pinecone/core-2020-05-10-deduplication huggingface dataset:
Hepatitis C Virus Quasispecies in Patients Infected with HIV-1: Correlation with Extrahepatic Viral Replication

AbstractHepatitis C virus (HCV) sequences recovered from serum, peripheral blood mononuclear cells (PBMCs), and various tissues from human immunodeficiency virus type 1 (HIV-1) positive patients were compared by single strand conformational polymorphism (SSCP) and sequencing. In five patients, paired serum and PBMCs samples were analyzed while in two other patients multiple autopsy tissues were studied. Sequences amplified from the NS5 and E2 regions were consistently identical in the same patient; however, three PBMCs samples and three different tissue samples (pancreas and adrenal gland in one patient and lymph node in the other patient) contained 5′ untranslated region (5′UTR) sequences that were different from circulating sequences. The presence of 5′ UTR sequences differing from circulating sequences correlated with the presence of HCV RNA negative strand, as the latter was detected by a Tth-based strand-specific assay in all but one of these samples. These two independent lines of evidence: viral sequence differences and the presence of RNA negative strand in the same tissues strongly argue for the genuine presence of extrahepatic HCV replication, at least in the setting of HIV-1 infection
Molecular Characterization of Tomato Spotted Wilt Virus Defective Interfering RNAs and Detection of Truncated L Proteins

AbstractJunction sites of 25 different defective interfering (DI) RNAs of tomato spotted wilt virus (TSWV) were characterized. The DI RNAs varied in size from 2.0 to 5.2 kilobases (kb) and contained a single internal deletion. The absence of DI RNAs smaller than 2 kb suggested a size constraint for the survival of TSWV DI RNAs. This hypothesis was reinforced by the finding of a dimeric DI RNA formed by two 1.6-long monomers linked head to tail. Three types of junction sites were found, one type originating from a simple deletion; the second type contained a few extra nucleotides of unknown origin; and the third type contained a stretch of three to five nucleotides, originally occurring at both sides of the deletion and of which one was deleted. In 19 of the 25 DI RNAs studied, the original reading frame was maintained, suggesting a selective preference of DI RNAs with translational potency. Truncated proteins encoded by these DI RNAs were indeed detected in the nucleocapsid preparations. Folding studies of the complete L RNA revealed that the calculated minimal energy of folding was at 16°C lower than at 23°, indicating a higher stability of this molecule at low temperatures. The results suggest an involvement of locally folded secondary structures in the process of deletion, rather than the requirement of certain sequences around the deletion point. The DI RNA generation in TSWV is essentially, as discussed, similar to the process of RNA recombination described in many viruses
Control of Baculovirus Polyhedrin Gene Expression by Very Late Factor 1

Abstractvlf-1is a baculovirus gene that regulates very late gene expression (J. R. McLachlin and L. K. Miller,J. Virol.,68, 7746–7756, 1994) and also plays a crucial role in the replication of the budded form ofAutographa californicanuclear polyhedrosis virus (AcMNPV) (S. Yang and L. K. Miller, “Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.”Virology,245, 99–109, 1998). To examine the influence ofvlf-1expression on baculovirus infection, we constructed recombinant viruses that expressed only low levels of VLF-1 and recombinants withvlf-1under the control of different promoters. Viruses with mutant alleles ofvlf-1that produced low levels of VLF-1 replicated the budded form of the virus normally but produced no occlusion bodies. Thus, a higher concentration of VLF-1 was needed to activate very late gene expression than was needed to support budded virus production. By altering the level and/or timing ofvlf-1expression, the timing of polyhedrin gene (polh) expression, which normally occurs very late in infection, could be advanced or delayed. Early overexpression ofvlf-1increased the level of expression from thepolhpromoter but caused premature cellular disintegration. The data indicate that VLF-1 is the limiting factor in very late gene expression and that the level of VLF-1 controls the onset of occlusion
Inhibition of Influenza Virus Hemagglutinin-Mediated Membrane Fusion by a Compound Related to Podocarpic Acid

AbstractEntry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformation that is fusogenic. A compound related to podocarpic acid (180299) was identified that inhibits multicycle replication of influenza A/Kawasaki/86 (H1N1) virus in culture. Treatment of Madin–Darby canine kidney (MDCK) cells with 180299 at 1 h before infection resulted in the inhibition of viral protein synthesis. Addition of 20 μg of 180299/ml at 1 h p.i. had no effect, indicating that 180299 affects an early step of the influenza viral replication cycle. Genetic analysis of reassortants between sensitive and resistant viruses demonstrated that hemagglutinin (HA) conferred the 180299-resistant (180299r) phenotype. Twelve independent isolates of influenza A/Kawasaki/86 were selected for resistance to 180299, and sequence analysis revealed that each of these viruses contained amino acid substitutions in the HA. These mutations are dispersed throughout the HA primary amino acid sequence and cluster in one of two regions: the interface between HA1and HA2and in a region near the fusion domain of HA2. When compared with the parent virus, the pH-of-inactivation of the resistant mutants was increased by 0.3 to 0.6 pH unit, suggesting that the mutant HAs undergo the conformational change at an elevated pH. Fusion of human erythrocytes to MDCK cells infected with parent influenza A/Kawasaki/86 was inhibited by 180299 (0.1–10 μg/ml) in a concentration-dependent manner, whereas fusion of erythrocytes to MDCK cells infected with 180299rmutants was not affected. These results suggest that 180299 interacts with the neutral pH conformation of influenza A HA and prevents the low-pH-induced change of HA to its fusogenic conformation
Differences in the Biological Phenotype of Low-Yielding (L) and High-Yielding (H) Variants of Swine Influenza Virus A/NJ/11/76 Are Associated with Their Different Receptor-Binding Activity

AbstractLow- (L) and high-yielding (H) variants of A/sw/NJ/11/76 influenza virus were compared for their growth properties in embryonated chicken eggs and MDCK cells and for their binding affinity for the membrane fractions prepared from cells of the chicken embryo allantoic membrane, MDCK, and swine tracheal cells, as well as for soluble sialic acid containing macromolecules and monovalent sialosides. We have shown, that during infection in MDCK cells and in eggs, the progeny of the L variant remain predominantly cell associated, in contrast to those of H. As a result, accumulation of the L mutant in allantoic or culture fluid is significantly slowed in comparison with the H variant. Visualization of the infectious foci formed by the viruses in MDCK cell monolayers and on the allantoic membrane revealed that L spreads predominantly from cell to cell, while the spread of H involves release of the virus progeny into solution and its rapid distribution over the cell monolayer via convectional flow of the liquid. In the binding assays, L displayed significantly higher binding affinity than H for cellular membranes, gangliosides, and sialylglycoproteins, however, the affinity of the variants for the monovalent sialic acid compounds was comparable. Unlike H, L bound strongly to dextran sulfate. The data obtained suggest that all distinctions of the L and H biological phenotypes reported previously [Kilbourne, E. D., Taylor, A. H., Whitaker, C. W., Sahai, R., and Caton, A. (1988) Hemagglutinin polymorphism as the basis for low-and high-yield phenotypes of swine influenza virus.Proc. Natl. Acad. Sci. USA85, 7782–7785] could be rationally explained by a more avid binding of the L variant to the surface of target cells, and that this effect is mainly due to enhanced electrostatic interactions
The Role of Low pH and Disulfide Shuffling in the Entry and Fusion of Semliki Forest Virus and Sindbis Virus

AbstractSemliki Forest virus (SFV), an enveloped alphavirus, infects cells via a membrane fusion reaction that is induced by the low pH in endocytic vesicles. The role of low pH in the entry of the alphavirus Sindbis virus (SIN) is unclear, and an alternative fusion mechanism involving receptor-induced disulfide bond rearrangements at neutral pH has been proposed. The entry properties of SFV and SIN were here compared in parallel using treatment with the weak base NH4Cl or the vacuolar ATPase inhibitors bafilomycin A-1 or concanamycin to neutralize endosome pH. Three membrane impermeant thiol modifying reagents, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB),p-chloromercuriphenylsulfonic acid (pCMBS), and monobromotrimethylammoniobimane (Thiolyte MQ), were used to inhibit thiol–disulfide exchange reactions. Primary infection by both SFV and SIN was inhibited by neutralization of endosome pH using NH4Cl, bafilomycin, or concanamycin. The concentration of NH4Cl or bafilomycin required for inhibition correlated with the pH dependence of membrane fusion for SFV, SIN, and a pH-shift mutant of SFV. SFV and SIN infection were partially inhibited by the thiol blocker DTNB, but not by pCMBS or Thiolyte MQ. Our data suggest that acidic endosomal pH induces the fusion activity of both SFV and SIN during virus infection
The RGD Sequence in Phage ø29 Terminal Protein Is Required for Interaction with ø29 DNA Polymerase

AbstractThe RGD (Arg-Gly-Asp) motif functions as a recognition site for adhesive proteins responsible for a number of cell–cell interactions. Certain viruses use this sequence as a receptor-binding site by interaction with cellular integrins. To elucidate the role of the RGD sequence of the ø29 terminal protein (TP), seven modified TPs were generated by site-directed mutagenesis. Most of the TP mutants were not efficiently used as primers, leading to a reduction of the TP-dAMP complex formation in the presence of the ø29 TP-DNA template. Moreover, these mutant TPs were poorly deoxyadenylylated by ø29 DNA polymerase in the absence of template. Analysis of primer TP/DNA polymerase complex formation showed that the modified TPs were affected in the formation of the heterodimeric complex. These results indicate that the RGD sequence present in ø29 TP is primarily involved in interaction with the viral DNA polymerase
Structure of Phage fr Capsids with a Deletion in the FG Loop: Implications for Viral Assembly

AbstractThe loop between β-strands F and G in the coat protein of small RNA bacteriophages forms the interactions at the fivefold and threefold (quasi-sixfold) icosahedral axes. In many cases, mutations in this region renders the coat protein unable to form capsids. This FG loop has therefore been suggested to be of major importance for the virus assembly process by guiding the assembly and helping to define the correct curvature of the virus shell. We have determined the crystal structure of a phage fr capsid where the coat protein has a four-residue deletion in the FG loop. This mutant retains the ability to form virus capsids of normal size but has a significantly lower temperature stability than the wild type. The structure reveals that the mutated loops are flexible and too short to interact with each other. This seems incompatible with a role of the FG loop in the regulation of capsid size
Coat Protein Interactions Involved in Tobacco Mosaic Tobamovirus Cross-Protection

AbstractTo investigate the molecular role of the tobacco mosaic tobamovirus (TMV) coat protein (CP) in conferring cross-protection, a potato X potexvirus (PVX) vector (S. Chapman,Plant J.2, 549–557, 1992) was used to systemically express a set of TMV mutant CPs inNicotiana benthamianaprior to challenge inoculation with TMV. PVX-expressed wild-type TMV CP delayed TMV accumulation for up to 2 weeks compared to unprotected plants or plants preinfected with the unmodified PVX vector. Similar delays in TMV accumulation were obtained using TMV CPs that were deficient in virion formation but competent to assemble into helical aggregates. In contrast, TMV CPs that were incapable of helical aggregation or unable to bind viral RNA did not delay the accumulation of TMV. Furthermore, TMV CPs with enhanced intersubunit interactions that favor helical aggregation produced significantly greater delays in the accumulation of challenge TMV than obtained from the wild-type CP. Thus the capabilities of TMV CP to interact with viral RNA and self-associate in a helical fashion appear to be essential to its ability to confer protection. Taken together, these findings support a model for CP-mediated resistance in which the protecting CP recoats the challenge virus RNA as it disassembles
Effects of Site-Directed Mutations of Transmembrane Cysteines in Sindbis Virus E1 and E2 Glycoproteins on Palmitylation and Virus Replication

AbstractThe two glycoproteins that form the external spikes of the alphaviruses are type 1 membrane proteins whose transmembrane domains of hydrophobic amino acids are close to the carboxyl termini of the polypeptides and anchor the proteins in the lipid bilayer. Most of the members of the alphavirus genus contain within this transmembrane sequence one or more highly conserved cysteines, which are positioned close to the cytoplasmic face of the lipid bilayer. Cysteines in the cytoplasmic domains of the alphavirus glycoproteins and other enveloped viruses have been shown to be modified by palmitylation. To determine whether the transmembranal cysteines in Sindbis virus also were palmitylated, we used site-directed mutation to change the single transmembranal cysteine in the E1 glycoprotein and two of the transmembranal cysteines in the E2 glycoprotein to alanines. Transfection of RNA transcribed from the differently mutated Sindbis virus cDNAs led to production of infectious virus. Cells infected with the mutant virions and labeled with [3H]-palmitic acid showed that the E1 mutant no longer contained fatty acid in the E1 glycoprotein and that the extent of palmitylation was reduced about twofold in the E2 glycoprotein of virions containing the E2 mutations. At early times postinfection, the mutants grew slightly slower than the wild type in cultures of chicken embryo fibroblasts and secreted about half the amount of virus particles as wild type, but little difference was found at later time points. A triple mutant containing both the E1 and E2 mutations formed virions deficient in palmitylation of both glycoproteins, and this mutant had growth properties that were similar to those of the independent E1 and E2 mutants. Virions with the mutated glycoproteins that were deficient in fatty acid were more susceptible than the wild-type virions to inactivation by the detergent Triton X-100
Interactions among HIV gp120, CD4, and CXCR4: Dependence on CD4 Expression Level, gp120 Viral Origin, Conservation of the gp120 COOH- and NH2-Termini and V1/V2 and V3 Loops, and Sensitivity to Neutralizing Antibodies

AbstractThe binding of HIV-derived recombinant soluble (s)gp120 to the CD4+/CXCR4+A3.01 T cell line inhibits the binding of the CXCR4-specific monoclonal antibodies 12G5, which interacts with the second extracellular loop, and 6H8, which binds the NH2terminus. We have used this as an assay to analyse the interaction of recombinant sgp120 from diverse viral origins with CXCR4. The strength of the interaction between sgp120 and CXCR4 correlated with sgp120 affinity for the CD4–CXCR4 complex, and the interaction of sgp120MNand sgp120IIIBwith CXCR4 was highly dependent on the level of CD4 expressed on a variety of different T cell lines. sgp120 from X4, R5X4, and R5 viruses interacted with CXCR4, although the R5 sgp120–CXCR4 interactions were weaker than those of the other gp120s. The interaction of sgp120IIIBor sgp120MNwith CXCR4 was inhibited by neutralizing monoclonal antibodies that prevent the sgp120-CD4 interaction but also by antibodies specific for the gp120 V2 and V3 loops, the CD4-induced epitope and the 2G12 epitope, which interfere weakly or not at all with CD4-sgp120 binding. The binding to A3.01 cells of wild-type sgp120HxB2, but not of sgp120 deleted in the COOH and NH2termini, interfered with 12G5 binding in a dose-dependent manner. Further deletion of the V1 and V2 loops restored CXCR4 binding activity, but additional removal of the V3 loop eliminated the gp120-CXCR4 interaction, without decreasing the affinity between mutated sgp120 and CD4. Taken together, these results demonstrate that the interactions between sgp120 and CXCR4 are globally similar to those previously observed between sgp120 and CCR5, with some apparent differences in the strength of the sgp120-CXCR4 interactions and their dependence on CD4
The Amino Terminus of Human CCR5 Is Required for Its Function as a Receptor for Diverse Human and Simian Immunodeficiency Virus Envelope Glycoproteins

AbstractThe chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins
The Matrix Protein of HIV-1 Is Not Sufficient for Assembly and Release of Virus-like Particles

AbstractThe matrix (MA) proteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are known to be important for the targeting and assembly of lentiviral proteins. The objective of the present study was to determine whether the MA protein of HIV-1 was sufficient for particle assembly and release. Eukaryotic expression of wild-type HIV-1 Gag-Pol, HIV-1 MA alone, or SIV MA alone was analyzed with radio-immunoprecipitation, density centrifugation, and a protease protection assay. Cells that expressed HIV-1 Gag-Pol or SIV MA alone released virus-like particles (VLPs) with sucrose gradient densities of 1.15 or 1.12 g/ml, respectively. The MA and/or capsid proteins in these particles were protected from protease degradation, indicating the presence of a protective outer membrane. Expression of HIV-1 MA protein alone resulted in release of MA which pelleted through a 20% sucrose cushion but failed to enter a 20–60% sucrose gradient and was not protected from protease degradation. The MA protein of SIV was previously reported to be sufficient for production of VLPs (S. A. Gonzalez, H, K, Affrachino, H. R. Gelderblom, and A. Burney.Virology194, 548–556, 1993; V. Liska, D. Spehner, M. Mehtali, D. Schmitt, A. Kirn, and A. M. Aubertin.J. Gen. Virol.75, 2955–2962, 1994). Our study confirmed that result, but indicated that the MA protein of HIV-1 was not sufficient to assemble and release VLPs
Human Papillomavirus Type 31b E1 and E2 Transcript Expression Correlates with Vegetative Viral Genome Amplification

AbstractHuman papillomavirus (HPV) genome replication is dependent on the expression of E1 and E2 proteins. The organotypic (raft) culture system was used to investigate changes in viral early gene expression and vegetative genome replication during the complete life cycle of HPV type 31b (HPV31b). We have previously shown the synthesis of HPV31b viral particles as early as 10 days of growth of CIN-612 9E raft tissues (Ozbun, M. A., and Meyers, C. (1997).J. Virol.71, 5161–5172). In the present study, we investigated the structures and temporal expression levels of HPV31b E1 and E2 transcripts, as well as the replication of the viral genome during the viral life cycle. The amplification state of the HPV31b genome was maximal at 10 days of raft tissue growth. Furthermore, the expression levels of E1 and E2 RNAs correlated with vegetative viral DNA replication. Levels of E1- and E2-specific transcripts were dissimilar throughout the viral life cycle. E2 RNA levels remained relatively constant, whereas E1 RNA levels were upregulated during the maximal amplification of viral genomes and the biosynthesis of virions. These data indicate that E1 may be the major regulator of viral genome amplification in preparation for DNA packaging and virion morphogenesis
Acylation of the Influenza Hemagglutinin Modulates Fusion Activity

The influenza virus hemagglutinin (HA) contains three highly conserved cysteine residues at positions 551, 559, and 562 close to the carboxyl-terminus of the HA2 subunit which serve as palmitylation sites. Wild-type HA of influenza virus A/FPV/Rostock/34 (H7N1) and HA permutated by exchange of the acylated cysteine to serine residues were expressed in CV-1 cells by a SV40 vector system. Since density of immunostained HA on the cell surface measured by flow cytometric analysis did not differ between wild-type and acylation mutants, it was possible to compare acylation mutants and wild-type HA for their capacity to induce membrane fusion at low pH. The following observations were made: (1) lateral diffusion of a lipid-like fluorophore (R-18) from the erythrocyte membrane to the plasma membrane of cells expressing HA on the surface occurred equally well with mutants and wild type. (2) Diffusion of a low-molecular-weight fluorescent water-soluble probe (calcein) from erythrocytes into the cytoplasm of HA-expressing cells was not altered either. (3) However, depending on the position and the number of the deleted acylation sites, the mutants showed a reduced ability to induce syncytia. The data indicate that deacylation of the cytoplasmic tail has no measurable effect on the capacity of HA to induce membrane fusion and pore formation but that it suppresses syncytia formation