pinecone/core-2020-05-10-deduplication huggingface dataset:
Determinants of Mouse Hepatitis Virus 3C-like Proteinase Activity

AbstractThe coronavirus, mouse hepatitis virus strain A59 (MHV), expresses a chymotrypsin-like cysteine proteinase (3CLpro) within the gene 1 polyprotein. The MHV 3CLpro is similar to the picornavirus 3C proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of Q/(S, A, G) dipeptide cleavage sites. However, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus 3C-like proteinases or of the precise coding sequence of 3CLpro within the gene 1 polyprotein. In this study, aspartic acid residues in MHV 3CLpro were mutated and the mutant proteinases were tested for activity in anin vitro transcleavage assay. MHV 3CLpro was not inactivated by substitutions at Asp3386(D53) or Asp3398(D65), demonstrating that they were not catalytic residues. MHV 3CLpro was able to cleave at a glutamine–glycine (QG3607-8) dipeptide within the 3CLpro domain upstream from the predicted carboxy-terminal QS3635-6cleavage site of 3CLpro. The predicted full-length 3CLpro (S3334to Q3635) had an apparent mass of 27 kDa, identical to the p27 3CLpro in cells, whereas the truncated proteinase (S3334to Q3607) had an apparent mass of 24 kDa. This 28-amino-acid carboxy-terminal truncation of 3CLpro rendered it inactive in atranscleavage assay. Thus, MHV 3CLpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3CLpro domain was required for activity. These studies suggest that the coronavirus 3CL-proteinases may have a substantially different structure and catalytic mechanism than other 3C-like proteinases
Alphavirus Budding Is Dependent on the Interaction between the Nucleocapsid and Hydrophobic Amino Acids on the Cytoplasmic Domain of the E2 Envelope Glycoprotein

AbstractThe interaction between the nucleocapsid core and the glycoprotein spikes is a critical component in the budding process of alphaviruses. A molecular model was previously proposed which suggested that this interaction was mediated by the binding of the cytoplasmic domain of glycoprotein E2 into a hydrophobic pocket found on the surface of the nucleocapsid protein [S. Lee, K. E. Owen, H.-K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn (1996)Structure4, 531–541; U. Skoging, M. Vihinen, L. Nilsson, and P. Liljeström (1996)Structure4, 519–529]. Two hydrophobic amino acids in the cytoplasmic domain of E2 were predicted to be important in the contact between the proteins. One of the residues, Y400 (Sindbis virus numbering), had previously been shown by mutational studies to be important in the budding of Semliki Forest virus [H. Zhao, B. Lindqvist, H. Garoff, C. H. von Bonsdorf, and P. Liljeström (1994)EMBO J.13, 4204–4211]. The role of the second residue, L402, had not been examined. By creating a panel of amino acid substitutions at this residue, followed by phenotypic analysis of rescued mutant viruses, we now show that L402 is critical for the production of Sindbis virus. Substitutions at this amino acid inhibit budding, and the data suggest that L402 plays an important role in the interaction between the glycoprotein and the nucleocapsid core. These data support the model and suggest that the proposed molecular interactions are important for the budding of alphaviruses from the cell
Spontaneous Occurrence of Early Region 1A Reiteration Mutants of Type 5 Adenovirus in Persistently Infected Human T-Lymphocytes

AbstractMutants of type 5 adenovirus (Ad5) with reiterated DNA sequences in the E1a region appeared in a human T-lymphocyte cell line, Molt-4, persistently infected with H5sub304, a deletion/substitution mutant that has a wild-type phenotype in viral replication. Endonuclease analyses and DNA sequencing revealed DNA reiteration in each mutant. In the four representative mutants investigated, the DNA reiterations all started within a six-base-pair consensus sequence, G(or C)CTGTG, located in the second exon of the E1a region (at nt 1333, 1367, or 1419). There was not any DNA homology between the breakpoints in the second exon and the inserting sequences (starting at nt 532, 710, or 792). Northern analyses suggested that the reiterated splicing sites of the representative mutants were all used in RNA splicing, and the closest donor and recipient joints were used most frequently. These observations imply that during persistent infection Ad5 underwent spontaneous mutations by sequence-specific breakage and nonhomologous end–end joining recombination events. These E1a reiteration mutants could be propagated in HeLa, A549, and KB cells; they were genetically stable; and they killed CREF cells at a strikingly high frequency. Preliminary observations tend to correlate this CREF cell killing with the accumulation of the early viral proteins and/or viral DNA in the infected cells. This degree of cell damage was not observed in Ad5wt or H5sub304 infection of CREF cells. The observed E1a reiterations provide a model to gain insight into understanding the evolutionary events of some, if not all, adenovirus types during many years of symbiotic, persistent relationship in human tonsils and adenoids and possibly other lymphoid organs
An Equine Herpesvirus-1 Gene 71 Deletant Is Attenuated and Elicits a Protective Immune Response in Mice

AbstractThe pathogenesis of pulmonary infection and the immune response following intranasal inoculation of mice with two equine herpesvirus type 1 (EHV-1) deletion mutants have been assessed. The mutants, ED71 and ED75, have deletions in genes 71 (EUS4) and 75 (10K), respectively. Deletions were replaced by theEscherichia coli lacZgene driven by the simian virus 40 (SV40) early promoter. It has previously been shown that the protein products of genes 71 and 75 are dispensablein vitrobut that removal of gene 71 results in a defect in virus maturation and capsid envelopment which impairs the ability of mutant virus to spread via release and readsorption. This study demonstrated that the 192-kDa gene 71 product is required for full expression of virulence in mice, whereas the putative 10-kDa product of gene 75 has minimal effect. Both mutants exhibited the same tissue and cytotropism as wild-type EHV-1 and induced both humoral and cell-mediated immune responses indistinguishable from those induced by the parental strain. Irrespective of the reduced pathogenicity of the gene 71 mutant, infected mice were protected against a challenge with wild-type EHV-1. These findings highlight the potential of ED71 as a vaccine candidate
Infection and Replication of Herpes Simplex Virus Type 1 in an Organotypic Epithelial Culture System

AbstractWe have used the organotypic culture system as a model to study the initial infectious process and spread of herpes simplex virus type 1 (HSV-1) in fully stratified and differentiated human epithelial tissue. The growth kinetics of HSV-1 were determined in organotypic tissues of human epidermal or ectocervical origin. Concurrently, we followed the spread of HSV-1 by immunostaining thin sections of infected organotypic tissue. After HSV-1 was applied to the top cornified epithelial layer, virus penetrated to the basal layer of replicating epithelium and grew to high titers. The virus was limited in its spread in that not all cells within the tissue had demonstrable infection. A ribonucleotide reductase mutant, ICP6Δ, could infect and replicate in basal layers of the organotypic tissues. However, we found that spread was limited in, and to, the basal cell layer. Peak ICP6Δ titers were 100-fold less than in cultures infected with wild-type HSV-1. Studies of HSV mutants should allow us to further define the role of specific viral genes which are associated with infection and spread in a tissue culture system that mimics the initial portal of entry for certain HSV infections
Human Immunodeficiency Virus Type 1 (HIV-1) Infection of Herpesvirus Saimiri-Immortalized Human CD4-Positive T Lymphoblastoid Cells: Evidence of Enhanced HIV-1 Replication and Cytopathic Effects Caused by Endogenous Interferon-γ

AbstractHerpesvirus saimiri (HVS) is a nonhuman primate gamma herpesvirus which can immortalize human T lymphocytes similar to Epstein–Barr virus immortalization of B cells. The HVS-immortalized T cell lines can be cloned and they remain functional, including susceptibility of CD4 expressing T cells to infection with human immunodeficiency virus type 1 (HIV-1). In this report, we have used five such HVS-transformed CD4-positive T cell clones to reevaluate the role of endogenous interferon gamma (IFNγ) in HIV-1 replication in T cells. All five clones had similar phenotypes; and four clones constitutively produced IFNγ and one clone did not. All five clones could be efficiently infected with HIV-1. HIV-1 infection of the IFNγ-positive cells also upregulated IFNγ mRNA production and IFNγ secretion but not production of IL-2 or IL-4. In contrast, infection of IFNγ-negative cells did not induce IFNγ, IL-2, or IL-4. Exposure to anti-IFNγ antibodies after HIV-1 infection significantly reduced virus production and inhibited virus-induced death of IFNγ-positive cells but had no effect on IFNγ-negative cells. We conclude that in CD4-positive T lymphocytes immortalized by HVS endogenous IFNγ does not inhibit HIV-1 but enhances HIV-1 replication and cytolysis. The potential augmenting effects of IFNγ on HIV-1 replication in CD4-positive T cells recommend caution in a therapeutic use of this cytokine in AIDS
Alternative Expression of a Chitosanase Gene Produces Two Different Proteins in Cells Infected withChlorellaVirus CVK2

AbstractSeveralChlorellavirus CVK2 proteins had chitosanase and/or chitinase activities. A gene coding for an ORF of 328 amino acids (aa) with a predicted molecular mass of 36,769 Da was cloned from the viral genome. The predicted amino acid sequence of an N′-portion (174 aa) of this gene product (vChta-1) showed 22 to 25% identity with various bacterial chitosanases. A glutathioneS-transferase (GST)–vChta-1 fusion protein had strong chitosanase activity. Western blot analysis with antisera raised against the vChta-1 protein identified two proteins of 37 and 65 kDa in virus-infectedChlorellacells beginning at 240 min postinfection and continuing until cell lysis. The larger protein was packaged in the virion, while the smaller one remained in the cell lysate. Both chitosanase proteins were produced from the single gene,vChta-1,by a mechanism of alternative gene expression
The Two P2 Ogr-like Domains of the Delta Protein from Bacteriophage P4 Are Required for Activity

AbstractThe satellite P4 phage Delta protein positively regulates the late genes of its helper bacteriophage P2, as well as its own late genes. Delta is a member of a class of activators associated with P2- or P4-like phages and is the largest member of this family. It resembles a covalently joined head-to-tail dimer of the other members of this family of activators. We have analyzed the requirement for both standard domains of Delta through the isolation of amber mutants and the insertion of amber linkers. We show that both domains of Delta are required for DNA bindingin vivoand for transcriptional activity. Proper spacing between the two domains is important for activity at two of the four P2 promoters. Expression of both domains from different plasmids causes activation of late gene transcriptionin vivoof all six late promoters of P2 and P4. A monomric Delta from another satellite phage, φR73, can function efficiently as a covalent dimer but when this Delta is made dimeric with the second half of P4 δ, it activates less efficiently
Mutations in the Region Encoding the Central Domain of Helper Component-Proteinase (HC-Pro) Eliminate Potato Virus X/Potyviral Synergism

AbstractCoinfection of tobacco plants with potato virus X (PVX) and any of several members of the potyvirus group causes a synergistic disease characterized by a dramatic increase in symptom severity correlated with a 3- to 10-fold increase in the accumulation of PVX in the first systemically infected leaves. We have recently shown that PVX/potyviral synergistic disease is mediated by expression of potyviral 5′-proximal sequences encoding P1, helper component-proteinase (HC-Pro), and a fraction of P3 (termed P1/HC-Pro sequence). Here we report the effect of mutations in this potyviral sequence on the induction of synergistic disease. Three transgenic tobacco lines expressing the tobacco etch potyvirus (TEV) P1/HC-Pro sequence with mutations within the P1 coding region were not impaired in their ability to mediate synergism when infected with PVX. In contrast, two of three transgenic lines with mutations in the HC-Pro coding region were unable to induce the synergistic increases in either symptom severity or PVX accumulation. Loss of synergistic function was associated with mutations within the region encoding the central domain of HC-Pro, while the ability to induce synergism was retained in a transgenic line expressing HC-Pro with an alteration in the amino-terminal “zinc-finger domain.” In coinoculation experiments, a TEV mutant lacking the sequence encoding the zinc-finger domain of HC-Pro induced a typical synergistic response in interaction with PVX. The results indicate that the zinc-finger domain comprising the first 66 amino acid residues of HC-Pro is dispensable for induction of synergistic disease and transactivation of PVX multiplication, while regions within the central domain of HC-Pro are essential for both of these responses
Inhibition of Cellular Cdk2 Activity Blocks Human Cytomegalovirus Replication

AbstractHuman cytomegalovirus is a herpesvirus that induces numerous cellular processes upon infection. Among these are activation of cyclin-dependent kinase 2, which regulates cell cycle progression in G1 and S phase. We report here that inhibition of cellular Cdk2 activity blocks HCMV replication. Inhibition of Cdk2 activity by roscovitine inhibits HCMV DNA synthesis, production of infectious progeny, and late antigen expression in infected cells in a dose-dependent manner. HCMV replication is also inhibited by the expression of a Cdk2 dominant negative mutant, whereas expression of wild-type Cdk2 has no effect on viral replication. These data indicate that activation of cellular Cdk2 is necessary for HCMV replication
Antigenically Distinct G Glycoproteins of BRSV Strains Share a High Degree of Genetic Homogeneity

AbstractBovine respiratory syncytial (BRS) virus can be divided into antigenic subgroups based on the reactivity of monoclonal antibodies (mAbs) to the attachment glycoprotein, G. Further, the polyclonal antibody response of calves vaccinated with recombinant vaccinia viruses expressing the G protein of a particular subgroup is also subgroup-specific. To investigate the genetic basis for the antigenic heterogeneity of the BRS virus G protein, the genes for the G protein from 6 BRS virus strains representative of the antigenic subgroups were cloned, sequenced, and compared with the prototype subgroup A strain, 391-2. There was only 10% nucleic acid difference and 15% amino acid difference between strains from different subgroups. These findings are in sharp contrast to the situation with human RS virus, where there is a 45% difference in amino acid identity between subgroups. In fact, the extent of amino acid difference between BRS virus subgroups is similar to the level of heterogeneity observed within human subgroups. Analysis of the reactivity of mAbs with peptides from the cysteine-rich region (174–188) of the G protein representing each antigenic subgroup indicated that amino acids at positions 180, 183, and possibly 184 are important in subgroup distinction. Taken together, these data suggest that although the genetic variation responsible for the antigenic differences determining subgroups among BRS viruses is more limited than that observed among human RS virus subgroups, the amino acid differences that exist have a profound effect upon antibody recognition
E7 Oncoprotein of Human Papillomavirus Type 16 Expressed Constitutively in the Epidermis Has No Effect on E7-Specific B- or Th-Repertoires or on the Immune Response Induced or Sustained after Immunization with E7 Protein

AbstractA line of FVB (H-2q) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the α-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development of inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive (“ignorant”) to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups of young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2qbackground of these mice, incorporation of a Dballele into the genome allowed comparison of Db-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization
Multiple Mechanisms for the Inhibition of Entry and Uncoating of Superinfecting Semliki Forest Virus

AbstractRecombinant Semliki Forest viruses (SFV) that express one or none of the viral structural proteins were used to infect cells and to analyze the fate of incoming superinfecting wild-type viruses. It was found that in addition to the previously described block in replication that superinfecting viruses encounter within 15 min of infection, other mechanisms of superinfection inhibition occurred at later times. Over a 6-hr infection period, inhibition was seen in binding of virus to the cell surface, in acid-activated penetration into the cytoplasm, and in uncoating of nucleocapsids. For each of these processes, the inhibitory mechanism was investigated. In summary, we found that infection evoked several independent mechanisms for blocking the entry and uncoating of superinfecting viruses. The results also offered new insights into the normal processes of penetration and uncoating of SFV
Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Nonstructural 5A Protein

AbstractHepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally, the inhibition of the PKR protein kinase by NS5A is the first described function for this HCV protein
The Apoptosis Pathway Triggered by the Interferon-Induced Protein Kinase PKR Requires the Third Basic Domain, Initiates Upstream of Bcl-2, and Involves ICE-like Proteases1

AbstractThe interferon-induced double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase which exerts antiviral and anticellular functions. The antiviral effect of PKR is mediated by the phosphorylation of the alpha subunit of the translational initiation factor eIF-2α, while it is not known whether the anticellular effect is due to phosphorylation of eIF-2α, IκB, or other unknown substrates. We have previously shown that activation of PKR during infection of cells with a vaccinia virus recombinant expressing the wild-type kinase resulted in a complete inhibition of viral and cellular protein synthesis and in the induction of apoptosis. Here, we report that expression of the human proto-oncogene bcl-2 blocks PKR-induced apoptosis but not PKR-induced inhibition of translation. In addition, PKR-induced apoptosis resulted in a cleavage of the death substrate poly(ADP-ribose) polymerase (PARP). Moreover, induction of apoptosis by PKR was not observed with a mutant lacking the third basic region (aa 234–272). Taken together, these results suggest that the third basic region of PKR is required for PKR-induced apoptosis, the process is initiated upstream of bcl-2 and involves activation of a cellular protease, CPP32, or its family members that cleave PARP
Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxvirus

AbstractWe have identified anAmsacta mooreientomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (kobs= 0.08 sec−1;Kcl= 0.22) are similar to those of the vaccinia virus enzyme