pinecone/core-2020-05-10-deduplication huggingface dataset:
Delineation of Regions Important for Heteromeric Association of Hepatitis C Virus E1 and E2

AbstractHepatitis C virus (HCV) is the major causative agent of blood-borne non-A non-B hepatitis. The persistence of HCV infection is believed to reflect escape from the host immunosurveillance system by mutations in hypervariable region 1 (HVR1) of the envelope protein 2 (E2). Two envelope proteins of HCV, E1 and E2, have been reported to form a heteromeric complex but the exact organization of the viral envelope proteins remains uncertain. We examined the interaction of E1 and E2 by far-Western blotting using the bacterial recombinant proteins and also by pull-down assay using mammalian expressed proteins. The major E1-interacting site of E2 was mapped within the N-terminal part of E2 (NCD1) (aa 415 to 500 of the polyprotein). Both HVR1 and HVR2, located at the N-terminal part of E2, were dispensable for the interaction. Although several discontinuous regions within NCD1 seemed to contribute to the strong binding to E1, the highly conserved amino acid sequences flanking HVR2 had the most significant effect. The amino acid residues “WHY” from 489 to 491 of E2 played an especially crucial role since the constructs with the internal deletion or substitution of the residues showed severely impaired E1-binding. The N-terminal part of E1 is important for the E2-binding as determined by far-Western blotting using the mammalian- and bacterial-expressed E2 proteins as probes. The mammalian-expressed, glycosylated forms of the E1 and E2 proteins exhibited E1–E2 binding activities similar to those of the bacterial-expressed, nonglycosylated forms in pull-down assays, suggesting that glycosylation is not prerequisite for the heteromeric complex formation of E1 and E2
Identification of Regions in HIV-1 Nef Required for Efficient Downregulation of Cell Surface CD4

AbstractDownregulation of cell surface CD4 is a characteristic property of all lentiviral Nef proteins. We have used mutational analysis to define regions within HIV-1 Nef that are critical for this biological activity. Two discontinuous regions in Nef, extending approximately from residues 96 to 144 and from residues 175 to 186, are reported to be essential for efficient CD4 downregulation. Interestingly, these sequences coincide with two conserved regions of the Nef protein that are juxtaposed to form a single surface on the known structure of Nef. A third, more amino terminal conserved region in Nef, previously reported to be important for Nef enhancement of virion infectivity, was found to be largely dispensable for CD4 downregulation. These data raise the possibility that Nef may contain two structurally distinct functional domains, only one of which contributes to the CD4 downregulation phenotype
Enhancement of HIV-1-Induced Syncytium Formation in T Cells by the Tyrosyl Kinase p56lck

AbstractThe CD4 glycoprotein is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1) and has also been reported to be physically associated with p56lck, a tyrosyl protein kinase. p56lckis a member of thesrcfamily of nonreceptor protein-tyrosine kinases and is expressed predominantly in T lymphocytes. Our objective was to study the effect of p56lckon the biology of HIV-1. For this purpose, we have stably transfected two human p56lck-negative T cell lines (C8166-45 and MT-2) with plasmids encoding for this cellular protein. Following coculture with HIV-1-infected cells or infection with cell-free virus, p56lck-expressing cell lines showed a greater propensity for virus-mediated syncytium formation than parental p56lck-negative cells. The enhancement of HIV-1-induced syncytium formation was not associated with the kinase activity of p56lck, as demonstrated by experiments using a kinase-deficient mutant. However, the physical interaction between CD4 and p56lckwas shown to be necessary to obtain the enhancement of syncytium formation since a mutated version of p56lck, which is deficient in its capacity to associate with CD4, did not lead to an increase in virus-mediated cell-to-cell fusion events. Finally, we determined that cells transfected with wild-type and kinase-negative mutant p56lckshowed a reduced rate of CD4 endocytosis compared to parental p56lck-negative cells. Together, these results suggest that p56lckcan be seen as an accessory molecule facilitating HIV-1-mediated syncytium formation in T cells by a mechanism involving the stabilization of the CD4 molecule at the cell surface
Adaptation of Measles Virus to Polarized Epithelial Cells: Alterations in Virus Entry and Release

AbstractWe have previously shown that the Edmonston strain of measles virus enters and is released preferentially at the apical surfaces of polarized epithelial cells. Small amounts of virus were found to be released at the basal surface. In the present study, we passaged the virus in polarized cells and characterized the passaged virus for its pattern of entry and release in epithelial cells as well as the ability to downregulate the receptor CD46. In contrast to the original stock virus, the passaged virus was found to be released at close to the same levels from both the apical and the basal surfaces. Accumulation of viral nucleocapsids and virus budding were observed at both membrane surfaces when cells were infected with the passaged virus. The passaged virus was also found to enter efficiently at the basal surface, unlike the original stock virus. Syncytial formation was observed at earlier times postinfection in cells infected with the passaged virus compared to cells infected with the stock virus. On Caco-2 cells, CD46 is found on both surfaces but is preferentially expressed on the apical membrane. The original Edmonston stock and two other wild-type strains, Chicago and Davis, were found to downregulate CD46 levels on the apical but not on the basolateral membrane of Caco-2 cells, while the passaged Edmonston measles virus did not downregulate CD46 on either surface. These data indicate that passage of measles virus through polarized epithelial cells results in selection of virus which exhibits a bidirectional pattern of entry and release through both the apical and the basolateral surfaces and which no longer downregulates CD46 expression on the cell surface
A Specific Interaction between Coat Protein and Helper Component Correlates with Aphid Transmission of a Potyvirus

AbstractSpecific binding between the coat protein (CP) and the helper component (HC) of the tobacco vein mottling potyvirus (TVMV) was characterized using a protein blotting–overlay protocol. In thisin vitroassay, HC interacted with either virions or CP monomers originating from the aphid-transmissible TVMV-AT but not from the non-aphid-transmissible TVMV-NAT. There was a strong correlation between the aphid transmissibility of a series of TVMV variants having mutations in the DAG motif of the CP and their ability to bind HC. Expression of TVMV CP derivatives in bacteria allowed a precise determination of the minimum domain mediating HC binding. This domain is composed of seven amino acids, including the DAG motif (DTVDAGK), located in the N-terminus of the TVMV CP at amino acid positions 2 to 8
AP-1cis-Response Elements Are Involved in Basal Expression and Vmw110 Transactivation of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10)

AbstractThe promoter of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) has two AP-1cis-response elements, respectively located at positions −62 and −94 relative to the transcription start site (Wymeret al.,1989,J. Virol.63, 2773–2784). Chloramphenicol acetyl transferase (CAT) analysis with hybrid constructions of the CAT structural gene and the ICP10 promoter or its mutants and gel retardation studies were used to examine the role of the AP-1cis-response elements in expression from the ICP10 promoter. Basal expression from the wild-type promoter was significantly (75–90%) reduced by mutation of the upstream or downstream AP-1 element. Mutation in the upstream AP-1 element also caused a 60% reduction in c-Jun-mediated activation. Activation was decreased 40% by mutation in the downstream AP-1 element and it was abrogated by mutation of both elements. Similar results were obtained for ACT-deleted mutants and mutants in which CT was mutated to AG. Thetrans-activation by Vmw110 was also reduced by mutation of the AP-1 elements (10- and 2-fold for the upstream and downstream element, respectively) and it was abrogated by mutation of both AP-1 elements. Mutation of nucleotides adjacent to the AP-1cis-response elements had no effect ontrans-activation. Gel retardation assays with a DNA probe representing the wild-type ICP10 promoter and nuclear extracts from HSV-1-infected cells identified one complex that was not seen with mock-infected cells or with cells infected with a Vmw110-deleted mutant. The complex was not seen when HSV-1-infected cells were reacted with an AP-1-mutant DNA probe, and its formation was competed by an AP-1 but not a mutant AP-1 oligonucleotide. The migration of this complex was retarded by c-Fos antibody, suggesting that both AP-1 and Vmw110 are involved in its formation. A mutant deleted in all sequences upstream of the TATA box was also activated by Vmw110, but this activation was only 2-fold lower than that seen for the wild type and significantly higher (10-fold) than that seen for the double AP-1 mutants. The data suggest that AP-1 elements play a crucial role in ICP10 gene expression/activation
The Human Cytomegalovirus UL97 Protein Is Phosphorylated and a Component of Virions

AbstractThe expression of the human cytomegalovirus (HCMV) UL97 open reading frame in infected or transfected cells in the presence of the antiherpes compound ganciclovir (GCV) results in the intracellular phosphorylation of GCV. There are conventional kinase domains within the UL97-encoded protein (pUL97). However, the role of pUL97 in the HCMV replication cycle, and the mechanism by which it causes phosphorylation of GCV, are currently unknown. Herein, the biosynthesis and biogenesis of pUL97 was studied in HCMV-infected cells. pUL97 is expressed with early-late kinetics and is posttranslationally modified by phosphorylation. This phosphorylation occurs within 1 hr after synthesis, affects the electrophoretic mobility of pUL97, and is independent of the presence of other HCMV proteins. pUL97 was localized to the nucleus of infected cells and found in the HCMV virions. Thus, pUL97 is a virion phosphoprotein, and a likely tegument component
Elevated Expression of the Human Parainfluenza Virus Type 1 F Gene Downregulates HN Expression

AbstractInteractions involved in the expression of parainfluenza glycoproteins were examined by expressing cDNA clones of theHNandFgenes from human parainfluenza virus type-1 (hPIV1) or Sendai virus (SV) in recombinant Semliki Forest virus (recSFV) or vaccinia-T7 expression vectors. We found that expression of a cloned F protein gene of hPIV1 resulted in downregulation of the HN proteins of hPIV1 or SV. Compared to the amount of HN expressed in the absence of F, coexpression of HN and F led to about 70% reduction in HN. This reduction of HN was observed in both total cell lysates and in protein localized on the cell surface. In contrast to hPIV1 F, SV F did not suppress the expression of HN. Northern blot analysis indicated that similar levels of HN mRNA accumulated in the absence or presence of hPIV1 F. The reduction of HN protein expression by hPIV1 F was detectable after as little as a 10-min labeling period, suggesting that downregulation occurred at the level of translation or at an early stage of protein folding. In hPIV1-infected cells, the amount of F protein synthesized was only about 15% of that of HN, whereas SV F is expressed at high levels. When the level of F in hPIV1-infected cells was artificially increased by recSFV, HN expression was suppressed. The reduction of F protein production in hPIV1-infected cells was regulated at the level of transcription. Characterization of mRNAs produced in hPIV1-infected cells showed that only 20% of the hPIV1 F mRNAs were monocistronic transcripts; 80% were bicistronic M-F readthrough mRNAs. Because proteins are suggested to be synthesized from only the first cistron of bicistronic mRNA in paramyxovirus (T. C. Wong and A. Hirano (1987)J. Virol.61, 584–589), production of F protein is likely suppressed by transcriptional regulation in hPIV1-infected cells. These results suggest that F is capable of downregulating the synthesis of HN, but that this is normally prevented in hPIV1-infected cells by suppression of F protein synthesis by transcriptional regulation
A Tobamovirus Genome That Contains an Internal Ribosome Entry Site Functionalin Vitro

AbstractMost eukaryotic mRNAs are translated by a “scanning ribosome” mechanism. We have found that unlike the type member of the genusTobamovirus,translation of the 3′-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhovet al.,1993; 1994) occurredin vitroby an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translatedin vitro:(i) “MP-CP-3′NTR” transcripts contained movement protein (MP) gene, CP gene and the 3′-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamovirusesin vivo.(ii) “ΔNPT-CP” transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) “CP-GUS” transcripts contained the first CP gene and the gene ofEscherichia coliβ-glucuronidase (GUS) at the 3′-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRESCP) promoting internal initiation of translationin vitro.Dicistronic IRESCP, containing chimeric mRNAs with the 5′-terminal stem–loop structure preventing translation of the first gene (MP, ΔNPT, or CP), expressed the CP or GUS genes despite their 3′-proximal localization. The capacity of crTMV IRESCPfor mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRESCP. It was concluded that integrity of IRESCPwas essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs
Binding of the Influenza A Virus to Cell-Surface Receptors: Structures of Five Hemagglutinin–Sialyloligosaccharide Complexes Determined by X-Ray Crystallography

AbstractThe structures of five complexes of the X-31 influenza A (H3N2) virus hemagglutinin with sialyloligosaccharide receptor analogs have been determined from 2.5 to 2.8 Å resolution by X-ray crystallography. There is well-defined electron density for three to five saccharides in all five complexes and a striking conformational difference between two linear pentasaccharides with the same composition but different linkage [α(2→6) or α(2→3)] at the terminal sialic acid. The bound position of the terminal sialic acid (NeuAc) is the same in all five complexes and is identical to that reported previously from the study of mono- and trisaccharides. The two oligosaccharides with NeuAcα(2→6)Gal linkages and GlcNAc at the third position have a folded conformation with the GlcNAc doubled back to contact the sialic acid. The pentasaccharide with a terminal NeuAcα(2→3)Gal linkage and GlcNAc at the third position has an extended (not folded) conformation and exits from the opposite side of the binding site than the α(2→6)-linked molecule of the same composition. The difference between the conformation of the pentasaccharide with a 2,6 linkage and the trisaccharide 2,6-sialyllactose suggests that 2,6-sialyllactose is not, as previously believed, an appropriate analog of natural influenza A virus receptors. The oligosaccharides studied are NeuAcα(2→3)Galβ(1→4)Glc, NeuAcα(2→6)Galβ(1→4)Glc, NeuAcα(2→3)Galβ(1→3)GlcNAcβ(1→3)Galβ(1→4)Glc, NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc, and [NeuAcα(2→6)Galβ(1→4)GlcNAc]2β(1→3/6)Gal-β-O-(CH2)5-COOCH3
HTLV-I Infection in Squirrel Monkeys (Saı&#x0308;miri sciureus) Using Autologous, Homologous, or Heterologous HTLV-I-Transformed Cell Lines

AbstractPeripheral blood mononuclear cells (PBMC) from three adult male squirrel monkeys (Saı&#x0308;miri sciureus) were transformed by human T-cell leukemia/lymphoma virus type I (HTLV-I) by cocultivation with lethally irradiated human MT-2 cells. Three permanent monkey T-cell lines producing HTLV-I were obtained and characterized. Six weeks after inoculation seroconversion was observed in three of three monkeys inoculated with autologous transformed T cells and in two of three monkeys receiving homologous cells. Proviral DNA was detected in their PBMC at various times after inoculation, with the highest proviral load and antibody titers being found in monkeys infected with homologous cells. Monkeys inoculated with heterologous MT-2 cells did not seroconvert, and HTLV-I provirus was detected only transiently in their PBMC. To determine whetherin vitroandin vivoHTLV-I infection of squirrel monkey cells led to a selection of monkey-adapted viral mutants, comparative sequencing of the proviral gp21envbetweenex vivomonkey HTLV-I-infected PBMC, the inoculum, and MT-2 cells was done and no significant differences were detected. The squirrel monkey, which is naturally free of simian T-cell leukemia/lymphoma virus, thus appears to be a suitable model for evaluating HTLV-I candidate vaccines and for studying the pathogenesis of HTLV-I
Emergence of Subtype Zaire Ebola Virus in Gabon

AbstractGabon has recently been struck three times by Ebola hemorrhagic fever. The first isolate originating from the 1994 outbreak has been subjected to molecular characterization of its GP and VP24 genes. Sequence analysis demonstrates that the agent, Gabon-94 virus, belongs to subtype Zaire of Ebola virus. The isolate is closely related to the Kikwit-95 isolate, and both viruses seem to have evolved from a progenitor virus different from that of the Zaire-76 isolates. The relatively close relationship of all subtype Zaire viruses isolated at different geographical locations and up to 20 years apart suggests an extreme conservation in the yet unknown natural reservoir of Ebola viruses. The level of genetic variability in the human host might be different as indicated by the comparison of isolates from a single outbreak (Mayinga-76 and Eckron-76), but needs further investigation on clinical material of patients by PCR since both isolates have different levels of passages in tissue culture
Genome Nucleotide Lengths That Are Divisible by Six Are Not Essential but Enhance Replication of Defective Interfering RNAs of the Paramyxovirus Simian Virus 5

AbstractFor some members of the Paramyxoviridae family of negative strand RNA viruses, efficient genome replication only occurs when the total genome length is a multiple of six (6Nlength, whereNis any integer). To determine if this “rule of six” requirement applied to the replication of the prototype paramyxovirus simian virus 5 (SV5), defective interfering (DI) RNA genomes were generated by sequential undiluted passage of virus in tissue culture. Molecular cloning and nucleotide sequence analysis of 10 RNA genomes revealed a series of copyback DI RNAs with chain lengths between 449 and 1365 bases, but only 4 of the 10 naturally occurring RNA genomes were of 6Nlength. Many of the cloned DI genomes could be grouped into two distinct nested sets, with the members of each set having the same polymerase crossover junctions and extent of terminal complementarity but differing from each other by internal deletions. One of these nested sets of genomes consisted of novel DI RNAs that contained a pentameric stretch of nontemplated adenosine residues inserted precisely at the polymerase crossover junction. A reverse genetics system was established in which SV5 DI genomes were replicatedin vivoentirely by cDNA-derived components. Using this system, two naturally occurring SV5 DI RNAs were examined in a mutational analysis to determine the role of genome length on SV5 RNA replication. The progressive insertion of one to six nucleotides into a 6Nlength DI genome (852 bases) resulted in a reduction in replication for RNAs that contained one to four additional bases (∼35–50% of WT levels), followed by an increase back to WT replication levels for genomes that were altered by five and six base insertions (∼70 and 100% of WT levels, respectively). An insertion of five nucleotides into a second non-6Nlength DI RNA (499 total bases) created a genome length that was a multiple of six (504 bases) and led to a ∼10-fold stimulation of replication over that of the unaltered genome. Together, these results indicate that there was a clear influence of 6Ngenome length on SV5 DI RNA replication, but the stringency of this replication requirement appeared to be less than that found previously for other paramyxoviruses. This work completes the testing of the rule of six replication requirement for representatives of each of the four genera of the Paramyxoviridae family and indicates that the preference for replication of 6Nlength RNA genomes varies between the individual paramyxoviruses
Preferential Translation of Reovirus mRNA by a σ3-Dependent Mechanism

AbstractWe have characterized reovirus strains that differ in the degree to which they inhibit cellular protein synthesis and used them to investigate mechanisms regulating gene expression in infected cells. A previous genetic study associated distinct effects of reovirus strains on cellular translation with polymorphisms in viral protein σ3. In cell extracts, σ3 sequesters double-stranded RNA (dsRNA) and blocks activation of the dsRNA-activated protein kinase (PKR), an interferon-induced enzyme that inhibits translational initiation by phosphorylating eIF-2α. We found that in infected cells, cellular protein synthesis is translationally regulated in a strain-specific manner. Using immunoprecipitation and indirect immunofluorescence we showed that the effect of a strain on cellular translation is not determined by the level of σ3, but appears to result from differences in σ3 localization. In cells infected with a strain that spares cellular translation, σ3 is present throughout the cytoplasm, whereas in cells infected with inhibitory strains, σ3 is restricted to perinuclear viral factories. Biochemical studies suggested that diffuse localization of σ3 is a consequence of low affinity for capsid protein μ1. Our findings are consistent with a model in which the efficiency of cellular translation is determined by the cytoplasmic level of σ3 that is not complexed with μ1