pinecone/core-2020-05-10-deduplication huggingface dataset:
Transcription, Translation, and Cellular Localization of ThreeAutographa californicaNuclear Polyhedrosis Virus Structural Proteins: ODV-E18, ODV-E35, and ODV-EC27

AbstractThis paper identifies two structural proteins of the occluded derived viral envelope ofAutographa californicanuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid
Analysis of a Salt Stable Mutant of Cowpea Chlorotic Mottle Virus

AbstractAn understanding of virion assembly and disassembly requires a detailed understanding of the protein–protein and protein–nucleic acid interactions which stabilize the virion. We have characterized a mutant of cowpea chlorotic mottle virus (CCMV) that is altered in virion stability. The mutant virions resist disassembly in 1.0MNaCl, pH 7.5, whereas the wild-type virions completely disassociate into RNA and capsid protein components. Sequence analysis of the mutant coat protein gene identified a single A to G nucleotide change at position 1484 of RNA 3 (position 134 of RNA 4), which results in a lysine to arginine change at position 42 of the coat protein. Introduction of the K42R mutation into wild-type CCMV coat protein results in a salt stable virion phenotype. Likewise, expression of the K42R mutant coat protein inEscherichia colifollowed byin vitroassembly produces virions that exhibit the salt stable phenotype. Analysis of this mutation demonstrates how a single amino acid change in the primary structure of the coat protein leads to tertiary interactions which stabilize the virion
Analyses of the Role of Structural Changes in the Regulation of Uncoating and Assembly of Alphavirus Cores

AbstractIn the late stages of alphavirus multiplication virus cores accumulate in the cytoplasm as stable structures, whereas they are unstable during the early stages of infection. Three types of explanation can be put forward to understand this phenomenon: (1) A cellular uncoating process is active early which is inactivated later, (2) the core structure differs between stable cores which accumulate and labile cores which are dissociated, (3) both mechanisms cooperate. A model based exclusively on the first principle involving core disassembly by cellular ribosomes has been proposed, but structural changes of the core may cooperate with the cellular uncoating process. We have therefore isolated cores of the Sindbis alphavirus from the cellular cytoplasm, from virus particles (vi-cores), and from low-pH-treated virus particles. Comparative analyses of the structure of these cores, using crosslinking and proteolytic digestion, and of the stability of the cores in the presence of ribosomesin vitrowere performed. The structural comparisons indicate that the interactions between the molecular components of these cores are very similar and probably identical. In the presence of ribosomes vi-cores were slightly more stable than the two other types of cores. Evidence for a cellular uncoating mechanism is furnished by experiments which analyze the stability of cores in the presence of postmitochondrial cytoplasm or of 60 S ribosomal subunits derived from either uninfected or Sindbis virus-infected cells. The results obtained indicate that structural alterations of the core do not play a role in the regulation of disassembly and assembly of alphavirus cores
Cloning of Rous Sarcoma Virus Enhancer Factor Genes. II. RSV-EF-II, Abundantly Expressed in Fibroblasts and Muscle Tissue, Binds to an Octamer Sequence, 5′-GTACCACC-3′, in the Noncoding Strand of RSV Enhancer

AbstractRous sarcoma virus (RSV) mainly replicates in avian fibroblasts, and the U3 enhancer region of the long terminal repeats of RSV contains the determinants for its tissue-tropic expression. We describe the cloning and characterization of an avian gene that encodes a protein capable of binding to the enhancer region of Rous sarcoma virus. A PCR-derived probe corresponding to the U3 region of RSV was used to isolate a cDNA clone by screening a chicken cDNA expression library. The cDNA is predicted to encode a polypeptide of 298 amino acids that is homologous to the Y-box (inverted CCAAT) family of DNA-binding transcription factors. This factor, which we refer to as Rous sarcoma virus enhancer factor-II (RSV-EF-II), shows 99% aa identity over a 105-amino-acid stretch that is highly conserved in all Y-box proteins, and is commonly referred to as the cold shock domain. RSV-EF-II selectively binds to single-stranded DNA, and the binding site, as determined by electrophoretic mobility shift assays, consists of the sequence 5′ GTACCACC 3′ located between nucleotides −112 to −119 in the noncoding strand of the RSV enhancer. Although RSV-EF-II shares considerable homology with the Y-box family of proteins, it does not bind to the inverted CCAAT boxes at positions −65 to −69 and −129 to −133 in the RSV LTR. Northern analysis indicates that RSV-EF-II-specific transcripts are expressed predominantly in avian fibroblasts and muscle tissue. The results of these binding and mRNA expression experiments suggest that RSV-EF-II may play an important role in tissue- and host-specific expression of RSV LTR-driven gene expression. Further, we show that RSV-EF-II acts as a repressor of transcription
Mutagenesis of the N-Linked Glycosylation Sites of the Yellow Fever Virus NS1 Protein: Effects on Virus Replication and Mouse Neurovirulence

AbstractThe flavivirus nonstructural glycoprotein NS1 is highly conserved and contains two N-linked glycosylation sites which are both utilized for addition of oligosaccharides during replication in cell culture. NS1 has been shown to contain epitopes for protective antibodies; however, its roles in virus replication and pathogenesis remain unknown. To study the function of NS1 during yellow fever virus replication, six mutant viruses which lack either one or both glycosylation sites and another one containing silent mutations at both sites were generated by site-directed mutagenesis. Mutants lacking the second glycosylation site and those bearing silent mutations were similar to the parental virus in their cell culture properties. Ablation of the first or both glycosylation sites generated mutants exhibiting small plaque phenotypes, decreased virus yields, reduced cytopathic effects, impaired NS1 secretion, and depressed RNA accumulation. In addition, mutants lacking the first or both glycosylation sites exhibited significant reduction in mouse neurovirulence after intracerebral inoculation. These defects appear to result from the lack of N-linked glycans rather than the introduction of deleterious amino acid substitutions or disruption ofcis-acting RNA elements important for RNA replication. These results suggest an important role for NS1 in flavivirus RNA replication and pathogenesis
In VitroTranscripts from Cloned cDNAs of the Lettuce Infectious Yellows Closterovirus Bipartite Genomic RNAs Are Competent for Replication inNicotiana benthamianaProtoplasts

AbstractFull-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication,Nicotiana benthamianaprotoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derivedin vitrotranscripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG)in vitrotranscripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only cappedin vitrotranscripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived cappedin vitrotranscripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectiousin vitrotranscripts can be derived from the cloned cDNAs of a closterovirus genome
Neutralization of SIVmac239/17E in Lymphocyte Cultures Involves Virus Strain-Specific Linear and Conformational Epitopes Encoded by Different Regions of theenvGene Including the “V3” Domain

AbstractSIVmac251 and its closely related derivatives SIVmac239 and SIVmac239/17E vary greatly in their susceptibility to neutralization with homologous and heterologous antisera. Whereas SIVmac251 induces homologous neutralizing antibodies, the antibodies induced by SIVmac239 rarely neutralize infectivity of this virus in lymphocyte cultures. In contrast, SIVmac239/17E is remarkably susceptible to neutralization with homologous and heterologous antisera induced by other strains of SIVmac. In this study, we studied the molecular basis for the neutralization of SIVmac239/17E. Using chimeric viruses in which different regions of theenvgene of both SIVmac239 and SIVmac239/17E were inserted into a background of either of the parental genomes, we showed that the newly acquired neutralization properties of SIVmac239/17E were attributable to amino acid substitutions between the V2 and V4 regions of gp120. Site-directed mutagenesis of theenvgene in this region showed that the arginine substitutions at positions 334 and/or 340 within the “V3” domain were fundamental to virus neutralization but other substitutions in the V2–V4 region added to the ease of its neutralization since it became neutralizable with much higher dilutions of serum. The molecular determinants for neutralization of this virus are distinct from those reported as responsible for neutralization of SIVmac251 and both are distinct from SIVmac239
Peptides Derived from the Interferon-Induced PKR Prevent Activation by HIV-1 TAR RNA

AbstractThe double-stranded RNA-dependent protein kinase (PKR) is believed to mediate cellular antiviral responses, function as a tumor suppressor, and regulate cell growth and differentiation. Its activation is dependent on double-stranded RNA (dsRNA) structures but these interactions are not fully understood. The possibility of direct interaction between dsRNA and the arginine and lysine-rich region of PKR (residues 54–74) was examined using synthetic peptides. We found that addition of a synthetic peptide corresponding to residues 54–74 of murine PKR or residues 60–80 of human PKR inhibited the autophosphorylation and activation of the kinase by either poly(I)–poly(C) or the 82-nucleotide-long TAR RNA. Gel-shift analysis indicated that the peptides disrupted the kinase–TAR complex by binding directly to TAR RNA. These findings delineate at least one dsRNA-binding domain in PKR which may be important for its cellular activation
CfMNPV BlocksAcMNPV-Induced Apoptosis in a Continuous Midgut Cell Line

AbstractMorphological and molecular changes produced byAutographa californicanuclear polyhedrosis virus (AcMNPV) infection in a permissive cell line, IPLB-SF-21AE (SF-21), ofSpodoptera frugiperdaand a nonpermissive cell line, FPMI-CF-203 (CF-203), ofChoristoneura fumiferanaare described. CF-203 cells inoculated withAcMNPV showed a DNA ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. Typical virus replication and occlusion body (OB) production were seen in SF-21 cells inoculated withAcMNPV and no apoptosis-like symptoms were observed. mRNA for the apoptosis suppressor gene p35 was detected 9 hr later inAcMNPV-inoculated CF-203 cells than in SF-21 cells. Only a trace amount of mRNA for theAcMNPV-inhibitor of apoptosis homologue (Ac-iap) gene and no mRNAs for the late genes,AcMNPV-polyhedrin (Ac-polh) andAcMNPV-p10 (Ac-p10), were detected inAcMNPV-inoculated CF-203 cells. Inoculation of CF-203 cells withCfMNPV at least 12 hr prior to inoculation withAcMNPV prevented apoptosis-like cell death, and mRNAs forAc-iap, Ac-polh,andAc-p10genes were expressed resulting in successful virus replication and OB production
In VitroCharacterization of Purified Human Thymic Dendritic Cells Infected with Human Immunodeficiency Virus Type 1

AbstractIn the thymus, dendritic cells (DC) are functionally associated with thymocytes and are recognized to play a major role in the intrathymic differentiation of T cells. Several studies have previously investigated the role of DC during HIV-infection, but the status of thymic DC in HIV-1 pathogenesis remains unclear. In this study, we investigated the susceptibility of purified human thymic DC to HIV-1 infectionin vitro.HIV-1 was not detected in cell-free supernatants collected from HIV-infected DC. However, these cultures were shown to transmit HIV-1 infection since coculture with permissive MT4 cells resulted in virus production. The exposure of DC in culture to HIV-1 was shown to promote severe DC morphological changes and killing. We also found that one or several heat labile soluble cytotoxic agents present in the HIV-1-infected DC supernatant mediated the killing of thymocytes. Our observations raise the possibility that (1) the HIV-1-induced DC killing, (2) the capacity of DC to transmit viral infection, and/or (3) the release of HIV-1-mediated cytotoxic agent(s) from DC may contribute to AIDS pathogenesisin vivo
Characterization of a 105-kDa Polypeptide Encoded in Gene 1 of the Human Coronavirus HCV 229E

AbstractGene 1 of the human coronavirus HCV 229E encompasses approximately 20.7 kb and contains two overlapping open reading frames, ORF 1a and ORF 1b. The downstream ORF 1b is expressed by a mechanism involving (−1) ribosomal frameshifting. Translation of mRNA 1, which is thought to be equivalent to the viral genomic RNA, results in the synthesis of two large polyproteins, pp1a and pp1ab. These polyproteins contain motifs characteristic of papain-like and 3C-like proteinases, RNA-dependent RNA polymerases, helicases, and metal-binding proteins. In this study, we have produced pp1ab-specific monoclonal antibodies and have used them to detect an intracellular, 105-kDa viral polypeptide that contains the putative RNA polymerase domain. Furthermore, usingtranscleavage assays with bacterially expressed HCV 229E 3C-like proteinase, we have demonstrated that the 105-kDa polypeptide is released from pp1ab by cleavage at the dipeptide bonds Gln-4068/Ser-4069 and Gln-4995/Ala-4996. These data contribute to the characterization of coronavirus 3C-like proteinase-mediated processing of pp1ab and provide the first identification of an HCV 229E ORF 1ab-encoded polypeptide in virus-infected cells
Mutations in the Endo Domain of Sindbis Virus Glycoprotein E2 Block Phosphorylation, Reorientation of the Endo Domain, and Nucleocapsid Binding

AbstractExposure of the carboxyl terminus (endo domain) of Sindbis virus membrane glycoprotein E2 to the cell cytoplasm is critical for the interaction of the nucleocapsid with viral envelope proteins in modified cell membranes. We have shown that the endo domain of PE2/E2 is initially translocated into membranes of the endoplasmic reticulum and subsequently drawn back into the cell cytoplasm during virus assembly. We suggested that phosphorylation of PE2/E2 might be responsible for the reorganization of the PE2/E2 carboxyl terminus. To test this hypothesis, two potential phosphorylation sites in the endo domain of E2, Thr398 and Tyr400, were changed by site-directed mutagenesis. Virus structural proteins are produced at normal levels in BHK-21 cells transfected with RNA containing the double mutation, nucleocapsids are formed, and the envelope proteins are exported from the endoplasmic reticulum; however, no virus is produced. The double mutation prevents phosphorylation of PE2/E2, and electron microscopy of cells transfected with the double mutant RNA reveals no attachment of nucleocapsids to cell membranes. The double mutation blocks exposure of the carboxyl terminus of E2 to the cytoplasm. Revertants of the double mutant to virus production all restored tyrosine at position 400 and restored the ability of the E2 protein to be phosphorylated. Although the threonine at position 398 is conserved among the alphaviruses, no revertant restored threonine at this position
Block of Tat-Mediated Transactivation of Tumor Necrosis Factor β Gene Expression by Polymeric-TAR Decoys

AbstractThetatgene product (Tat) of human immunodeficiency virus type 1 (HIV-1) is an early regulatory protein which transactivates HIV-1 gene expression by interacting with thetrans-activation response element (TAR) present in the HIV-1 long terminal repeat (LTR). In HIV-1-infected cells Tat can also activate the expression of tumor necrosis factor (TNF). Recent results indicate that essential for this effect is the interaction of Tat with a TAR-like structure present in the TNFβ messenger RNA leader region that closely resembles the TAR of the HIV-LTR. Here we show that because of this similarity of mechanisms, the expression of an RNA species encoding polymeric-TAR sequences and known to inhibit Tat-mediated HIV-1 gene expression also blocks TNF gene expression in response to Tat, but not TNF promoter activation induced by human T cell leukemia/lymphotropic virus type I Tax protein. Since TNF is increased in HIV-1-infected individuals and can activate HIV-1 gene expression or rescue Tat-defective HIV-1 proviruses, activation of TNF by Tat may be part of a complex pathway in which HIV-1 uses its own expression to increase infectivity and to induce disease. This study shows a dual role for the polymeric-TAR construct in inhibiting HIV-1 replication and strengthens the potential use of this protective gene in gene therapy for AIDS
Molecular Cloning of the Human Immunodeficiency Virus Subtype 2 Strain HIV-2UC2

AbstractAn infectious molecular clone was derived from the HIV-2UC2isolate that previously was found to persistently infect and induce an AIDS-like disease syndrome in baboons. The molecularly cloned virus (HIV-2UC2mc) showedin vitroproperties similar to those of the parental isolate with regard to T-cell tropism, cytopathicity, and the ability to infect primary baboon PBMC. Nevertheless, when inoculated into two baboons, the cloned virus showed a limited ability to replicate in these animals. DNA sequence analysis revealed a defectivevprgene in the UC2mc as well as in the pathogenic parental UC2 strain. Thus, thevprgene is not required for the induction of disease in baboons. The attenuated infectious molecular clone of UC2 should be useful for future studies designed to map the genetic determinants of HIV-2 pathogenesis in the baboon model and to evaluate vaccine strategies
The Cytoplasmic Domain of Bovine Herpesvirus 1 Glycoprotein B Is Important for Maintaining Conformation and the High-Affinity Binding Site of gB

AbstractBovine herpesvirus 1 (BHV-1) glycoprotein B (gB) has been shown to interact with two types of receptor on Madin Darby bovine kidney cells. The first receptor is heparan sulfate proteoglycan, whereas the second high-affinity receptor remains unknown. In order to study the structural requirement for gB's high-affinity binding activity, different forms of the gB ectodomain were expressed and compared with authentic gB. By using chemical cross-linking and sucrose gradient centrifugation, we found that BHV-1 gB was able to form dimers. A region between the cleavage site and the transmembrane anchor region, residues 506 to 763, was found to be required for gB oligomerization. Although the purified gBt and gBtM, two truncated forms of gB, formed oligomers, they did not block the high-affinity cellular receptor, suggesting that oligomerization was not the reason for the loss of the high-affinity binding site on gB. However, an N-terminal juxtamembrane region-located epitope recognized by a monoclonal antibody, designated epitope I, was lost from gBt and gBtM, indicating that both truncated gBs are conformationally changed. Therefore, the structure around this particular region may be required for the existence of the gB high-affinity binding site
Fetal or Neonatal Infection with Attenuated Simian Immunodeficiency Virus Results in Protective Immunity against Oral Challenge with Pathogenic SIVmac251

AbstractWe have reported that infection of fetal or neonatal rhesus macaques with attenuated SIVmac1A11 results in transient viremia, anti-SIV antibody responses, weak or absent cytotoxic T-lymphocyte responses, and no clinical disease. In light of these results, we hypothesized that congenital infection with SIVmac1A11 produced immune tolerance to SIV. To test this hypothesis, at approximately 1 year of age, five rhesus macaques infected with SIVmac1A11 as fetuses (n= 3) or newborns (n= 2) and five naive juvenile rhesus macaques were challenged orally with pathogenic SIVmac251. The five naive animals became persistently viremic after oral SIVmac251 inoculation. In contrast, one of three monkeys inoculated with SIVmac1A11in uteroand one of two animals inoculated with SIVmac1A11 at birth were virus culture negative. Virus was isolated from PBMC of the other animals infected with SIVmac1A11in uteroor at birth. However, one animal had a substantially lower viral load than the control animals. These results suggest that SIV-specific immunity rather than tolerance results from congenital infection with attenuated SIVmac and that this immunity is sufficient to provide some protection from pathogenic virus challenge. These results also demonstrate that SIV can be transmitted orally in 6- to 17-month-old rhesus monkeys
Target-Sequence Preferences of HIV-1 Integration Complexesin Vitro

AbstractIntegration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes