pinecone/core-2020-05-10-deduplication huggingface dataset:
Expression of the Barley Stripe Mosaic Virus RNAβ “Triple Gene Block”

AbstractGenomic RNAβ of barley stripe mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (βa) and a “triple gene block” consisting of the βb, βc, and βd ORFs that overlap one another. Two subgenomic β RNAs (sgRNAβ1 and sgRNAβ2) with sizes of 2.5 and 0.96 kb were identified in BSMV-infected protoplasts, and their transcription initiation sites were mapped to nucleotides 789 and 2327, respectively, of RNAβ by primer extension experiments. In a cell-free wheat germ translation system, genomic RNAβ served as a mRNA only for the 22-kDa coat protein, and sgRNAβ1 directed synthesis of only the 58-kDa βb protein. However, with sgRNAβ2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14- and the 23-kDa proteins were recognized by the βd antibodiesin vitroandin vivo.These results demonstrated that the 14-kDa protein was encoded by the βd ORF and suggested that the 23-kDa protein, designated βd′, is a readthrough product of the amber stop codon of the βd ORF. Mutagenesis of sgRNAβ2 revealed that the 17-kDa protein was a product of the βc ORF. Expression of sgRNAβ1 and sgRNAβ2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs α and γ plus chimeric RNAβ derivatives containing the CAT gene in-frame with the βb, βc, βd, or βd′ ORFs. Elimination of the sgRNAβ1 promoter abolished CAT expression from the βb-CAT chimeric RNA, and removal of the sgRNAβ2 promoter prevented CAT expression from the βc-CAT, βd-CAT, and βd′-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNAβ, whereas sgRNAβ1 serves as a messenger for translation of the βb protein, and sgRNAβ2 functions as a messenger for translation of βc and βd and the newly discovered βd′ protein. Additional mutagenesis experiments indicate that βc is translated by a leaky scanning mechanism
Actin Binding and Proteolysis by the Baculovirus AcMNPV: The Role of Virion-Associated V-CATH

AbstractInfection of larvae byAutographa californicaM nuclear polyhedrosis virus (AcMNPV) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. InSpodoptera frugiperdatissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. The first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. Cable formation requires release of the budded virus (BV) nucleocapsid from the endosome, but does not require new protein synthesis, suggesting that the nucleocapsid contains the activity necessary to induce cable formation. We have identified two distinct BV-associated actin-targeting activities. The first, a nucleocapsid-associated actin-binding activity, enabled actin copelleting and may also induce actin polymerization and cable formation. The second activity, associated with the nucleocapsid and envelope fractions of BV, was a protease that specifically degraded actin. This protease was identified as V-CATH, a cathepsinL-like protease that is a product of the AcMNPVv-cathgene
Immunity Specificity Determinants in the P4-like Retronphage φR73

AbstractRetronphage φR73 exhibits extensive sequence homology to the satellite bacteriophage P4. Bacteriophage P4 superinfection immunity is elicited by a small RNA (CI RNA) that causes premature transcription termination within the operon coding for the P4 replication functions. This control is exerted via interaction of the CI RNA with two complementary target sites on the untranslated leader RNA of the replication operon. We found that φR73 is endowed with a similar immunity system but is heteroimmune to P4. The heteroimmunity is due to six base differences in the CI RNA and to compensatory base substitutions in the target sequences. The sequence differences in the CI RNA are all located in single-stranded regions, which appear to play a predominant role in the interaction with the target sites. Analysis of phage carrying a hybrid immunity system indicates that, although two target sequences are required for the establishment of lysogeny, a single site is sufficient to make a phage sensitive to the prophage immunity
Characterization of Puumala Virus Nucleocapsid Protein: Identification of B-Cell Epitopes and Domains Involved in Protective Immunity

AbstractB-cell epitopes in the nucleocapsid protein (N) of Puumala (PUU) virus were investigated by use of truncated recombinant proteins and overlapping peptides. Six of seven epitopes, recognized by bank vole monoclonal antibodies, were localized within the amino-terminal region of the protein (aa 1–79). Polyclonal antibodies from wild-trapped or experimentally infected bank voles identified epitopes located over the entire protein. Antibody end-point titers to different N fragments indicated that the amino-terminal region is the major antigenic target in PUU virus-infected bank voles. To investigate the role of PUU virus N in protective immunity, we analyzed the immunogenicity of truncated recombinant N and developed an animal model based on colonized bank voles. No PUU virus N antigen, nor any glycoprotein-specific antibodies, could be detected after virus challenge in animals immunized with an amino-terminal fragment (aa 1–118), a fragment covering two thirds of the protein (aa 1–267), or total N, indicating that a complete protection was evoked by the recombinant proteins. Two of eight animals immunized with shorter N fragments displayed either N antigen, or glycoprotein-specific antibodies, suggestive of partial protection. Prechallenge sera from all groups of immunized animals were found negative or only weakly positive for neutralizing antibodies when assayed by focus reduction neutralization test, which indicated an important role for cell-mediated immunity in protection
Mapping of the Red Clover Necrotic Mosaic Virus Subgenomic RNA

AbstractThe red clover necrotic mosaic dianthovirus capsid protein is expressedin vivofrom a subgenomic RNA (sgRNA) identical to the 3′-terminal 1.5 kb of RNA-1. The 5′ leader sequence of the capsid protein sgRNA is 62 nucleotides, contains a 14-nucleotide putative promoter sequence homologous to the RNA-1 5′ terminus, and exhibits a high level of similarity with the tobacco mosaic virus 5′ leader translational enhancer element Ω. Analysis of the RNA-1 secondary structure, in the region adjacent to the position where the 5′ end of the sgRNA was mapped, predicts a stable stem–loop which includes the putative sgRNA promoter element. It is suggested that this structure is important for recognition of the sgRNA transcriptional initiation from the full-length negative-sense RNA-1
Inhibition of HIV-1 Replication in Viral Mutants with Altered TAR RNA Stem Structures

AbstractHuman immunodeficiency virus type 1 (HIV-1) Tat-mediatedtrans-activation requires the structural integrity of TAR RNA and the cooperative interaction of human host cell proteins. The TAR domain, minimally required fortatresponse, includes the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop sequences. However, little is known about the significance of the 5′-stem structure of TAR in the regulation of viral growth. We designed viral mutations, specifically in the TAR RNA lower stem structure, and studied their effects on the kinetics of viral growth in T-lymphocyte cell lines and in activated human peripheral blood mononuclear cells. Mutations that destabilized the lower TAR stem structure inhibited viral growth to various degrees in different[formula]T-cells. These results suggest that the structural integrity of the lower stem structure of TAR plays an important role in viral growth, presumably by binding to specific host cell proteins that stabilize Tat–TAR interactions
Mutational Analysis of Interactions between the Gag Precursor Proteins of Murine Leukemia Viruses

AbstractThe yeast two-hybrid system was used to test for interactions among the Gag precursor proteins of three members of the murine leukemia virus family. These Gag proteins all interact with each other in all combinations, but do not interact with the distantly related HIV-1 Gag. A series of deletion mutants of Moloney MuLV were examined to determine the minimal interaction domain. Either one of two regions of Gag was independently sufficient to mediate homodimerization, suggesting multiple points of contact in the precursor. Analysis of a set of point mutations in the CA region revealed a complex pattern of effects on Gag–Gag interactions
Hepatitis B Virus X Protein Partially Substitutes for E1A Transcriptional Function during Adenovirus Infection

AbstractLack of anin vitroculture system for human hepatitis B virus has hampered the ability to address fundamental questions regarding the viral life cycle and the effect of viral gene products during productive infection. To study the activity of HBV X protein (HBx) in the context of a viral infectious cycle, we provided HBxin transduring adenovirus infection of liver-derived cells. In hepatoma cells infected with adenovirus mutants deficient in expression of various E1A products, HBx was able to partially substitute for the transcriptional activation function of E1A. HBx also activated adenovirus replication, but to a lesser extent than the activation of transcription. Adenovirus genes transcribed by either RNA polymerase II or RNA polymerase III were activated by HBx during infection. These results suggest that HBx and E1A activate transcription by a similar mechanism and that this viral infection system will be useful for characterization of the functional activities of HBx
Cloning, Sequence Analysis, and Expression of the Major Capsid Protein of the Iridovirus Frog Virus 3

AbstractThe nucleotide sequence of the gene encoding the major capsid protein (MCP) of frog virus 3 (FV3) has been determined and compared to other iridovirus capsid genes. Nucleotide sequence and S1 nuclease analysis showed that the FV3 MCP gene encoded a transcript of 1452 nucleotides containing a 12 nucleotide AU-rich 5′ nontranslated region (NTR) and a 50-nucleotide 3′ NTR whose terminus was predicted to fold into a hairpin of moderate stability. An open reading frame initiating from the 5′-most AUG codon encoded a protein of 463 amino acids with a predicted molecular weight of 49,860. Expression of the putative FV3 MCP genein vitroconfirmed that it encoded the major capsid protein of FV3 and supported the suggestion that translation initiated at AUG-1. Pairwise amino acid alignments detected a high degree of sequence identity between the FV3 MCP and other iridoviruses. These results indicate that iridoviruses possess an evolutionarily related major capsid protein and provide information useful not only in studies of viral gene expression, but also in characterizing newly isolated iridoviruses
Increased Transcomplementation Properties of Plasmids Carrying HSV-1 Origin of Replication and Packaging Signals

AbstractThis report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use of complementing plasmids containing a virus origin of replication (oriS) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 CgalΔ42 virus, a UL42 null mutant, by amplicon plasmids carrying either wild-type (pA-UL42) or mutated alleles (pA-ΔUL42) of the HSV-1 UL42 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective CgalΔ42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-ΔUL42 amplicon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess functional activity of an HSV-1 essential gene in a one-step experiment and (ii) oriS-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors
Simian Immunodeficiency Virus (SIV) gp130 Oligomers Protect Rhesus Macaques (Macaca mulatta) against the Infection with SIVmac32H Grown on T-Cells or Derivedex Vivo

AbstractThe efficacy of three SIVmac32H gp130 vaccines was compared in rhesus monkeys. Three rhesus monkeys were each immunized over a period of 20 weeks with a total of 600 μg virion-derived gp130 oligomers (O-gp130) mixed with keyhole limpet hemocyanin and emulsified with incomplete Freund's adjuvant. Three other monkeys were infected with 5 × 108PFU of vaccinia virus wild type (VV-wt) while three additional animals received an equivalent dose of VV expressing the gp130 of SIVmac (VV-gp130). At Week 8, the two VV-wt animals received an additional immunization with 100 μg O-gp130 each. All VV-infected animals then received booster immunizations at Weeks 12, 16, and 20 with a total of 300 μg O-gp130 per animal. All animals along with two controls were challenged iv with 50 MID50of T-cell-grown SIVmac32H at Week 22. Four weeks after the challenge and thereafter, both controls and one animal from either VV group were infected as demonstrated by polymerase chain reaction (PCR), virus isolation, and antibody response. In contrast, all O-gp130 animals and one animal each from the VV-wt and the VV-gp130 group were completely protected as shown by negative PCR and virus reisolation. One animal of the VV-gp130 group was partially protected, since it remained virus isolation negative but became PCR positive. All protected animals did not develop a secondary antibody response. Six months after the first challenge, the five completely protected animals were reimmunized twice 4 weeks apart with a total of 200 μg O-gp130 per animal. Two weeks later, all animals were challenged with 5 MID50of the SIVmac32H/spI prepared from the spleen of an immunized, but unprotected SIV-infected rhesus monkey. After the second challenge, all three control animals and one of the vaccinees became productively infected. In contrast, two animals were completely protected, one from the former O-gp130 and one from the former VV-gp130 group. One animal from the former VV-wt group was only DNA–PCR positive and thus partially protected. Therefore, immunization with virion-derived gp130 oligomers of SIVmac32H can confer protection against the infection with T-cell-grown SIVmac32H as well as theex vivoisolate SIVmac32H/spI
A Second Double-Stranded RNA Virus from Yeast

AbstractTwo double-stranded RNA viruses exist as permanent persistent infections of the yeastSaccharomyces cerevisiae:ScVL1 and ScVLa. Both belong to the Totiviridae, which include a number of fungal and protozoan double-stranded RNA viruses. Although ScVL1 and ScVLa share the same genomic organization and mode of expression and coexist in the same cells, they show no evidence of recombination: with one limited exception, sequence conservation is detectable only in regions conserved in all totiviruses. Both have two open reading frames on their single essential RNAs:cap(encoding a capsid polypeptide) andpol(encoding an RNA-dependent RNA polymerase). The ScVLa virus, like ScVL1, appears to express its Pol domain by a −1 translational frameshift
Construction and Analysis of a Bacteriophage φ6 Gene 14 Nonsense Mutant

AbstractThe production of a small low-abundance protein encoded by the large dsRNA genome segment of bacteriophage φ6 in extracts of phage-infectedPseudomonas phaseolicolahas been reported previously. Construction and analysis of a phage containing a nonsense mutation in this gene, designated gene 14, indicates that its product is not essential for growth in the laboratory. However, since early protein synthesis is delayed, burst sizes are reduced, and plaques are smaller than wild type, P14 is needed for optimal phage development
Synthesis and Analysis of a 640-bppolRegion of Novel Human Endogenous Retroviral Sequences and Their Evolutionary Relationships

AbstractNucleotide sequence comparisons of thepolgene among 47 retroelements identified two very conserved regions, separated by a span of approximately 640 bp, that have not been previously reported. A set of mixed oligonucleotide primers, 5′-MOP-2 and 3′-MOP-2, homologous to these two conservedpolregions was constructed for use in detection of retroelements. When MOPs-2 were employed in PCR amplification studies, products of about 0.64 kb in size were amplified from human and mouse genomic DNAs and from HIV-1 proviral DNA, but not from negative control plasmid DNAs. The PCR products amplified with MOPs-2 from human LuC-1 teratocarcinoma cell DNA were subcloned and sequenced. Five clones of approximately 0.64 kb in size were identified, and sequence comparisons with all entries in GenBank indicated that these five clones have highest homology, in a range of 64.31 to 98.65%, with the correspondingpolregion of HERV-K10 and HM-16 of the human endogenous retrovirus-K (HERV-K) family. Southern hybridizations at high stringency demonstrated that these five clones are present in all human DNAs tested. The evolutionary relationships of these clones with the equivalentpolregion of other retroelements were defined by phylogenetic analyses that placed three clones into the HERV-K family and two clones into a new family of human endogenous retroelements. In addition, clone HERV-(K)73 contains the smaller PV1bpolsegment that was reported to be selectively expressed in blood leukocytes of patients with polycythemia vera
Recombinant Adenovirus Deleted of All Viral Genes for Gene Therapy of Cystic Fibrosis

AbstractRecombinant adenoviruses are being developed for gene therapy of inherited disorders such as cystic fibrosis because they efficiently transduce recombinant genes into nondividing cellsin vivo.First generation recombinant adenoviruses, rendered defective by deletion of sequences spanning E1a and E1b, express low levels of early and late viral genes that activate destructive cellular immune responses. Current strategies for improving recombinant adenoviruses attempt to inactivate other essential genes through deletion and growth in new packaging cell lines or incorporation of temperature sensitive mutations which allow propagation of the virus in available packaging cell lines at permissive temperatures. We describe in this report a new type of recombinant adenovirus that is deleted of all viral open reading frames. This recombinant (called ΔrAd), which contains only the essentialciselements (i.e., ITRs and contiguous packaging sequence), is propagated in 293 cells in the presence of E1-deleted helper virus. Concatamers of the monomeric vector genome were passaged and capable of transduction. The ΔrAd genome is packaged into virions that sediment at a lower density than the helper virus in cesium gradients forming the basis for a purification protocol. A fully deleted recombinant adenovirus that expresses human cystic fibrosis transmembrane conductance regulator was produced and used to transduce human airway epithelial cells derived from a cystic fibrosis patient. Packaging and propagation of a fully deleted adenovirus is an important step toward the development of a safer vector. Improved production and purification strategies need to be developed before this new vector system can be evaluatedin vivo
E1A Second Exon Requirements for Induction of Target Cell Susceptibility to Lysis by Natural Killer Cells: Implications for the Mechanism of Action

AbstractThe E1A oncogene of adenovirus type 5 induces susceptibility of mammalian cells to lysis by natural killer lymphocytes (NK cells). It is unknown whether sensitization to NK killing is mediated directly by targeting effects of interactions between E1A peptides and cell surface MHC molecules or indirectly by an E1A activity that requires structural integrity of the oncoprotein. To discriminate between these hypotheses, rat and hamster cells expressing wild type E1A were contrasted with those expressing truncated products resulting from E1A termination or deletion mutations. Transfected rat cells, expressing truncated proteins from the E1A first exon that encodes MHC-binding peptides, remained resistant to lysis by NK cells, whereas cells expressing full-length E1A protein were highly susceptible to lysis. Studies of infected hamster cells showed that addition of either of two, nonoverlapping, second exon regions reconstituted cytolytic susceptibility induction by E1A. The results do not support the E1A-peptide-MHC hypothesis, since no single E1A peptide coding region was sufficient to convey cytolytic susceptibility to expresser cells. The data indicate that coordinate functions of the E1A first exon and redundant accessory regions in the second exon are required for E1A-induced susceptibility to NK killing
Genomic Cloning and Characterization of the Nonoccupied Allele Corresponding to the Integration Site of Human Papillomavirus Type 16 DNA in the Cervical Cancer Cell Line SiHa

AbstractHuman papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR