pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterization of Human Immunodeficiency Virus Type 1 vif Gene in Long-Term Asymptomatic Individuals

AbstractWe have determined the sequence of the human immunodeficiency virus type 1 (HIV-1) vif genes from a cohort of 42 long-term nonprogressors (LTNP) and compared these sequences to those of 8 late progressors. The coding potential of the vif open reading frame directly derived by nested PCR from uncultured peripheral blood mononuclear cell DNA was conserved in all 50 individuals. The nucleotide distances between vif sequences were not significantly different between LTNP and late progressors, indicating similar selections of viruses within both types of long-term HIV-1-infected subjects. However, a statistically significant correlation between an amino acid signature at position 132 of Vif and the viral load was found within LTNP. Namely, amino acid Ser was associated with low viral load and amino acid Arg with high viral load. This signature was also observed when LTNP with low viral load were compared to progressors. The Ser132 signature was introduced in place of Arg132 present in the HIV-1 YU-2 Vif prototype into chimeric viruses to assess the impact of Vif signature on the virus. While the replication properties in the SupT1 cell line were unmodified, the mutagenized virus revealed a fivefold decreased replication in activated PBMC, suggesting a possible role of this Vif signature for viral production in vivo
Suppressor Mutations That Allow Sindbis Virus RNA Polymerase to Function with Nonaromatic Amino Acids at the N-Terminus: Evidence for Interaction between nsP1 and nsP4 in Minus-Strand RNA Synthesis

AbstractThe alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30°C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40°C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40°C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis
Mutations in the E2 Glycoprotein of Venezuelan Equine Encephalitis Virus Confer Heparan Sulfate Interaction, Low Morbidity, and Rapid Clearance from Blood of Mice

AbstractThe arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20–31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin–agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0–50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate
Functional Features of Hepatitis C Virus Glycoproteins for Pseudotype Virus Entry into Mammalian Cells

AbstractWe have previously reported the generation of pseudotype virus from chimeric gene constructs encoding the ectodomain of the E1 or E2 glycoprotein of hepatitis C virus (HCV) genotype 1a appended to the trans membrane domain and cytoplasmic tail of the vesicular stomatitis virus (VSV) G protein. Sera derived from chimpanzees immunized with homologous HCV glycoproteins neutralized pseudotype virus infectivity (L. M. Lagging et al., J. Virol. 72, 3539–3546, 1998). We have now extended this study to further understand the role of HCV glycoproteins in pseudotype virus entry. Although a number of mammalian epithelial cells were susceptible to VSV/HCV pseudotype virus infection, plaquing efficiency was different among host cell lines. Pseudotype virus adsorption at low temperature decreased plaque numbers. Treatment of E1 or E2 pseudotype virus in media between pH 5 and 8 before adsorption on cells did not significantly reduce plaque numbers. On the other hand, treatment of cells with lysosomotropic agents or inhibitors of vacuolar H+ ATPases had an inhibitory role on virus entry. Concanavalin A, a plant lectin, exhibited neutralization of both HCV E1 and E2 pseudotype virus infectivity. However, mannose binding protein, a C-type mammalian lectin, did not neutralize virus in the absence or presence of serum complement. Pseudotype virus infectivity was only partially inhibited by heparin, a highly sulfated glycosaminoglycan, in a saturable manner. Additional studies suggested that low-density lipoprotein receptor related molecules partially inhibit E1 pseudotype virus infectivity, while CD81 related molecules interfere with E2 pseudotype virus infectivity. A further understanding of HCV entry and strategies appropriate for mimicking cell surface molecules may help in the development of new therapeutic modalities against HCV infection
Satellite and Defective RNAs of Cryphonectria hypovirus 3-Grand Haven 2, a Virus Species in the Family Hypoviridae with a Single Open Reading Frame

AbstractCryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66–73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3′-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5′-terminal 1.4 kb derived from the 5′-end of dsRNA 1 and the 3′-terminal 2.2 kb from the 3′-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A)+. The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5′-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5′-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40–70 residues
The GroEL Protein of the Whitefly Bemisia tabaci Interacts with the Coat Protein of Transmissible and Nontransmissible Begomoviruses in the Yeast Two-Hybrid System

AbstractWe have previously suggested that a GroEL homolog produced by the whitefly Bemisia tabaci endosymbiotic bacteria interacts in the insect hemolymph with particles of Tomato yellow leaf curl virus from Israel (TYLCV-Is), ensuring the safe circulative transmission of the virus. We have now addressed the question of whether the nontransmissibility of Abutilon mosaic virus from Israel (AbMV-Is) is related to a lack of association between GroEL and the virus coat protein (CP). Translocation analysis has shown that, whereas TYLCV-Is DNA is conspicuous in the digestive tract, hemolymph, and salivary glands of B. tabaci 8 h after acquisition feeding started, AbMV-Is DNA was detected only in the insect digestive tract, even after 96 h. To determine whether AbMV-Is particles were rapidly degraded in the hemolymph as a result of their inability to interact with GroEL, we have isolated a GroEL gene from B. tabaci and used a yeast two-hybrid assay to compare binding of the CP of TYLCV-Is and AbMV-Is to the insect GroEL. The yeast assay showed that the CPs of the two viruses are able to bind efficiently to GroEL. We therefore suggest that, although GroEL-CP interaction in the hemolymph is a necessary condition for circulative transmission, the nontransmissibility of AbMV-Is is not the result of lack of binding to GroEL in the B. tabaci hemolymph, but most likely results from an inability to cross the gut/hemolymph barrier
Ecotropic Murine Leukemia Virus Receptor Is Physically Associated with Caveolin and Membrane Rafts

AbstractWe used a Sindbis virus expression system to stably express a chimeric ecotropic murine leukemia virus (MLV) receptor gene, CAT1, fused to green fluorescent protein (gfp) in BHK cells. The chimeric gene was expressed on the cell surface and functioned as an MLV receptor. Using gfp as an epitope tag, we found that CAT1 cross-immunoprecipitated with caveolin, a cellular protein associated non-clathrin-coated endocytic vesicles. Biochemical studies showed that CAT1 copurified with caveolin in a detergent-insoluble membrane fraction that forms cholesterol-rich “rafts” on the cell surface. Disruption of rafts by methyl-β-cyclodextrin, a drug that extracts cholesterol, reduced susceptibility to MLV without decreasing surface CAT1. The results indicate that association of the MLV receptor with cholesterol-rich rafts is important for an early step in virus infection
Assembly of Sendai Virus: M Protein Interacts with F and HN Proteins and with the Cytoplasmic Tail and Transmembrane Domain of F Protein

AbstractSendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain
Expression and Coreceptor Function of APJ for Primate Immunodeficiency Viruses

AbstractAPJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity
Two Amino Acid Substitutions in the SIV Nef Protein Mediate Associations with Distinct Cellular Kinases

AbstractA functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity
Inhibition of the Constitutive and Induced IFN-β Production by IL-4 and IL-10 in Murine Peritoneal Macrophages

AbstractWe had previously reported that freshly harvested peritoneal macrophages (PM) are in a type I IFN-mediated antiviral state, which is lost during in vitro culture of PM, concomitantly with a progressive decline in the expression of IFN-β. We report herein that in vitro culture of PM in the presence of IL-4 or IL-10 results in an enhanced decay of the IFN-β-mediated antiviral state to vesicular stomatitis virus (VSV). Moreover, IL-4 and IL-10 inhibited the production of type I IFN induced by LPS or NDV infection, as assessed by IFN production and induction of IFN-mediated antiviral state. The accumulation and physiological turnover of IFN-β mRNA was not affected by IL-4 or IL-10. Finally, neither IL-10 nor IL-4 exerted any inhibitory effect on the antiviral activity induced by exogenous type-I IFN. These results suggest that Th2 cytokines, such as IL-4 and IL-10, act as negative regulators of the type I IFN-mediated antiviral response in PM and may represent stop signals for the constitutive or induced type I IFN expression in PM
Pseudosubstrate Inhibition of Protein Kinase PKR by Swine Pox Virus C8L Gene Product

AbstractThe interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2α on Ser-51 to inhibit protein synthesis in virally infected cells. Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2α. We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells. In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells. The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2α and the C8L and K3L gene products. Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR
NSAID Treatment Suppresses VSV Propagation in Mouse CNS

AbstractCyclooxygenase (COX) is the key enzyme in the conversion of arachidonic acid to prostaglandins. COX has two isoforms: COX-1, the constitutively expressed form, and COX-2, the inducible form. Prostaglandins are mediators of many critical physiological and inflammatory responses, but little is known about their roles during a viral infection in the central nervous system (CNS). We used non-selective inhibitors of COX, aspirin and indomethacin, and a selective antagonist of COX-2, celecoxib, to study the role of prostaglandins in Vesicular Stomatitis Virus (VSV) induced encephalitis. We found that the inhibition of COX antagonizes VSV propagation both in vitro and in vivo. In addition, aspirin and celecoxib both prevented the disruption of the blood brain barrier in VSV-infected mice. In vitro experiments showed that the effect of COX inhibition was at least partially mediated by increased production of Nitric Oxide (NO), a molecule known to inhibit VSV replication. When NO production was inhibited by Nω-nitro-L-methyl-arginine-ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, the difference in viral titer between aspirin (or celecoxib)-treated and the control cells was abolished. VSV-infected mice treated with celecoxib expressed more NOS-1 and produced more NO in their CNS compared to the controls. Our data suggest that the product(s) of COX have antagonistic effect(s) on NO production in the mouse CNS
Hantavirus Pulmonary Syndrome in Panama: Identification of Novel Hantaviruses and Their Likely Reservoirs

AbstractHantavirus pulmonary syndrome (HPS), a severe respiratory disease with high mortality caused by rodent-borne hantaviruses, has previously been identified in the United States and Canada as well as central and southern South America. In late 1999 and early 2000, an outbreak of acute illness compatible with HPS was reported in Los Santos, Panama, with the death of 3 of the 12 (25%) suspected cases. Hantavirus-specific antibodies were detected in patient sera, and virus RNA was detected by reverse transcriptase-polymerase chain reaction. Sequence analysis of virus genome N-, G1-, and G2-encoding fragments showed this to be a novel hantavirus, Choclo virus. Serologic and virus genetic analyses of rodents trapped in the area showed Oligoryzomys fulvescens to be the likely reservoir for the HPS-associated Choclo virus. In addition, Zygodontomys brevicauda rodents were shown to harbor another genetically unique hantavirus, Calabazo virus
Evaluation of Recombinant Vaccinia Virus—Measles Vaccines in Infant Rhesus Macaques with Preexisting Measles Antibody

AbstractImmunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication-defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants