pinecone/core-2020-05-10-deduplication huggingface dataset:
Pleiotropic Mutations in the HIV-1 Matrix Protein That Affect Diverse Steps in Replication

AbstractThe matrix domain of the Gag precursor protein, and the mature matrix protein, which is derived from processing of the Gag precursor, functions in several steps of the human immunodeficiency virus type-1 (HIV-1) life cycle. We made numerous mutations throughout the matrix protein and identified three mutants in the N-terminal portion of the matrix that drastically diminish the ability of the virus to replicate. Each of these replication-defective mutants was unable to acquire efficiently the envelope glycoprotein of HIV-1. To determine whether these same mutations affect other steps in viral replication we pseudotyped mutant particles with the envelope glycoprotein from an amphotropic murine leukemia virus. Each of these mutants was also hampered in other steps in virus replication. Two mutants were defective in entry or uncoating, and the third was hampered in a step following reverse transcription. Since viral replication was analyzed under conditions in which the nuclear localization function of the matrix protein is not required, the matrix protein may be required for an additional replication step following reverse transcription
Characterization of Host Range Factor 1 (hrf-1) Expression inLymantria disparM Nucleopolyhedrovirus- and RecombinantAutographa californicaM Nucleopolyhedrovirus-Infected IPLB-Ld652Y Cells

AbstractWe previously identified a gene, host range factor 1 (hrf-1), inLymantria disparM nucleopolyhedrovirus (LdMNPV) which promotedAutographa californicaM nucleopolyhedrovirus (AcMNPV) replication in a nonpermissive cell line IPLB-Ld652Y (Ld652Y). A recombinant AcMNPV, vAcLdPS, that borehrf-1controlled by two synthetic baculovirus late promoters and that replicated in Ld652Y cells was constructed. In this study, we constructed a new recombinant AcMNPV, vAcLdPD, bearing onlyhrf-1controlled by its own promoter. vAcLdPD replicated in Ld652Y cells in the same manner as vAcLdPS, confirming thathrf-1alone was sufficient to promote AcMNPV replication in Ld652Y cells.hrf-1was transcribed as a delayed early gene in LdMNPV but as an immediate early gene in both recombinant AcMNPVs. Primer extension analysis showed that the initiator sequence TCAGT was used as the transcription start site in both LdMNPV and recombinant AcMNPVs. Additional sequencing revealed several regulatory motifs in thehrf-1upstream region.hrf-1transcripts in LdMNPV- and vAcLdPS-infected Ld652Y cells terminated near the polyadenylation signal at the end ofhrf-1ORF while in vAcLdPD, thehrf-1transcripts terminated at a downstream polyadenylation signal at the end of ORF 603. Using Western blot analysis, we detected HRF-1 expression in both recombinant AcMNPV-infected Ld652Y cells but not in LdMNPV-infected Ld652Y cells
Hepatitis C Virus NS5B Protein Is a Membrane-Associated Phosphoprotein with a Predominantly Perinuclear Localization

AbstractHepatitis C virus NS5B protein is an RNA-dependent RNA polymerase. To investigate the properties and function of this protein, we have expressed the NS5B protein in insect and mammalian cells. NS5B was found to be present as fine speckles in the cytoplasm, particularly concentrated in the perinuclear region, suggesting its association with the nuclear membrane, the endoplasmic reticulum, or the Golgi complex. This conclusion was supported by the biochemical demonstration that NS5B was associated with the membranes in the cells. Furthermore, it was shown that NS5B protein is a phosphoprotein. These properties may be related to its function as an RNA polymerase
Proviral Organization and Sequence Analysis of Feline Immunodeficiency Virus Isolated from a Pallas' Cat

AbstractThe nucleotide sequence and genomic organization have been determined for a highly cytopathic feline immunodeficiency virus (FIV) isolated from a Pallas' cat. The 9747-bp provirus of this virus, FIV-Oma, has typical lentivirus organization with LTRs,gag, pol,andenvopen reading frames (ORFs), putativevifandrevORFs, and an ORF similar to ORF2/ORFA of domestic cat FIV isolates. Although the FIV-Oma provirus is 300 to 600 bp longer than other FIV proviruses, these additional bases are distributed throughout the genome. Phylogenetic analysis of a conserved region of thepolgene suggests that FIV-Oma is more closely related to some of the puma and lion lentiviruses than it is to domestic cat FIV isolates; however, many regions of the genome exhibit extensive nucleotide sequence divergence. None of the eight molecular proviral clones isolated from a genomic library are infectious, but we have constructed an infectious, cytopathic clone of FIV-Oma from subcloned and PCR-amplified fragments of these proviral clones. This clone will be useful for identifying the genetic determinants of FIV-Oma's biological activities
Hepatitis C Virus Genomic Variability in Untreated and Immunosuppressed Patients

AbstractTo investigate whether immune pressure enhances the genetic diversity of the hepatitis C virus (HCV) hypervariable region 1, nucleotide sequences were compared in multiple sera, collected longitudinally, from three untreated patients and four patients undergoing liver transplantation for HCV-related cirrhosis. A minor variant became dominant in three of three patients following transplantation and persisted unchanged for months. Compared with untreated HCV carriers, transplant recipients had fewer quasispecies, fewer nucleotide changes (1.61 and 2.58/month), fewer amino acid sequence changes (0.40 and 1.94/month), as well as higher ratio of transitional to transversional mutations (2.57 and 0.98,P< 0.02) and lower replacement to silent mutations (1.33 and 8.21,P< 0.01). The two patients with the least genomic variation died of HCV graft infection. The data suggest that HCV variants which infect the graft are selected by recipient immune pressure at the time of transplant and that preferential replication in the graft is enhanced by routine immunosuppression
Interaction of Transcription Factors RFX1 and MIBP1 with the γ Motif of the Negative Regulatory Element of the Hepatitis B Virus Core Promoter

AbstractThe negative regulatory element (NRE) of the hepatitis B virus (HBV) core promoter contains three subregions which act synergistically to suppress core promoter activity. One of these subregions, NREγ, is active in both HeLa cervical carcinoma cells and Huh7 hepatoma cells and was found to be bound by a protein factor present in both cell types. Here we show that the transcription factor RFX1 can bind to NREγ and transactivate the core promoter through this site. Mutations which abrogated the gene-suppressive activity of NREγ prevented RFX1 from binding to NREγ. In addition, RFX1 can bind simultaneously, most likely as a heterodimer, with the transcription factor MIBP1 to NREγ. In the absence of a cloned MIBP1 gene for further studies, we hypothesize that RFX1 acts with MIBP1 to negatively regulate the core promoter activity through the NREγ site. The ability of RFX1 to transactivate the core promoter raises the possibility that RFX1 may play a dual role in regulating HBV gene expression
Autographa californicaNuclear Polyhedrosis Virusp143Gene Product Is a DNA-Binding Protein

AbstractWe have identified the protein product of theAutographa californicanuclear polyhedrosis virus (AcMNPV)p143gene by constructing a recombinant baculovirus overexpressing the gene product P143. The overexpressed protein exhibited a relative mobility of approximately 140 kDa and was stable for at least 12 hr after synthesis. Immunoblotting using a monoclonal antibody developed against the overexpressed protein identified a similar polypeptide in AcMNPV-infected cells which was detectable by 4 hr postinfection. P143 was present within infected cell nuclei at relatively constant amounts until at least 72 hr after infection, suggesting that P143 may perform other functions at late times after infection. P143, purified from infected cell nuclei by chromatography over hydroxylapatite and DNA cellulose, bound in a sequence-independent fashion to double-stranded but not to single-stranded DNA to form a ladder of retarded protein–DNA complexes. Together, these data are consistent with the essential role of P143 for viral DNA replication and suggest that P143 may function by direct binding to DNA
The Morphology of the Immature HIV-1 Virion

AbstractNewly released HIV-1 particles exhibit an immature morphology, previously reported to be characterized by a doughnut/ring-shaped structure. In this study we showed that among immature extracellular virus particles not only were particles with doughnut-shaped morphology present, but particles with a crescent morphology were also observed. These particles occurred with different frequencies, depending on whether they were in the cell or in cell-free fractions. The crescent-shaped particles were more abundant in the cell-free fractions, whereas the particles in the cell fraction mainly exhibited doughnut-shaped morphology. The crescent-shaped structure may represent an assembly intermediate
Identification of Mutations in a Sindbis Virus Variant Able to Establish Persistent Infection in BHK Cells: The Importance of a Mutation in the nsP2 Gene

AbstractSindbis virus is a positive strand RNA virus that has provided a valuable model for studying virus structure and replication. It is also being developed as a vector for the expression of heterologous proteins. Many studies with this virus are carried out in cultured BHK cells where infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weisset al.had established a persistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) particles and had isolated an attenuated virus, SIN-1 virus, from the culture [Weisset al.(1980)J. Virol.33, 463–474]. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cells. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5′ terminus and the four nonstructural protein genes from SIN-1 virus. A single coding mutation in the nsP2 gene (a predicted change of Pro-726 → Ser) produced a virus that was able to establish persistent infection in BHK cells. Additional mutations in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 virus. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, β-galactosidase, than that obtained with the original Sindbis virus replicon
Characterization of JKT-7400, an Orbivirus Which Grows inAedes albopictusMosquito Cells: Evidence Pointing to a Minor Virion Protein, VP6, as the RNA Guanylyltransferase

AbstractJKT-7400 virus, an orbivirus originally isolated fromCulexmosquitos, was plaque purified and adapted toAedes albopictusmosquito cells. Conditions which enhance viral cytopathic effect and optimize plaque formation are described. In contrast to bluetongue virus, the prototype orbivirus, no replication of JKT-7400 virus in vertebrate cells was observed. The core particle of JKT-7400 virus contains 10 segments of dsRNA and three minor proteins, VP1, VP4, and VP6. The inner shell contains two major proteins, VP2 and VP7, and the outer shell consists of the other two major proteins, VP3 and VP5. Evidence is presented suggesting that the viral protein associated with the capping of virus mRNA, i.e., the guanylyltransferase, is VP6, one of the core proteins
In VitroMutational and Inhibitory Analysis of thecis-Acting Translational Elements within the 5′ Untranslated Region of Coxsackievirus B3: Potential Targets for Antiviral Action of Antisense Oligomers

AbstractThe 5′ untranslated region (5′UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5′UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5′UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5′UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem–loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem–loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings,in vitrotranslation inhibition assays using RNA fragments of the 5′UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these twocis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5′UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. Thesecis-acting translational elements may serve as potential targets for the antiviral action of oligomers
Both Pre-S1 and S Domains of Hepatitis B Virus Envelope Proteins Interact with the Core Particle

AbstractThe three envelope proteins of the hepatitis B virus (HBV) are encoded by a single open reading frame in the genome containing three separate in-phase AUG codons. This organization defines three protein domains (pre-S1, pre-S2, S) which form the small (S), middle (M, pre-S2/S), and large (L, pre-S1/pre-S2/S) proteins. Mature virions are generated by the budding of preformed nucleocapsids through endoplasmic reticulum (ER) membranes containing S and L proteins, whereas the M protein is not necessary. This suggests an important function for the pre-S1 domain. To investigate the protein–protein interactions involved during the maturation process of the HBV virion, we studiedin vitrothe binding affinity to purified HBV core particles of various synthetic peptides identical to regions of the envelope proteins. Data previously obtained with deletion mutants were confirmed and refined. The 13 C-terminal amino acids of pre-S1 bound efficiently to core particles, whereas other pre-S domains did not. Moreover, the amino acid sequence 56–80 in the cytosolic loop of S bound efficiently to the HBV core. This double interaction between the HBV capside and both S and pre-S1 domains may be required for virion morphogenesis
Genome Amplification and Long-Distance Movement Functions Associated with the Central Domain of Tobacco Etch Potyvirus Helper Component–Proteinase

AbstractThe tobacco etch potyvirus (TEV) helper component–proteinase (HC–Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC–Pro coding sequence. The resulting mutants were analyzed with respect to HC–Pro proteolytic activityin vitro,genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC–Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC–Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC–Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC–Pro suppliedin transwas able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC–Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC–Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC–Pro functions involved in these processes is at least partiallytrans-active. Additionally, the long-distance movement properties of a previously characterized HC–Pro-defective mutant (TEV–GUS/CCCE) were characterized further using grafted nontransgenic and HC–Pro-expressing transgenic plants. The results indicated that HC–Pro is required in both inoculated and noninoculated tissues to complement the TEV–GUS/CCCE movement defects
Expression of Epstein–Barr Virus Latent Membrane Protein 1 Protects Jurkat T Cells from Apoptosis Induced by Serum Deprivation

AbstractIt has been generally accepted that inhibition of apoptosis is important in the development of malignancy. To determine whether Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1), the virus-coded transforming oncogene product, has an anti-apoptotic function in non-B-cells, Jurkat T cells were transfected with the LMP1-expression vector pSV2gptMTLM consisting of the human metallothionein promoter and were selected for mycophonolic acid resistance. LMP1-expressing clones of Jurkat cells showed resistance to apoptosis induced by serum deprivation. In LMP1-expressing clones, although the levels of Bcl-2 and Bax were similar to those in the clones of vector transfectants or parental cells, c-Myc expression was significantly depressed. Down-regulation of c-Myc by LMP1 was confirmed by using LMP1-expressing clones treated with CdCl2. Addition of c-mycantisense oligonucleotides to Jurkat cells specifically inhibited apoptosis induced by serum deprivation at the concentrations which suppressed c-Myc expression. These results suggest that LMP1 expression and subsequent down-regulation of c-Myc protect Jurkat T cells from apoptosis induced by serum deprivation. The significance of the anti-apoptotic function of LMP1 in non-B, Jurkat T cells is discussed in relation to the pathogenesis of EBV malignancy
Binding of the Ubiquitous Cellular Transcription Factors Sp1 and Sp3 to the ZI Domains in the Epstein–Barr Virus Lytic Switch BZLF1 Gene Promoter

AbstractInduction of the Epstein–Barr virus lytic cycle in latently infected B cells requires the expression of the immediate-early lytic gene BZLF1. We have previously identified severalcis-elements within the BZLF1 promoter that are required for induction by known inducers of the lytic cycle [E. Flemington and S.H. Speck (1990)J. Virol.64, 1217–1226]. These include four elements termed the ZI domains (ZIA, ZIB, ZIC, and ZID) that share extensive homology and that have recently been shown to bind several cellular transcription factors [A. M. Borras, J.L. Strominger, and S.H. Speck (1996)J. Virol.70, 3894–3901]. Here Sp1 and Sp3 are identified as the cellular factors present in crude B cell nuclear extract preparations that bind to the ZIC domain. In addition, three of the four complexes observed in electrophoretic mobility shift analyses employing probes containing either the ZIA or the ZID domains also represent Sp1 or Sp3 binding. Binding of Sp1 and Sp3 to the ZI domains was shown to be significantly weaker than binding of these factors to a consensus Sp1 site. A heterologous promoter construct containing three repeats of a consensus Sp1 site, cloned upstream of a single copy of the ZII (CREB/AP1) element from the BZLF1 promoter linked to the β-globin TATA box, exhibited phorbol ester inducibility. The latter observation was consistent with the functional behavior exhibited by a heterologous promoter construct containing multiple copies of the ZIC domain liked to the ZII element. However, the basal activity of the heterologous promoter construct driven by the consensus Sp1 sites was ca. 10-fold higher than that of the heterologous reporter construct containing multimerized ZIC sites. Thus, the low affinity of Sp1 binding to the ZI domains may contribute to the low-level basal activity of the BZLF1 promoter
Functional Analysis of the Transmembrane Anchor Region of Bovine Herpesvirus 1 Glycoprotein gB

AbstractIn herpesviruses, homologues of glycoprotein B (gB) are essential membrane proteins which are involved in fusion. However, there is no clear evidence regarding the location of the fusogenic domain on gB. By using bovine herpesvirus 1 (BHV-1) as a model, we studied the relationship between the structure and the fusogenic activity of gB. This was achieved by expressing genes of different gB derivatives containing specific truncations at the end of segments 2 or 3 of the transmembrane region in Madin–Darby bovine kidney cells under the control of the bovine heat-shock protein hsp70A gene promoter. All expressed gB products were structurally similar to authentic gB. One truncated form of gB, gBt, which contains residues 1–763, was efficiently secreted. However, gBtM (residues 1–807), which includes the first two segments at the carboxyl terminus, showed unstable retention on the cell surface, whereas gBtMA (residues 1–829), which contains all three membrane-spanning segments, was mostly intracellularly retained with some unstable surface anchorage. Another truncated gB, gBtDAF, which has gB residues 1–763 (gBt) and a human decay-accelerating factor (DAF) carboxyl tail, was also expressed. The DAF fragment provided a signal for the addition of a glycosyl phosphatidylinositol-based membrane anchor, which could target the gBt chimeric protein on the cell membrane. Immunofluorescence staining and pulse–chase kinetic studies support the theory that gBtM, gBtMA, and gBtDAF are retained on nuclear and cellular membranes via different segments of the transmembrane region or the DAF fragment, respectively. For the cells expressing gBt or gBtM, no cell fusion was observed, whereas cells expressing gBtMA clearly showed fusion. However, in gBtDAF cells, the overexpression and cellular accumulation of recombinant gB products did not cause fusion either, which supports our contention that the fusion phenomenon in gBtMA cells is caused by the fusogenic activity of the expressed gBtMA. With the help of sequence analysis, our results indicate that segment 2 of the transmembrane anchor region might be a fusogenic domain, whereas the real anchor is segment 3