pinecone/core-2020-05-10-deduplication huggingface dataset:
Fine Mapping of a C-Terminal Linear Epitope Highly Conserved among the Major Envelope Glycoprotein E2 (gp51 to gp54) of Different Pestiviruses

AbstractEnvelope glycoprotein E2 (gp51 to gp54) is the major neutralizing antigen of pestiviruses, which include classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV), and border disease virus (BVD). Previous studies carried out using a panel of monoclonal antibodies raised against CSFV strain Brescia have revealed the existence of four antigenic domains, A to D, of the E2 protein, all of which are located at the N-terminal half of the molecule. Here we report the detailed mapping, using three complementary techniques, of a novel linear epitope located at the C-terminal part of the molecule, which reacted with a monoclonal antibody (4-9D4) as well as polyclonal animal sera. This epitope is highly conserved in the three different members of pestiviruses and hence can be used as a genus-specific diagnosis tool. The observation that this epitope is not accessible on the native virus surface, together with its C-terminal location, supports a recently proposed structural model, indicating that the C-terminal part of E2 is membrane-bound while the N-terminal half of the molecule is exposed on the virus surface
Recognition of Human Cytomegalovirus Gene Products by HCMV-Specific Cytotoxic T Cells

AbstractThe antigen specificity of HCMV-specific cytotoxic T cells (CTL) was investigated in healthy adult seropositive donors by analyzing the frequency of CTL precursors (CTLp) against six different HCMV-encoded proteins. HCMV-specific, CD8+ CTL clones from two donors were also characterized. The precursor frequency analysis showed that a major phosphoprotein of HCMV, pp65 (UL83), was the immunodominant CTL target antigen in healthy seropositive adults. The frequency of CTLpspecific for different HCMV proteins ranged from 1/12,000 for pp65 to >1/100,000 for gB (UL55), gH (UL75), pp50 (UL44), and pp28 (UL98). Characterization of the HCMV-specific CTL clones from two donors revealed that the majority of clones lysed targets expressing pp65 and that none of the clones recognized other HCMV proteins
Induction of Systemic and Mucosal Immune Responses in Cotton Rats Immunized with Human Adenovirus Type 5 Recombinants Expressing the Full and Truncated Forms of Bovine Herpesvirus Type 1 Glycoprotein gD

AbstractWe generated both replication-incompetent (HAd5-gD-E1 and HAd5-tgD-E1) and replication-competent (HAd5-gD-E3 and HAd5-tgD-E3) human adenovirus type 5 (HAd5) recombinants expressing the full (gD) or truncated form (tgD) of the glycoprotein gD gene of bovine herpevirus type 1 (BHV-1). Recombinant gD and tgD expressed by HAd5-gD-E1 and HAd5-gD-E3 and by HAd5-tgD-E1 and HAd5-tgD-E3, respectively, were recognized by gD-specific monoclonal antibodies (MAbs) directed against linear and conformational epitopes, suggesting that antigenicity of recombinant gD and tgD was similar to that of the native gD expressed in BHV-1 infected cells. In HAd5-gD-E1- or HAd5-gD-E3-inoculated cotton rats there was a strong gD- and HAd5-specific IgG and IgA antibody response. The immune response was significantly lower in animals similarly immunized with HAd5-tgD-E1 or HAd5-tgD-E3, indicating that live adenovirus vaccine vectors may be better suited to the full-length form of glycoprotein gD than its truncated form. After a BHV-1 challenge, no infectious BHV-1 virions were isolated from the trachea of cotton rats previously immunized with HAd5-gD-E1 or HAd5-gD-E3. These results suggest that adenovirus E1 insertion (replication-incompetent) and E3 insertion (replication-competent) vectors have excellent potential for use in developing live recombinant virus vaccines and provide evidence that the cotton rat model can be used in BHV-1 vaccination–challenge trials
Rat Cytomegalovirus Has a Unique Immediate Early Gene Enhancer

AbstractThe immediate early (IE) enhancer/promoter regions of human cytomegalovirus (HCMV), simian cytomegalovirus (SCMV), and murine cytomegalovirus (MCMV) share the following characteristics: (1) they demonstrate high-level transcriptional properties, (2) multiple repetitive elements are found throughout the enhancer region, and (3) consensus binding sites for cellular transcription factors are frequently located within the repetitive elements. Here we characterize the enhancer/promoter region of the major IE gene locus for rat cytomegalovirus (RCMV). The RCMV IE enhancer/promoter region is able to activate transcription at the same level as HCMV or MCMV IE enhancer/promoter regions. In contrast to HCMV, MCMV, and SCMV, the RCMV IE enhancer/promoter region is almost completely lacking in repetitive elements and the only recognized consensus binding sites for cellular transcription factors recognized by sequence are three CTF, two AP1, and one NF-κB binding sites. However, enhancing activity does not appear to be dependent on these individual sites in transient transfection experiments, but is additive across the enhancer region
Mapping Functional Domains in AcMNPV pp31

AbstractAutographa californicanuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1–4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein
A Neural Substrate of Hyperactivity in Borna Disease: Changes in Brain Dopamine Receptors

AbstractRats experimentally infected with the neurotropic RNA virus, Borna disease virus, have a hyperactive movement disorder. Because locomotor activity is modulated by the nucleus accumbens (N. Acc.) dopamine (DA) system, high-affinity DA uptake, DA D1, D2, and D3 receptor binding sites were examined in N. Acc. subregions of normal and infected rats by quantitative receptor autoradiography. The N. Acc. of infected rats had decreased mazindol and D2 and D3 radioligand binding in the core and decreased D3 radioligand binding in rostral subregions. The abnormalities observed in the N. Acc. DA system of infected rats may offer insights into the potential viral pathogenesis of psychiatric conditions with a dopaminergic substrate such as schizophrenia and affective disorders
Amino Acid Substitutions in the CA Protein of Moloney Murine Leukemia Virus That Block Early Events in Infection

AbstractThe capsid domain (CA) of the retroviral Gag protein is a major constituent of the virion core. To examine the role of this protein in M-MuLV morphogenesis and replication, a series of substitution mutations affecting the central region of CA were introduced into an infectious proviral DNA. The altered DNAs were introduced into cells, and the resulting lines were analyzed for production of infectious virions. Only one of the replication defective mutants analyzed was blocked in virion assembly. The remaining mutant DNAs induced the formation and release of particles containing genomic RNA and polymerase protein. The reverse transcriptase associated with these mutant virions was capable of transcribing both minus strand strong stop and extended DNA products using the endogenous genomic RNA as templatein vitro.Upon infection of fresh cells, however, no viral DNA synthesis could be detected either by Southern analysis or by an RNase protection assay developed specifically to detect intermediate products of reverse transcription. The results indicate that the bulk of the CA mutants are blocked before reverse transcription of the viral genome and suggest an important role for the capsid protein in an early stage of viral replication
Epstein–Barr Virus Single-Stranded DNA-Binding Protein: Purification, Characterization, and Action on DNA Synthesis by the Viral DNA Polymerase

AbstractEpstein–Barr virus (EBV) BALF2 gene product is one of the essential components in the lytic phase of the EBV DNA replication. The BALF2 protein was purified to near homogeneity from the nuclear extract of B95-8 cells with virus productive cycle induced by chemical agents. SDS–polyacrylamide gel electrophoresis showed the presence of a single polypeptide with a molecular weight of 130 K, which was identified as BALF2 protein by Western immunoblot analysis. On Superose 6 HR 10/30 gel filtration the BALF2 protein eluted at a position corresponding to an apparent molecular mass of approximately 128 K, indicating that the BALF2 protein behaves as a monomer in solution. The purified BALF2 protein bound to single-stranded DNA preferentially over double-stranded DNA or single-stranded RNA. Replication of singly primed M13 single-stranded DNA by the EBV DNA polymerase complex in the absence of the BALF2 protein exhibited a highly processive mode of replication and generated full length products in addition to some bands of pausing sites. Although the addition of the BALF2 protein did not affect the replication rate, the average chain length of the replication products was slightly increased with eliminating bands of pausing sites. Similar effects were observed with the reconstituted polymerase complex composed of the BALF5 and BMRF1 Pol subunits. On the other hand, in the absence of the BALF2 protein, the BALF5 Pol catalytic subunit alone extended the primer slightly and paused at specific sites on M13 ssDNA template where stable secondary structure is predicted. However, addition of the BALF2 protein, in contrast to the case of herpes simplex virus ICP8 which does not affect the overall distribution of length of the replication products synthesized by the HSV Pol catalytic subunit (Gottliebet al.,1990,J. Virol.64, 5976–5987), stimulated DNA synthesis and yielded a distribution of replication products with long lengths in addition to full length products. Although the BALF2 protein behaved as if it converts a low processive enzyme of the EBV Pol catalytic subunit to a highly processive form like the BMRF1 Pol accessory subunit, challenger DNA experiments revealed that the EBV Pol catalytic subunit is transferred to challenger DNA even in the presence of the BALF2 protein. It is therefore likely that the EBV BALF2 protein functions to melt out the regions of secondary structure on the single-stranded DNA template, thereby reducing and eliminating pausing of the EBV DNA polymerase at specific sites. These properties indicate that the EBV BALF2 protein acts as a single-stranded DNA-binding protein during lytic phase of EBV DNA replication
Simian Virus 40 Large T Antigen Binds to Topoisomerase I

AbstractBinding of simian virus 40 (SV40) large T antigen to human and calf thymus topoisomerase I (topo I) was readily detected by using modified enzyme-linked immunosorbent assays and immunoblots. In addition to WT T antigen, binding could also be readily demonstrated with T antigen fragments from the amino-terminal region as well as with fragments missing this region, but much less so with small t antigen or with human p53. Antibody-blocking experiments showed that a monoclonal antibody that binds to the N-terminal region and several antibodies that recognize the central region of T antigen interfere with the binding to topo I. Our data are consistent with the existence of two separate topo I-binding regions in T antigen, one mapping within residues 82 to 246 and an apparently weaker one present after residue 246. By comparing the binding of T antigen to topo I with that of T antigen to DNA polymerase α or RPA, a single-stranded DNA-binding protein, it was determined that the T antigen–topo I interaction is much stronger and that the binding sites for topo I and DNA polymerase overlap, whereas the one for RPA differs. Several unwinding-defective mutants of T antigen were partially defective in their binding to topo I, suggesting that the binding to topo I is required for unwinding circular DNA. Finally, immunoprecipitation experiments demonstrated that T antigen can interact with DNA-bound topo I, indicating that such an interaction may take place during SV40 DNA replication
Intracellular andin Vitro-Translated 27-kDa Proteins Contain the 3C-like Proteinase Activity of the Coronavirus MHV-A59

AbstractThe coronavirus mouse hepatitis virus-A59 (MHV-A59) encodes a serine-like proteinase (3C-like proteinase or 3CLpro) in ORF 1a of gene 1 between nucleotides 10209 and 11114. We previously have demonstrated that proteins expressedin vitrofrom a cDNA clone of the 3CLpro region possess proteinase activity, and that the proteinase is able to cleave substratein trans.We sought to determine if the 27-kDain vitrocleavage product (p27) was an active form of the 3CLpro and whether this was consistent with the 3CLpro expressed in virus-infected cells. Antibodies directed against the 3CLpro domain detected 27-kDa MHV proteinsin vitroand in MHV-A59-infected cells. The 27-kDa proteins were able to cleave substratein transwithout other protein cofactors or supplemental membranes, and the p27 proteinase activity was retained after purification by immunoprecipitation and gel electrophoresis. When p27 was expressedin vitrowith portions of the amino- and carboxy-terminal flanking domains (MP1 and MP2), p27 was not liberated byciscleavage. The proteolytic activity of the 27-kDa proteins was inhibited by a variety of cysteine and serine proteinase inhibitors, and was eliminated by the cysteine proteinase inhibitor E64d. These results indicate that the 27-kDa protein is a mature proteinase in MHV-A59-infected cells, and that appropriate processing of this molecule occursin vitro
The Sendai Virus V Protein Interacts with the NP Protein to Regulate Viral Genome RNA Replication

AbstractThe interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathioneS-transferase (gst)–viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P–L (RNA polymerase), NP0-P (encapsidation substrate), and P–P complexes and now demonstrate NP–NP and NP0–V protein interactions. Expression of gstP and P proteins and binding to glutathione–Sepharose beads as a measure of complex formation confirmed the P–P interaction. The P–gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345–411. We also show that full-length and deletion gstV and gstW proteins bound NP0protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78–316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replicationin vitro,showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NP0– P encapsidation substrate
The Mutation Rate of Human Immunodeficiency Virus Type 1 Is Influenced by thevprGene

AbstractA system has been designed to study thein vivoforward rate of mutation of human immunodeficiency virus type 1 (HIV-1) during one round of replication. A HIV-1 shuttle vector was used that contained thelacZα peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 × 10−5mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 reverse transcriptase with sense-strand RNA and DNA templates of thelacZα peptide gene in a cell-free system. To test the hypothesis that thevprgene product might, at least in part, account for the lower mutation rate observedin vivo,a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-typevprgene product. Avpr−shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of thevpr−vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence ofvpron the mutation rate. Replication of avpr−shuttle vector in the presence of a wild-typevprexpression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that thevprmutation could be complementedin trans.Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence ofvpron the mutation rate
Mutations in Both the U5 Region and the Primer-Binding Site Influence the Selection of the tRNA Used for the Initiation of HIV-1 Reverse Transcription

AbstractThe initiation of HIV-1 reverse transcription is primed by a cellular tRNALys,3molecule which is bound to a complementary sequence near the 5′ end of the viral RNA genome designated as the primer-binding site (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNALys,3primer used to initiate reverse transcription. To further explore the role that the A-loop plays in reverse transcription, we have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3′-terminal 18 nucleotides of tRNAIle, tRNAPro, or tRNATrp[pHXB(Ile), pHXB(Pro), or pHXB(Trp), respectively]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNAIle[pHXB(Ile-AC)], tRNAPro[pHXB(Pro-AC)], or tRNATrp[pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in production of infectious virus. PCR was used to amplify the PBS regions which were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pHXB(Pro), and pHXB(Trp) reverted to be complementary to tRNALys,3. The PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be complementary to tRNALys,3; the A-loop region was still complementary to tRNAIle. In contrast, viruses derived from transfection of pHXB(Pro-AC) initially maintained a PBS complementary to tRNAPro. Upon extended culture, we identified proviruses which contained PBSs complementary to two additional tRNAs: tRNAIleand tRNALys,3. Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNALys,3and a second complementary to tRNAProor tRNAIle. Viruses derived from transfection of pHXB(Trp-AC) were the most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs were complementary to tRNATrp. After furtherin vitroculture, proviruses were identified with a PBS complementary to a new tRNA, tRNAMet. Finally, upon extended culture, the viruses derived from the transfection of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutations upstream from the PBS in U5 that created a stretch of 3 adenine nucleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-1 reverse transcription
Use ofcis- andtrans-Acting Viral Regulatory Sequences to Improve Expression of Human Immunodeficiency Virus Vectors in Human Lymphocytes

AbstractWe compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1tatgene andpolsequences reported to exertcis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing thepolsequences and was only slightly less than that seen for atrans-complementedenv-deleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible
Mutagenesis of a Hepatitis B Virus Reverse Transcriptase Yields Temperature-Sensitive Virus

AbstractReplication of the hepadnavirus genome is catalyzed by a multifunctional reverse transcriptase (thepolprotein) that exhibits DNA polymerase and DNA priming activities and has the ability to transfer RNA and DNA strands across the viral genome. A salient feature of this enzyme is the ability to prime RNA-directed DNA synthesis with protein rather than with RNA. This is reflected in its unique physical make up, which includes an amino-terminal (TP) domain that is separated by a spacer from the reverse transcriptase (RT) domain. To establish a structure–function relationship for thepolprotein, we examined 52 mutants for their ability to replicate viral DNAin vitroand in cultured cells. We demonstrated that the role of the TP domain is limited to the early steps of viral DNA synthesis including RNA packaging and protein priming. Both the TP and the RT domains are required for the interaction with epsilon RNA, which is the template for the protein-priming reaction and serves as the RNA packaging signal. In addition, we report the isolation of a thermosensitive variant of a hepadnavirus that will permit investigations of individual steps of the viral replication cycle under synchronized conditions