites other than this single promoter. This study therefore provided an insight into thecis-elements required for HDV RNA replication and transcription and further contributed to our understanding of HDV life cycle
Analysis of a Temperature-Sensitive Vaccinia Virus Mutant in the Viral mRNA Capping Enzyme Isolated by Clustered Charge-to-Alanine Mutagenesis and Transient Dominant Selection

AbstractWe have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994)Proc. Natl. Acad. Sci. USA91, 4554–4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis.In vitroassays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis
Patterns of Amino Acid Variability in NSI-like and SI-like V3 Sequences and a Linked Change in the CD4-Binding Domain of the HIV-1 Env Protein

AbstractThe V3 domain plays a central role in the biology of the HIV-1 Env glycoprotein gp120 as a dominant target for neutralizing antibodies for some HIV-1 isolates, and as a major determinant in the switch from the nonsyncytium-inducing (NSI) to the syncytium-inducing (SI) form of HIV-1 that is associated with accelerated disease progression. Basic amino acid substitutions are known to play an important role in the SI phenotype. We have used the presence of basic amino acid substitutions in V3 sequences to divide sequences in a large data base into SI-like and NSI-like. We found significant differences in features of sequence variability between these two groups of sequences. Of the thirty-six most frequent substitutions in V3, twenty appear disproportionately among either the SI-like sequences or the NSI-like sequences. The fourteen favored substitutions among the SI-like sequences account for 50% of the twofold increased variability seen in this group. In addition, we found a linked change within the CD4-binding domain of gp120 downstream of V3. These differences are interpreted in the context of structure, function, and selective pressure. An understanding of these patterns of sequence variability offers the possibility of designing a degenerate SI-specific V3 immunogen to use as a therapeutic vaccine with the hope of slowing or preventing the appearance of SI variants
Basic Amino Acid Residues at the Carboxy-Terminal Eleven Amino Acid Region of the Phosphoprotein (P) Are Required for Transcription but Not for Replication of Vesicular Stomatitis Virus Genome RNA

AbstractThe phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein Nin vivo.We have shown that the deletion of 11 amino acids from the C-terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesisin vitroin a transcription reconstitution reaction as well as transcriptionin vivoof a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) bothin vitroandin vivo,suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N proteinin vivo,indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNAin vivo.Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively
Interaction of the Human Protein Kinase PKR with the Mouse PKR Homolog Occurs via the N-Terminal Region of PKR and Does Not Inactivate Autophosphorylation Activity of Mouse PKR

AbstractThe RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes,HIS3 andlacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of theHIS3 andlacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathioneS-transferase (GST) fusions inEscherichia coliwere purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1–184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR