pinecone/core-2020-05-10-deduplication huggingface dataset:
Paracrine Activation of the HIV-1 LTR Promoter by the Viral Tat Protein Is Mechanistically Similar toTrans-Activation within a Cell

AbstractThe HIV-1 Tat protein activates transcription of the viral LTR promoter through interaction with the nuclear transcription machinery of the host cell. Tat can also activate the LTR promoter in a paracrine orinter-cellular manner by a yet unknown mechanism. One possibility is that Tat protein itself is secreted by cells and taken up by other cells. According to this mechanism,inter-cellular transcriptional activation by Tat should be very similar tointra-cellulartrans-activation in Tat-producing cells. A large number of cytokine genes was recently reported to be Tat-responsive, raising the possibility that such cytokines and the corresponding cellular transduction pathways are involved ininter-cellular Tat action. The transcriptional events in such an indirect route are likely to differ fromintra-cellular Tat action. To discriminate between a direct or indirect mechanism ofinter-cellular Tat action, we compared the activity of a set of Tat mutants and different promoter constructs ininter-cellular andintra-cellular transcriptional activation. Identical results were obtained in both assays, suggesting that Tat protein itself is exported by one and transported into the nucleus of another cell. The demonstration that Tat antibodies specifically inhibit theinter-cellular route is also consistent with cell-to-cell transport of the Tat protein. Furthermore, we found that the second Tat coding exon, including the RGD motif that has been proposed to interact with an integrin receptor, is not required for cellular uptake of the Tat protein
Enhancement of Hepatitis C Virus NS3 Proteinase Activity by Association with NS4A-Specific Synthetic Peptides: Identification of Sequence and Critical Residues of NS4A for the Cofactor Activity

AbstractThe NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-freetrans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase domain was expressed inEscherichia coli,purified, denatured, and refolded to an enzymatically active form. We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid substitution within the designated peptide identified residues I29and I25as critical for potential cofactor activity. We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid peptide (aa 22–33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development
Immunogenicity of JHM Virus Proteins: Characterization of a CD4+T Cell Epitope on Nucleocapsid Protein Which Induces Different T-helper Cell Subsets

AbstractThe CD4+T-cell-restricted recognition of the structural proteins in the JHM strain of MHV (JHMV) was compared between two mouse strains. Following immunization with inactivated JHMV, all proteins elicited an immune response in both C57BL/6 and BALB/c mouse strains, except the nucleocapsid (N) protein, which was not immunogenic in C57BL/6 mice. T-cell lines and clones were derived from BALB/c mice immunized with UV-inactivated JHMV, to determine the epitope(s) for CD4+T cells. The N protein appears immunodominant, since all T-cell lines and clones derived therefrom recognized this protein. To locate epitope-containing domains within the N protein, truncated N-protein fragments expressed in vaccinia constructs were used to stimulate the T-cell clones. Five independent T-cell clones recognized three separate N-protein domains: 1–133, 134–249, and 250–306. Since three of five clones recognized the last domain, its sequence was studied in more detail by constructing overlapping synthetic peptides covering this region. A single epitope was localized within the peptide comprising the N-protein residues 266–279. Its restriction element was identified as I-Edusing mAb to I-Edand I-Ad. In addition, peptide N266–279 contains the motif for binding to I-Ed. This peptide elicited proliferative responses following immunization with JHMV, confirming its recognition in the complete virus. In addition, peptide N266–279 was recognized by T-cell clones that express differences in cytokine profile as well as in TCR Vβ usage
Partial Substitution of the Functions of the Herpes Simplex Virus 1 UL13 Gene by the Human Cytomegalovirus UL97 Gene

AbstractThe predicted amino acid sequence of the human cytomegalovirus UL97 protein bears partial homology to the herpes simplex virus 1 UL13 protein, especially in regions that are homologous to conserved domains characteristic of protein kinases. Earlier studies showed that UL13 mediated the posttranslational processing of several herpes simplex virus 1 proteins including ICP22. Whereas no kinase activity has been specifically attributed to UL13, it has been shown that UL97 can phosphorylate ganciclovir. To examine whether UL97 can substitute for UL13, we constructed a herpes simplex virus 1 recombinant virus, R4970, in which UL13 was replaced by UL97 and in addition, the thymidine kinase gene was deleted. Characterization of this recombinant virus showed the following: (1) The recombinant virus grew as well as the wild-type virus in BHKTK+ cells, which restricted the growth of the UL13−virus. (2) UL97 could partially mediate the posttranslational modification of HSV-1 ICP22. This modification correlated with the restoration of the amounts of ICP0 and US11 proteins, which were down regulated in the UL13−virus-infected cells. (3) The recombinant virus was sensitive to ganciclovir in Vero- and KHOS-infected cells but not in the 143 thymidine kinase minus cells derived from KHOS cells. Vero cells infected with this recombinant virus phosphorylated ganciclovir. We conclude that UL97 partially compensates for UL13 functions
Sequence Analysis of the Bicistronic Drosophila X Virus Genome Segment A and Its Encoded Polypeptides

AbstractDrosophila X virus represents the entomobirnavirus genus of the Family Birnaviridae. Segment A of this bisegmented dsRNA containing virus was cloned and sequenced. The 3360-bp-long nucleotide sequence revealed the presence of two open reading frames (ORFs). A large ORF of 3096 nucleotides, which is flanked by a 107-bp 5′ and a 157-bp 3′-untranslated region, and a 711-nucleotide-long small ORF located within the carboxy half of the large ORF but in a different reading frame. The large ORF encodes a 114-kDa polyprotein which is cotranslationally processed by the virus-coded protease VP4 to generate preVP2, VP3, and VP4 (VP1 is encoded by genome segment B). N-terminal amino acid sequencing of VP3 and VP4 established the order NH2-preVP2-VP4-VP3-COOH within the polyprotein. The small ORF straddles the VP4/VP3 junction and is capable of encoding a basic, arginine-rich 27-kDa polypeptide which so far has not been detected in infected cells. The amino acid sequences specified by the two ORFs were compared to those of infectious pancreatic necrosis virus (IPNV) and infectious bursal disease virus (IBDV) that represent the two other genera (aquabirnavirus and avibirnavirus) of the Birnaviridae family. Significant sequence homology among the three viruses was found to be restricted to the amino and carboxy regions of preVP2 and to a small 21-residue-long domain near the carboxy terminal region of VP3. Significant sequence homology is exhibited by the small ORFs of the three viruses
Overexpression of Actin in AcMNPV-Infected Cells Interferes with Polyhedrin Synthesis and Polyhedra Formation

AbstractAutographa californicaM nuclear polyhedrosis virus (AcMNPV) terminates host protein synthesis during the late stage of infection, at approximately 14 hr postinfection (hpi). If infection takes place in the presence of cytochalasin D (CD), however, host actin synthesis is transiently stimulated and continues to be synthesized until approximately 30 hpi, and the hyperexpression of polyhedrin is delayed from about 20 hpi until about 36 hpi (S. N. Talhouk and L.E. Volkman,Virology182, 626–634, 1991; N. Wei and L.E. Volkman,Virology191, 42–48, 1992). To investigate whether these events are causally related, i.e., whether actin synthesis negatively affects polyhedrin synthesis, we constructed recombinant viruses that expressed actin at various levels during infection. We found that the expression of actin by a strong promoter interfered with polyhedrin synthesis at a posttranscriptional level. It also interfered with polyhedra formation, which may suggest a mechanism for the observed paucity of polyhedra in infected midgut columnar epithelial cellsin vivo
Rates of Shutdown of HIV-1 into Latency: Roles of the LTR andtat/rev/vpuGene Region

AbstractCEM T-cells chronically infected with most HIV-1 isolates gradually cease virus production over a 4–6 week period. This is due to slow shutdown of virus replication in the majority of the cells, leading to latent infections. We identified one HIV-1 isolate (HIV213) which shut down into latency at a rate much slower than most HIV strains, requiring more than 12 weeks for the majority of the cells to become nonproductive. This indicated that genes of the virus influence the rate of shutting down, or alternatively, the length of time chronically infected cells produce virus. The viral gene(s) influencing differential rates of shutdown were mapped using chimeric viruses composed of the HIV213genome substituted with various restriction fragments from HIVMCK, which rapidly progresses into latency. We found that the 3′ region of the LTR was the major determinant influencing the rate of shutdown, but thetat/rev/vpuregion also slightly influenced this phenotype. These data show that at least these two genomic regions can influence the duration of virus production in chronically infected cells and that polymorphisms in these regions result in phenotypically divergent viruses which go into latency at different rates. It is also possible that this viral property may be an important determinant of clinical outcomes
Activation of the Hepatitis B Virus S Promoter by Transcription Factor NF-Y via a CCAAT Element

AbstractThe middle and small surface proteins of hepatitis B virus are translated from 5′-heterogeneous transcripts specified by the S promoter. We have generated a series of linker-substitution mutants that encompass the 130 base pairs comprising this promoter and measured the amount of transcripts and protein products synthesized from each mutant. The results confirm our previous finding that a CCAAT element is an important up-stream activating element for this promoter, as mutation of this element leads to a >20-fold decrease in promoter activity.In vitrobinding assays showed that the cellular transcription factor NF-Y (CCAAT-binding factor) binds to this element, and expression of a dominant-negative NF-Y subunit in transfected cells specifically reduced surface protein expression from the S promoter via the CCAAT element. In addition, two Sp1 sites also contribute to S promoter activity by a total of approximately 6-fold. Therefore, the S promoter is activated by both NF-Y and Sp1, but more strongly by the former factor
Phylogenetic and Epidemiologic Analysis of the Walleye Dermal Sarcoma Virus

AbstractWalleye dermal sarcoma virus (WDSV) is a newly described retrovirus that is etiologically associated with a multifocal skin tumor of a fish common in North America, the walleye. Tumor prevalence ranges from 27% of adult walleyes in a densely populated lake, Oneida Lake, New York, to 1% in less populated waters. Phylogenetic analysis of the surface (SU) domain of the WDSV envelope gene of isolates from different regions of North America showed that viral isolates formed distinct clusters according to their geographic origin, except viral isolates from Oneida Lake, which were also much more variable. Viral clones isolated from an individual tumor had identical nucleotide sequences. This finding is consistent with tumors developing from single infected dermal cells, and supports the etiological role of this virus in tumor development. Like in other retroviruses, the SU domain of the WDSVenvgene was more variable thangag,and the ratio of nonsynonymous over synonymous mutations was comparable to that of the V3 loop of HIV-1. These findings indicate that WDSV SU is the object of strong selective immunologic pressures, like the SU domain of other retroviruses
Productive and Persistent Infection of Human Thymic Epithelial Cellsin Vitrowith HIV-1

AbstractInfection with HIV-1 results in a disruption of the thymic microenvironment and the presence of HIV-1 in thymic epithelial cells has been demonstratedin vivo.In the present study, we examined the susceptibility of a highly enriched culture of thymic epithelial cells (TEC) to infectionin vitroby HIV-1 laboratory strains and primary isolates. Replication in TEC is shown to depend on the virus and on the expression of CD4 molecules that are found to be expressed at a low density on the plasma membrane. Our results are consistent with infection of TEC controlled by the efficiency of the interactions between the envelope glycoprotein of the virus and the cell surface molecules. As a consequence, certain HIV-1 viruses induce a productive and persistent infection in TEC without damaging the cells. Altogether these results support the idea that TEC may act as a reservoir for HIV-1 in the thymus but are probably destroyed by an indirect mechanism involving infection of thymocytes
Nucleotide Sequence Analysis of the Respiratory Syncytial Virus Subgroup A Cold-Passaged (cp) Temperature Sensitive (ts)cpts-248/404 Live Attenuated Virus Vaccine Candidate

AbstractThe complete nucleotide sequence of the RSVcpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passagedcp-RSV, andcpts-248 virus, which constitute the series of progenitor viruses. RSVcpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36°) than itscpts-248 parent virus (shut-off temperature 38°) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited bycp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for thetsand attenuation phenotypes of its chemically mutagenized derivatives,cpts-248 andcpts-248/404. Compared with itscpts-248 parent, thecpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, thecpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of thecpts-248/404 in tissue culture. Biologically cloned derivatives of RSVcpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of thecpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans
Vaccinia Virus Replication Is Independent of Cellular HSP72 Expression Which Is Induced during Virus Infection

AbstractHSP72 is dramatically induced in the ovaries of vaccinia virus (VV)-infected mice and associates with VV proteins. In order to investigate the role of HSP72 during vaccinia virus replication, we have constructed a recombinant vaccinia virus encoding the major inducible cellular HSP72 (VV-HSP72+) and examined the replication characteristics of this virus. VV-HSP72+ exhibited growth kinetics identical to and peak titers very similar to those of control viruses, bothin vitroandin vivo.In particular, replication of VV-HSP72+ was identical to that of control viruses in the HSP72-negative cell line Y3.Ag.1.2.3, and overexpression of HSP72 had no effect on the virulence of VV infection in normal or immunocompromised mice. We conclude that while VV infection results in the induction of the major inducible 72-kDa HSP, VV replication proceeds normally in the absence of this protein. It is unclear whether another celluar chaperone is required to facilitate virus replication in place of HSP72 in Y3.Ag.1.2.3 cells or whether HSP expression plays no role in virus replication, but is simply a component of the generalized stress response to virus infection
Initiation of (−)-Strand RNA Synthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis of Oligonucleotides

AbstractRNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (−)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (−)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (−)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (−)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRpin vitro.Addition of heparin or Mn2+differentially affected production of the 8-mer and full-length (−)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (−)-strand RNA synthesis
Complement-Activating Antibodies in Sera from Infected Individuals and Vaccinated Volunteers That Target Human Immunodeficiency Virus Type 1 to Complement Receptor Type 1 (CR1, CD35)

AbstractComplement receptor type 1 (CR1) plays a central role in clearing immune complexes from the circulation and probably contributes to the retention of immune complexes on the surface of follicular dendritic cells. Virus-specific, complement-activating antibodies can target human immunodeficiency virus type 1 (HIV-1) to CR1-bearing cells but the potential impact that these antibodies have on HIV-1 pathogenesis is unknown. To study these antibodies, an assay was developed in which immune complexes containing HIV-1, antibody, and complement were formedin vitroand captured on the surface of 96-well immunoplates coated with recombinant soluble human CR1 (rsCR1). Captured virus was detected by p24 immunoassay or by infection of human CD4+lymphocytes. Two laboratory strains of HIV-1 (IIIB and MN) and primary isolates could be captured using sera from infected individuals or vaccinated volunteers as a source of complement-activating antibodies. HIV-1 immune complexes captured by solid-phase rsCR1 could be transferred to MT-2 cells for productive infection. Antibodies had no activity in this assay when the normal human serum used as a source of complement had been heat-inactivated or depleted of complement component C3, confirming a requirement for complement. These complement-activating antibodies in sera from infected individuals showed strong cross-reactivity with HIV-1 IIIB, MN, and a heterologous primary isolate, but reacted poorly with the autologous isolate obtained at the time of serum collection. Average titers of these antibodies measured with HIV-1 IIIB were moderately lower in HIV-1-infected progressors compared to nonprogressors. In contrast to sera from infected individuals, sera from gp160IIIB-vaccinated volunteers showed specificity for the vaccine strain of virus. These results provide supporting evidence that envelope-specific, complement-activating antibodies induced by infection or gp160 immunization can target HIV-1 immune complexes to CR1. In addition, they demonstrate that such antibodies may sometimes be type-specific and that HIV-1 immune complexes bound to CR1 are infectious
Modified Entry and Syncytium Formation by Herpes Simplex Virus Type 1 Mutants Selected for Resistance to Heparin Inhibition

AbstractHerpes simplex virus type 1 (HSV-1) mutants were selected by passage of HSV-1(KOS) in HEp-2 cells such that binding and penetration occurred in the presence of heparin. Analysis of selected uncloned virus pools revealed that approximately 95% of virus formed syncytia and greater than 58% were gC-negative. Plaque-purified gC-negative syncytial mutants were more resistant than HSV-1(KOS) to heparin inhibition, as was an engineered nonsyncytial recombinant deleted for gC, ΔgC6. Thus, absence of gC was sufficient to explain the enrichment for gC-negative mutants. The syncytial phenotype of most mutants mapped to a mutation in gK. Transfer of this mutation to HSV-1(KOS) resulted in a recombinant that induced fusion of Vero cells but not HEp-2 cells and was more sensitive to heparin inhibition of entry, revealing a previously undescribed phenotype of mutations in gK. Engineered gC-negative virus containing the gK syncytial mutation induced fusion of both cell lines and was as resistant to heparin inhibition as was ΔgC6. Because deletion of gC reduces infectivity of HSV-1 in the absence of heparin, mutations in gC combined with the syncytial mutation could have provided a selective advantage. Thus, absence of gC reduced heparin inhibition of binding and penetration while the combination of the gC and gK mutations enhanced spread through the HEp-2 cell monolayer by cell fusion. Because extreme selective pressure was required to favor these mutations and such mutations are rare in clinical isolates, the wild-type forms of gC and gK must provide for optimal viral replication and propagation in cell culture as well asin vivo,despite the view that gC is dispensable in cultured cells