pinecone/core-2020-05-10-deduplication huggingface dataset:
Phosphorylation of Canine Distemper Virus P Protein by Protein Kinase C-ζ and Casein Kinase II

AbstractTranscription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-ζ) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-ζ has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-ζ has only minor activity. Selective inhibition of PKC-ζ activity within CV1 cells eliminated permissiveness to CDV replication, indicating anin vivorole for PKC-ζ in the virus replication cycle. The broad tissue expression of PKC-ζ parallels the pantropic nature of CDV infections, suggesting that PKC-ζ activity is a determinant of cellular permissiveness to CDV replication
Regulation of a Geminivirus Coat Protein Promoter by AL2 Protein (TrAP): Evidence for Activation and Derepression Mechanisms

AbstractTomato golden mosaic virus (TGMV) is a bipartite member of the subgroup III Geminiviridae. Like all geminiviruses, TGMV replicates in the nucleus of susceptible cells by rolling circle replication (RCR). Double-stranded replicative form DNA generated during RCR serves as template for the transcription of viral genes by RNA polymerase II and the associated cellular transcription machinery. Previous studies in tobacco protoplasts andNicotiana benthamianaleaf discs have shown that the viralAL2gene product transactivates expression of the coat protein (CP) andBR1movement protein genes, and that activation occurs at the level of transcription. Because of its function and properties, we propose the name TrAP, transcriptional activator protein, for theAL2gene product. Using transgenes consisting of complete and truncated versions of theCPpromoter fused to the GUS reporter gene, we show in the studies presented here that TrAP is required forCPgene expression in both mesophyll and phloem tissues. Surprisingly, TrAP appears to induceCPexpression by different mechanisms in different cell types: it may activate theCPpromoter in mesophyll cells, and acts to derepress the promoter in phloem tissue. In addition, TrAP is clearly capable of inducing the expression of responsive chromosomal promoters and could, in principle, activate host genes. Distinct viral sequence elements mediate expression and derepression in phloem and activation in mesophyll, suggesting that TrAP interacts with different components of the cellular transcription machinery to accomplishCPgene expression in different cell types, and underscoring the intricacy and complexity of virus–host interactions
Analysis of the Two Subgenomic RNA Promoters for Turnip Crinkle Virusin Vivoandin Vitro

AbstractInfection of plants or protoplasts with turnip crinkle virus (TCV), a monopartite RNA virus, results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs. The transcription start site for the 1.45-kb sgRNA was previously mapped to position 2606 (J. C. Carrington, T.J. Morris, P.G. Stockley, and S.C. Harrison, (1987).J. Mol. Biol.194, 265–276) corresponding to position 2607 in the TCVms isolate and the start site for the 1.7-kb sgRNA has now been mapped to position 2333 in TCVms. A 96-base sequence (90 bases upstream and 6 bases downstream) encompassing the transcription start site for the 1.45-kb sgRNA was sufficient for full promoter activity. Similarly, a 94-base sequence (90 bases upstream and 4 bases downstream) encompassing the start site was required for full activity of the 1.7-kb sgRNA promoter. The 1.45-kb sgRNA promoter, but not the 1.7-kb sgRNA promoter, was able to direct synthesis of a nontemplate RNAin vitrousing partially purified TCV RNA-dependent RNA polymerase. Computer generated secondary structures for the two sgRNA promoters revealed an extensive hairpin just upstream from the transcription start site. Comparisons of corresponding sequences from related viruses indicates higher sequence conservation for the 1.45-kb sgRNA promoter compared with the 1.7-kb sgRNA promoter, despite the latter's location within the RNA-dependent RNA polymerase open reading frame
Neutralization Determinants of Laboratory Strains and Field Isolates of Equine Arteritis Virus: Identification of Four Neutralization Sites in the Amino-Terminal Ectodomain of the GLEnvelope Glycoprotein

AbstractThe N-terminal hydrophilic ectodomain of the GLenvelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding GLenvelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 10B4, indicating that they also recognize epitopes on the GLprotein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified-live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the GLprotein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1variable region (amino acids 61–121) within the second half of the N-terminal hydrophilic ectodomain of the GLprotein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV
Particle Bombardment-Mediated DNA Vaccination with Rotavirus VP6 Induces High Levels of Serum Rotavirus IgG but Fails to Protect Mice against Challenge

AbstractThe rotavirus inner capsid protein VP6 contains conserved epitopes that are potential targets for eliciting protective immunity against different serotypes within the same group of rotavirus. In order to determine whether VP6 alone can induce protective immunity, an expression vector pcDNA1/EDIM6 containing gene 6 of rotavirus EDIM strain was constructed and used as a vaccine in an adult mouse model. Cloned gene 6 was determined to be 1356 nucleotides long and contained a 5′ noncoding region of 23 nucleotides, a 3′ noncoding region of 139 nucleotides, and a coding frame of 1194 nucleotides for a polypeptide of 397 amino acid residues. Recombinant VP6 was expressed in rabbit reticulocyte lysate and the heat-denatured recombinant VP6 migrated in SDS-gels with an apparent molecular weight of approximately 43 kDa. Five additional polypeptide bands corresponding to oligomers of recombinant VP6 were observed when the expressed product was not heat denatured. To determine the immunogenicity of recombinant VP6, female BALB/c mice were injected intramuscularly or intradermally with pcDNA1/EDIM6, or were inoculated epidermally with plasmid-coated gold beads using the Geniva Accell particle delivery device. Only intradermal injection and particle delivery elicited measurable serum anti-rotavirus IgG responses, but responses developed following particle delivery were significantly (P< 0.001) greater. However, none of the delivery methods induced serum or stool anti-rotavirus IgA responses and, when challenged with EDIM no protection against infection was observed in the immunized mice. Therefore, parenteral immunization with VP6 alone elicited large anti-rotavirus IgG responses but did not elicit protection against murine rotavirus infection in this model
A Specific Host Cellular Protein Binding Element Near the 3′ End of Mouse Hepatitis Virus Genomic RNA

AbstractA distinct host cellular protein binding element was mapped within a 38-nucleotide (nt) sequence 166–129 nucleotides upstream of the 3′ end of the MHV-JHM genome using a RNase T1protection/gel mobility shift electrophoresis assay. The resultant RNA–protein complex contains six host cellular proteins, one protein of 120-kDa molecular mass, two poorly resolved species approximately 55 kDa in size, a second pair of poorly resolved 40-kDa proteins, and a minor component of 25 kDa. A series of RNA probes containing deletions or clustered transversion mutations were tested for their ability to form complexes with mock- and MHV-JHM-infected cytoplasmic extracts. Three mutant RNA probes (mA, mB, and mC) with deletions at 154–140, 139–129, and 128–118, respectively, expressed 4, 37, and 94% of the host protein binding activity exhibited by the wild-type RNA. Defective interfering (DI) RNAs (DImA, DImB, and DImC) containing corresponding deletions at 154–140, 139–129, 128–118, and another DI RNA (DImD) with a deletion at nucleotides (nts) 112–102, a region which did not affect RNA–protein interactions, were transfected into MHV-JHM-infected 17CL-1 cells to assay the effects of these mutations on DI RNA replication. All of these mutations had an adverse effect on DI RNA replication. However, analysis of negative strand mutant DI RNAs revealed that two mutants (DImC and DImD) carrying deletions having little or no effect on RNA–protein interaction in our RNA–protein binding assays maintained their mutant sequences. In contrast, the other two mutants (DImA and DImB) containing deletions that dramatically decreased RNA–protein binding activity did not maintain their mutations; wild-type sequences were restored in the majority of the progeny negative strand molecules. These data indicate that the 26-nucleotide sequence at positions 154–129 from the 3′ end of viral genome is important to both RNA–protein binding and viral replication. This protein binding element contains an 11-nt sequence (UGAGAGAAGUU, positions 139–129) very similar to a more 3′ sequence (UGAAUGAAGUU) previously implicated in host protein binding and viral RNA replication (Yu and Leibowitz, 1995a and 1995b)
Second-Site Reversion of a Dysfunctional Mutation in a Conserved Region of the Tobacco Mosaic Tobamovirus Movement Protein

AbstractThe N-terminal two-thirds of tobamovirus movement proteins (MPs) contain two well conserved regions. Within region I (amino acids 56–96) is an area predicted by computer analysis to have loop secondary structure (amino acids 76–87). A single or two double amino acid mutations were introduced into the loop in region I of the TMV MP to destabilize the structure. The three mutant MPs were defective in movement function. The single amino acid mutation resulted in a Pro81→Ser substitution. The mutant virus, TP81S, containing the Pro81→Ser substitution, was propagated on a transgenic line ofNicotiana tabacumthat expresses the sunn-hemp mosaic tobamovirus MP. Inoculation of virus progeny from the transgenic plants onto hypersensitiveN. tabacumindicated the presence of infectious virus at a low frequency. Necrotic lesions were detected at 4 days postinoculation, 2 days later than those induced by wild-type TMV. Inoculation of virus extracted from necrotic lesions ontoN. tabacumresulted in a delayed and attenuated systemic infection relative to that induced by TMV, indicating that a second-site mutation restored movement function rather than a reversion of the original mutation. Sequence analysis revealed that the revertant MP gene had two additional amino acid substitutions, a Thr104→Ile and a Arg167→Lys. Introduction of the amino acid substitutions individually or in combination into the MP of TP81S indicated that both substitutions were required for the revertant phenotype. The data indicate that structure within region I is important in maintaining an active conformation for functional MP, that changes outside region I can compensate for alterations within the region, and suggest that region I may interact with a distal portion of the protein
Oat Blue Dwarf Marafivirus Resembles the Tymoviruses in Sequence, Genome Organization, and Expression Strategy

AbstractThe complete nucleotide sequence and genome organization of oat blue dwarf marafivirus (OBDV) were determined. The 6509 nucleotide RNA genome encodes a putative 227-kDa polyprotein (p227) with sequence motifs similar to the methyltransferase, papain-like protease, helicase, and polymerase motifs present in the nonstructural proteins of other positive strand RNA viruses. The 3′ end of the open reading frame (ORF) that encodes p227 (ORF 227) also encodes the two capsid proteins: a 24-kDa capsid protein is presumably cleaved from the p227 polyprotein, whereas the 21-kDa capsid protein appears to be translated from a subgenomic RNA (sgRNA). Encoded amino acid and nucleotide sequence comparisons, as well as the OBDV genome expression strategy, show that OBDV closely resembles the tymoviruses. OBDV differs from the tymoviruses in its general biology, in its lack of a putative movement gene that overlaps the replication-associated genes, and in its fusion of the capsid gene sequences to the major ORF. OBDV also possesses a 3′ poly(A) tail, as compared to the tRNA-like structures found in most tymoviral genomes. Due to the strong similarities in genome sequence and expression strategy, OBDV, and presumably the other marafiviruses, should be considered a member of the tymovirus lineage of the alpha-like plant viruses
Attenuation of B5R Mutants of Rabbitpox Virusin VivoIs Related to Impaired Growth and Not an Enhanced Host Inflammatory Response

AbstractThe rabbitpox virus (RPV) B5R protein, synthesized late in infection, is found as a 45-kDa membrane-associated protein of the envelope of infectious extracellular enveloped virus (EEV) and as a 38-kDa protein secreted from the cell by a process independent of morphogenesis. The protein is not found associated with intracellular mature virus (IMV). Deletion of the gene attenuates the virus (RPVΔB5R) in animals (mice and rabbits), has relatively little effect on formation of IMV, prevents EEV formation in some but not all cells, and leads to a reduced host range. Analysis of the sequence of the protein suggests relatedness to factor H of the complement cascade. Collectively, these observations suggest that attenuation of the virusin vivocould be linked to an inhibition of the inflammatory response, a deficiency in growth, or both. In this report we have analyzed the behavior of RPVΔB5R in infected mice and rabbits and conclude that attenuation of the mutant virus likely results from simple failure to grow within the infected animal and that the inflammatory response probably contributes little to the observed attenuation
Cycloheximide Inhibition of Delayed Early Gene Expression in Baculovirus-Infected Cells

AbstractThe baculovirus protein IE1 is required for the transactivation of many early viral genes in transient expression assays. However, cycloheximide inhibition studies have failed to reveal a dependence of early gene transcription on expression of IE1 in infected cells. We show here that synthesis of IE1 was not effectively inhibited by the addition of 100 μg/ml cycloheximide, the concentration routinely used in these studies. However, when cycloheximide was added at 250 μg/ml, IE1 synthesis was repressed to less than 5% of control levels. These more stringent conditions were used to discriminate between immediate early and delayed early genes. Transcription of three immediate early genes (ie1, ie2,andie0) was increased by the addition of high concentrations of cycloheximide. However, transcription of three other early genes (39k, p35,andlef-3), which are known to be dependent on IE1 transactivation, was significantly reduced by the addition of 250 μg/ml cycloheximide. Immunoblot analyses also revealed a difference between the immediate and delayed early class of viral genes. Synthesis of IE1, IE2, and IE0 was resistant to cycloheximide treatment, while translation of SSB/LEF-3 and pp31 was strongly inhibited even at the lower concentration of cycloheximide. Although cycloheximide was shown to be useful in defining early temporal classes, it induced apoptosis in both uninfected and infected Sf9 cells when used at the inhibitory concentration
Host Site Selection for Concerted Integration by Human Immunodeficiency Virus Type-1 Virionsin Vitro

AbstractHost site selection for full-site integration by human immunodeficiency virus type-1 (HIV-1) integrase (IN) from nonionic detergent-lysed virions was investigated. Linear retrovirus-like DNA (469 bp) possessing 3′ OH recessed long terminal repeat termini was efficiently inserted by a bimolecular donor reaction into a supercoiled DNA target (2867 bp), producing the HIV-1 5-bp host site duplication. Sequence data were analyzed from 193 donor–target recombinants obtained from the linear 3.8-kb DNA product. The selection of host target sites appeared randomly distributed and was independent of lysis and assay conditions. The fidelity of the 5-bp duplications in comparison to other size duplications was highest (94%) with high-salt (300 mMNaCl) lysis of the virions and 60 mMNaCl for strand transfer using Mg2+as the divalent cation. Base sequence analysis demonstrated some biases in the 5-bp duplications at the sites of strand transfer and at the immediate host sequences surrounding the duplications. In addition to the observed duplications, ∼30% of the recombinants isolated from the linear 3.8-kb DNA product contained specific and repetitive small-size deletions. No deletions smaller that 17 bp were observed and the distance between the deletion sets had a periodicity of ∼10 bp. The mechanisms involved in how HIV-1 IN produces the 5-bp duplications and the repetitive host site deletions are discussed
Challenge of Chimpanzees Immunized with a Recombinant Canarypox-HIV-1 Virus

AbstractTo evaluate the potential protective efficacy of a live recombinant human immunodeficiency virus type 1 (HIV-1) canarypox vaccine candidate, two chimpanzees were immunized five times with ALVAC-HIV-1 vCP250, a recombinant canarypox virus that expresses the HIV-1IIIB(LAI)gp120/TM,gag,and protease gene products. One month after the last booster inoculation, the animals were challenged by intravenous injection of cell-associated virus in the form of peripheral blood mononuclear cells from an HIV-1IIIB(LAI)-infected chimpanzee. One chimpanzee with a neutralizing antibody titer to HIV-1IIIB(LAI)of 128 at the time of challenge was protected, whereas both the second animal, with a neutralizing antibody titer of 32, and a naive control animal became infected. At 5 months after challenge, the protected chimpanzee and a third animal, previously immunized with various HIV-1MNantigens, were given a booster inoculation. The two animals were challenged intravenously 5 weeks later with twenty 50% tissue culture infectious doses of cell-free HIV-1DH12, a heterologous subtype B isolate. Neither chimpanzee had neutralizing antibodies to HIV-1DH12, and neither one was protected from infection with this isolate. The immune responses elicited by vaccination against HIV-1IIIB(LAI)or HIV-1MNdid not, therefore, protect the animals from challenge with the heterologous cell-free HIV-1DH12
Synthesis and Processing of the Equine Herpesvirus 1 Glycoprotein M

AbstractIn a previous report, the function of the equine herpesvirus 1 (EHV-1) glycoprotein M (gM) homolog was investigated. It was shown that EHV-1 gM is involved in both virus entry and direct cell-to-cell spread of infection (N. Osterriederet al., J. Virol.70, 4110–4115, 1996). In this study, experiments were conducted to analyze the synthesis, posttranslational processing, and the putative ion channel function of EHV-1 gM. It was demonstrated that EHV-1 gM is synthesized as anMr44,000 polypeptide, which is cotranslationally N-glycosylated to anMr46,000–48,000 glycoprotein. TheMr46,000–48,000 gM moiety is processed to anMr50,000–55,000 glycoprotein, which is resistant to treatment with endoglycosidase H, indicating that processing occurs in the Golgi network. EHV-1 gM forms a dimer in infected cells and the virion, as was demonstrated by the presence of anMr105,000–110,000 gM-containing band in electrophoretically separated lysates of infected cells and purified extracellular virions. TheMr105,000–110,000 protein band containing gM was also observed in lysates of cells that had been transfected with EHV-1 gM DNA. The translation of EHV-1 gM is initiated at the firstin-framemethionine of the gM open reading frame as shown by transient transfection experiments of full-length gM and a truncated gM lacking the aminoterminal 83 amino acids. Functional expression of EHV-1 gM inXenopus laevisoocytes together with voltage-clamp analyses demonstrated that gM per se does not exhibit ion channel activity as had been speculated from the predicted structure of the polypeptide
Bovine Herpesvirus 1-Induced Apoptosis Occurs at the G0/G1 Phase of the Cell Cycle

AbstractWe have previously shown that bovine herpesvirus 1 (BHV-1), even when inactivated, induces apoptotic cell death in mitogen-stimulated bovine peripheral blood mononuclear cells (PBMCs) (Hanonet al.,1996,J. Virol.70, 4116–4120). In order to gain insight into this process, we have investigated the cell cycle phase at which BHV-1 induces apoptosis in PBMCs. Our results show that the percentage of cells that progress through the S phase was always lower in BHV-1-infected PBMCs than in control cells. This effect was not due to a defective activation of mitogen-stimulated PBMCs since BHV-1 only slightly affected the percentage of cells expressing BoCD25, a well-known lymphocyte activation marker. Furthermore, mimosine and cyclosporine A, two chemicals that inhibit entry into the S phase of the cell cycle by different pathways, did not affect the ability of BHV-1 to induce apoptosis. BHV-1-induced apoptosis also occurred in unstimulated PBMCs and interestingly, this was associated with the expression of c-mycand BoCD25 proteins both of which are related to cell cycle progression. All together, these data provide evidence demonstrating that BHV-1-induced apoptosis occurs at the G0/G1 phase of the cell cycle
Phosphorylation of the Adeno-Associated Virus Replication Proteins

AbstractThe adeno-associated virus (AAV) replication proteins Rep78 and Rep68 regulate viral gene expression and DNA amplification. Their effects on both processes suggest that they play roles in all phases of the virus life cycle. We have investigated Rep protein phosphorylation to determine if this modification might alter Rep function. All four Rep proteins were found to be phosphorylated in AAV and adenovirus co-infected cell cultures, and Rep proteins contained phospho-serine whereas no phospho-threonine or -tyrosine was detected. We also observed that when viral DNA synthesis was inhibited, there was a significant decrease in the level of Rep78/68 phosphorylation. Our results suggest a plausible mechanism whereby AAV Rep78/68 function may be regulated by phosphorylation