pinecone/core-2020-05-10-deduplication huggingface dataset:
Complete Genomic Sequence of the Lytic Bacteriophage DT1 ofStreptococcus thermophilus

AbstractStreptococcus thermophiluslytic bacteriophage DT1, isolated from a mozzarella whey, was characterized at the microbiological and molecular levels. Phage DT1 had an isometric head of 60 nm and a noncontractile tail of 260 × 8 nm, two major structural proteins of 26 and 32 kDa, and a linear double-stranded DNA genome with cohesive ends at its extremities. The host range of phage DT1 was limited to 5 of the 21S. thermophilusstrains tested. UsingS. thermophilusSMQ-301 as a host, phage DT1 had a burst size of 276 ± 36 and a latent period of 25 min. The genome of phage DT1 contained 34,820 bp with a GC content of 39.1%. Forty-six open reading frames (ORFs) of more than 40 codons were found and putative functions were assigned to 20 ORFs, mostly in the late region of phage DT1. Comparative genomic analysis of DT1 with the completely sequencedS. thermophilustemperate phage O1205 revealed two large homologous regions interspersed by two heterologous segments. The homologous regions consisted of the early replication genes, the late morphogenesis genes, and the lysis cassette. The divergent segments contained the DNA packaging machinery, the major structural proteins, and remnants of a lysogeny module
Interaction of the Capsid Protein p24 (HIV-1) with Sequence-Derived Peptides: Influence on p24 Dimerization

AbstractProtein–protein interactions of the p24 (HIV-1) capsid protein play an essential role in the production of infectious virus particles. To map the putative p24 dimerization site, a set of overlapping peptides spanning the p24 sequence was prepared using spot synthesis on a cellulose membrane and probed with recombinant p24 (rp24). Three sequence regions interacting with rp24 were identified. Peptides from each region were synthesized, but only one peptide was effectively able to inhibit rp24 dimerization in solution. Amino acids that were exposed in the corresponding p24 region were mutated in rp24, resulting in a significant decrease of rp24 dimerization. Thus, participation of this region in virus capsid assembly can be assumed
Satellite RNA Is Essential for Encapsidation of Groundnut Rosette Umbravirus RNA by Groundnut Rosette Assistor Luteovirus Coat Protein

AbstractGroundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenicNicotiana benthamianaplants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed
Movement of Hordeivirus Hybrids with Exchanges in the Triple Gene Block

AbstractThe barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infectedNicotiana benthamiana,a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (βb gene) indicated that (i) sequence-independent binding of βb to viral RNAs could occur during formation of βb-RNA complexesin vivo,and that (ii) the βb MP is involved in virus long-distance movement, for which homologous N- and C-terminal βb domains are required
Characterization of the Surface Proteins of Influenza A (H5N1) Viruses Isolated from Humans in 1997–1998

AbstractInfluenza A (H5N1) viruses infected humans in Hong Kong between May and December, 1997. Sixteen viruses, including 6 from fatal cases, were isolated during this outbreak. Molecular analysis of the surface proteins genes encoding the hemagglutinin (HA) and neuraminidase (NA) of these H5N1 isolates, of a subtype not previously known to infect humans, are presented. The 16 human H5 HA sequences contain multiple basic amino acids adjacent to the cleavage site, a motif associated with highly pathogenic avian influenza A viruses. The phylogenetic relationship among both avian and human H5 hemagglutinins indicates that the human isolates are related directly to isolates that circulated among chickens in the live poultry markets in Hong Kong prior to and during the outbreak in humans. HA sequences from the human isolates and a recent chicken isolate represent a separate clade, within which there are two subgroups that are distinguishable antigenically and by the presence of a potential glycosylation site. Likewise the N1 neuraminidases of the human H5 isolates represent a clade that is evolutionarily distinct from previously characterized N1 neuraminidases. The recent human H5N1 virus NA genes are avian-like, indicating direct introduction from an avian source rather than evolution of a human N1 NA. All of the 16 human NA genes encode a shortened stalk due to a 19-amino acid deletion, also found in the recent avian H5N1 isolates from Hong Kong. Two unique amino acids were identified in the N1 NAs of the recent human isolates; however, it is not known if these residues influence host range. Neither the HA nor the NA genes of the human H5N1 virus isolates show evidence of adaptive changes during the outbreak. Although analyses of the surface protein genes of the H5N1 viruses from this outbreak did not provide immediate answers regarding the molecular basis for virulence, the analyses provided clues to potentially important areas of the genes worth further investigation
Host Range of Human T-Cell Leukemia Virus Type I Analyzed by a Cell Fusion-Dependent Reporter Gene Activation Assay

AbstractWe constructed a sensitive and quantitative assay system to examine human T-cell leukemia virus type I (HTLV-I) envelope (env) glycoprotein-mediated cell fusion in which T7 RNA polymerase in donor cells coexpressing env glycoproteins activates a reporter gene in recipient cells upon cell fusion. An efficient expression of HTLV-I env glycoproteins (gp46 and gp21) was observed in 293T cells transfected with an expression plasmid by both immunoblot and immunofluorescence analyses. The cells expressing env glycoproteins also exhibited self-fusion. By cocultivating the donor cells with recipient cells transfected with a reporter plasmid possessing the luciferase gene under the T7 promoter, the expression of luciferase was observed upon cell fusion. The activation of the luciferase gene was inhibited by either anti-env neutralizing antibody or synthetic peptide corresponding to env gp21, thus indicating the cell fusion to be specifically mediated by the HTLV-I env glycoproteins expressed in the donor cells. A broad range of cell lines exhibited susceptibility to HTLV-I env-mediated cell fusion by this assay. This newly established assay system may thus provide an efficient way both to study the fusion mechanisms mediated by HTLV-I env glycoproteins and to identify the HTLV-I receptor(s)
Paramyxovirus Fusion Protein: Characterization of the Core Trimer, a Rod-Shaped Complex with Helices in Anti-Parallel Orientation

AbstractThe fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, α-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998).Virology 248,20–34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an α helix. Mutations at heptad repeataanddresidues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein
A Short Region in the Genome of Hepatitis B Virus Is Critical for Maintenance of High Transcript Levels

AbstractThe majority of hepatitis B virus (HBV) transcripts are not normally spliced during the viral life cycle, but several splice donor and acceptor sites are conserved on HBV transcripts. In particular, the genome region between nt 450 and 500 of the HBV genome appears to be rich in such sequences. In this study we deleted a short 30-nt sequence between a conserved splice donor site at HBV genome position 462 and a splice acceptor site at position 491, thus deleting the surface/polymerase open reading frames by 10 amino acid residues. At the transcriptional level, this deletion led to >99% reduction of the 2.1-kb class of subgenomic transcripts in transfected cells. Nuclear run-on experiments revealed that the transcription rate of the deleted 2.1-kb transcript is unchanged when compared with the wildtype, suggesting a posttranscriptional mechanism for the downregulation of the deleted transcript. In addition, experiments with a replication-competent HBV mutant containing the 30-nt deletion showed that the corresponding 10-amino-acid sequence within the reverse transcriptase domain of the polymerase protein appeared to be nonessential
Treatment of Influenza Virus-Infected SCID Mice with Nonneutralizing Antibodies Specific for the Transmembrane Proteins Matrix 2 and Neuraminidase Reduces the Pulmonary Virus Titer but Fails to Clear the Infection

AbstractAntibodies (Abs) can contribute to the cure of a viral infection, in principle, in two ways by: (1) binding to infected cells and thereby reducing the production of progeny virus [here termed cell-targeting (CT) activity] and (2) reacting with released progeny virus and thereby inhibiting the spread of the infection [termed virus neutralizing (VN) activity]. We have previously shown that a pulmonary influenza virus infection in severe combined immunodeficient mice could be cured by treatment of these mice with hemagglutinin (HA)-specific monoclonal Abs (mAbs) that mediated both of the above activities. Although the therapeutic activity of these mAbs correlated with their VN activity, it remained unclear how much their CT activity contributed to the Ab-mediated recovery process. To clarify this point, we tested the therapeutic efficacy of two mAbs of IgG2a isotype that mediated CT but no VN activity: one specific for the viral neuraminidase and the other for matrix protein 2. Both mAbs reduced pulmonary virus titers by 100- to 1000-fold but they failed to clear the infection, even when administered in combination and at therapeutically saturating concentrations. The results suggest that CT activity contributes significantly also to the therapeutic activity of HA-specific mAbs and further support the notion that VN-activity is required for Ab-mediated virus clearance
Recombinant Vesicular Stomatitis Virus Expressing Respiratory Syncytial Virus (RSV) Glycoproteins: RSV Fusion Protein Can Mediate Infection and Cell Fusion

AbstractThe genes encoding the respiratory syncytial virus (RSV) attachment (G) and fusion (F) envelope glycoproteins were expressed separately as additional genes in recombinant vesicular stomatitis viruses (VSV). Cells infected with the VSV-RSV F recombinant formed large syncytia illustrating the fusion activity of F in absence of other RSV proteins. Both F and G glycoproteins were expressed at the cell surface and incorporated into virions. Incorporation of these proteins did not require cytoplasmic tail sequences of VSV G. Using a compound, ammonium chloride, that raises the endosomal pH, we showed that presence of the RSV F glycoprotein in the envelope of recombinant VSV allowed for infectivity through a low-pH-independent pathway. Recombinant VSV expressing RSV glycoproteins could be useful as an RSV vaccine
Mechanism of Interferon Action: Functional Characterization of Positive and Negative Regulatory Domains That Modulate Transcriptional Activation of the Human RNA-Dependent Protein KinasePkrPromoter

AbstractThe PKR protein kinase is an important regulator of viral mRNA translation. A ∼50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). ThePkrpromoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bpIFN-stimulatedresponseelement (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5′ flanking region of the humanPkrgene to define how the novel KCS element acts to affect basal as well as IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) revealed that nuclear proteins bound selectively to the KCS element in a manner that was not dependent upon either IFN treatment or protein binding at the adjacent ISRE element. KCS protein binding activityin vitrocorrelated with activation of transcriptionin vivoin transient transfection assays. Competitionsupershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Sp1. In addition to the positive regulatory domain containing the KCSISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned ∼400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affectedPkrtranscription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within thePkrpromoter that function through the KCS element to affect basalIFN-inducible transcription ofPkr
Minimal Sequence and Structural Requirements of a Subgenomic RNA Promoter for Turnip Crinkle Virus

AbstractInfection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997).Virology232, 174–186]. Secondary structure-sensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure conforms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggest that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerasein vivo
Internal Processing of Hepatitis C Virus NS3 Protein

AbstractHepatitis C virus (HCV) NS3 protein contains at least three enzymatic activities: NS2-3 protease, NS3 serine protease, and NTPase/RNA helicase. It has been shown that NS2/3 cleavage is mediated by NS2-3 protease, whereas NS3 serine protease is responsible for the other four cleavage sites of the nonstructural (NS) region. In this study, we showed that the internal cleavage of NS3 protein produced two products of 49 kDa (NS3a) and 23 kDa (NS3b) when the entire NS3 region (aa 1027–1657) or the whole open reading frame (aa 1–3010) was expressed in mammalian and insect cells. By means of site-directed mutagenesis, we demonstrated that NS3a/NS3b cleavage occurs within the RNA helicase sequence motif that is highly conserved in theFlaviviridaefamily and that neither NS2-3 protease nor NS3 serine protease was responsible for this cleavage. The NS3 protease of flaviviruses, dengue virus type 2, for example, has been shown to mediate the internal cleavage of NS3. The NS3 proteins of HCV and dengue virus may thus be cleaved internally at the same sequence by different mechanisms of proteolysis. Also discussed is a possible role for the internal processing of HCV NS3 in the viral life cycle and its pathogenesis
An Internal Polypyrimidine-Tract-Binding Protein-Binding Site in the Hepatitis C Virus RNA Attenuates Translation, Which Is Relieved by the 3′-Untranslated Sequence

AbstractHepatitis C virus (HCV) RNA binds to several cellular proteins, which may regulate translation or replication of viral RNA. One of these is polypyrimidine tract-binding protein (PTB), which binds to the 5′-untranslated region (UTR) and the 3′-end 98 nucleotides (nt) (X region) of HCV RNA. Both of these PTB-binding sites regulate HCV translation. In this study, we further investigated the nature of PTB binding on HCV RNA. UV cross-linking studies using HeLa cell extracts and a recombinant PTB showed that the PTB–5′-UTR binding was much weaker than the PTB–3′-UTR binding. Unexpectedly, we found an even stronger PTB-binding site in the core-protein-coding region of HCV RNA. The binding domain was mapped to the 3′-end of this region, which contains a pyrimidine-rich sequence highly conserved among HCV isolates. Using a set of synthetic HCV RNAs with or without this sequence inin vitrotranslation studies, we showed that the PTB-binding sequence in the core-coding region strongly inhibited translation of HCV RNA. This inhibition was relieved by the presence of the X region at the 3′-end. Furthermore, the previously reported translational enhancement by the HCV 3′-UTR was more pronounced when this PTB-binding site was present in the RNA. These results suggest that PTB binding to an internal site of HCV RNA provides another mechanism for regulation of HCV translation
Complementarity between 3′ Terminal Nucleotides of tRNA and Primer Binding Site Is a Major Determinant for Selection of the tRNA Primer Used for Initiation of HIV-1 Reverse Transcription

AbstractThe initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIleor tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3′ terminal 18 nucleotides of the cognate tRNA. However, uponin vitroculture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIleor tRNAHis. The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place ofenv.Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIleor tRNAHisis three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe. The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS
Analysis of the Site Occupancy Constraints of Primary Amino Acid Sequences in the Motif Directing Palmitylation of the Vaccinia Virus 37-kDa Envelope Protein

AbstractVaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A(Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1–12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines;A,aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)Amotif is required for efficient palmitylation of p37