pinecone/core-2020-05-10-deduplication huggingface dataset:
Catalytic Efficiency and Phenotype of HIV-1 Proteases Encoding Single Critical Resistance Substitutions

AbstractWe have shown that a bacteriophage lambda genetic screen system may be useful in predicting the activity and phenotype of HIV-1 protease in the course of viral infection and antiretroviral therapy. This simple and rapid genetic screening system has been used here to characterize HIV-1 proteases encoding single primary resistance substitutions. Except for proteases with amino acid changes at positions 46 and 84, proteases containing single-resistance substitutions displayed a lower catalytic efficiency than the WT enzyme. Single mutants could be identified by their efficiency, demonstrating that modest differences in protease activity can be monitored with this simple assay. Overall, drug susceptibility could be reduced by introduction of single mutations. However, high-level protease inhibitor (PI) resistance was only achieved by multiple mutated proteases. The small but reproducible increase in resistance displayed by single mutants also demonstrated the ability of this genetic screen system for detecting minor reductions in drug susceptibility. These results show that the bacteriophage lambda genetic screen system used here is a useful tool in the analysis of specific contribution of mutations in the HIV protease-coding region or in specific cleavage sites that affect the process of PI resistance
Role of Nucleotide Sequences in the V3 Region in Efficient Replication of CCR5-Utilizing Human Immunodeficiency Virus Type 1 in Macrophages

AbstractMacrophages express both CXCR4 and CCR5 coreceptors, but restrict X4 HIV-1 replication unless the Env-V3 region, a major determinant of cell tropism, is exchanged with that of R5 HIV-1. As the V3 exchange concomitantly alters the nucleotide sequences, we introduced silent mutations in the V3 or C2 region of macrophage-tropic R5 JRFL without changing the amino acids. Immunoblot analysis confirmed that viral proteins including Env-gp120 were similarly incorporated in wild-type (wt) and mutant virions. The silent mutants infected CCR5-positive MAGIC5 cells but not CCR5-negative MAGI cells, as productively as wt viruses, indicating that the silent mutations did not alter coreceptor utilization. In contrast, two of three silent V3-mutant viruses failed to replicate efficiently in primary macrophages, whereas other V3- or C2-mutants and wt JRFL infected macrophages productively. Furthermore, synthesis of the full-length viral DNA of the aberant V3-mutant was largely reduced in macrophages. These results suggest that V3 nucleotide sequences may be one of the postentry factors restricting HIV-1 replication in macrophages
Construction of a Novel JCV/SV40 Hybrid Virus (JCSV) Reveals a Role for the JCV Capsid in Viral Tropism

AbstractJC virus (JCV) is a common human polyomavirus that infects greater than 70% of the general population worldwide. JCV is also the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the CNS. Currently, little is known about the mechanisms that restrict JCV tropism to a few human cell types and tissues. In vivo, JCV can be detected in oligodendrocytes and astrocytes in the CNS of patients with PML. The virus can also be detected in kidney, tonsil, and B lymphocytes of patients both with and without PML. In vitro, JCV can only be propagated in cultures of human fetal glial cells or in cell lines derived from this tissue. In contrast, the closely related monkey polyomavirus, SV40, has a broad tropism for primate cells, including those cells that are also susceptible to infection by JCV. We hypothesized that one potential block to infection is at the level of virus entry. To examine this, we constructed a JCV-SV40 chimeric viral genome that contains the regulatory region and the early genes of SV40 and the late structural genes of JCV. The hybrid virus (JCSV) induced SV40-like cytopathic effect in human glial cells and hemagglutinated human type O red blood cells similar to JCV. More importantly, the hybrid virus maintained the host range of JCV, suggesting that interactions between the virus capsid and host cell receptors contribute to JCV tropism
Upregulation of the Golgi Protein GP73 by Adenovirus Infection Requires the E1A CtBP Interaction Domain

AbstractGP73 is a novel type II Golgi transmembrane protein that is expressed at high levels in the hepatocytes of patients with viral hepatitis (R. D. Kladney, G. A. Bulla, L. Guo, A. L. Mason, A. E. Tollefson, D. J. Simon, Z. Koutoubi, and C. J. Fimmel, 2000, Gene 249, 53–65) and is induced in cultured cells by infection with viruses including adenoviruses. Its biological function and the mechanisms by which its expression may be regulated by viral infection are unknown. Here we report that GP73 is induced at the RNA and protein level in human Hep3B hepatoma cells infected by human Ad5 and Ad2. Hep3B cells were infected with wild-type or mutant adenoviruses. GP73 expression was measured by RNase protection assay, immunoblotting, or immunofluorescence microscopy. GP73 RNA and protein levels were strikingly induced following infection. The rise in GP73 expression coincided with the appearance of the adenovirus E1A and DBP proteins and preceded the expression of the fiber protein, a marker of the late phase of infection. Infection did not affect the expression of giantin, GPP130, or golgin-84, three integral Golgi membrane proteins with structural similarities to GP73. Mapping studies using a panel of mutant adenoviruses demonstrated that the E1A C-terminus, specifically its CtBP interaction domain (CID), is required for GP73 expression. Subsequently, Hep3B cells were transiently transfected with plasmids expressing wild-type or mutant E1A proteins. These studies confirmed that E1A induced GP73 expression via the CID. Our studies establish GP73 as a novel adenovirus-induced cellular protein whose expression is regulated through the CID of the E1A protein
Phosphorylation of Hepatitis C Virus Core Protein by Protein Kinase A and Protein Kinase C

AbstractHepatitis C virus (HCV) core protein can form capsid-like particles and is believed to be the viral capsid protein. Besides its structural functions, this protein is also known to possess multiple regulatory functions. In this article, we have studied the possible phosphorylation of HCV core protein in two different human liver-derived cell lines Huh7 and HepG2. Our results indicated that the HCV core protein could be phosphorylated, albeit inefficiently, independent of its downstream E1 protein in these two cell lines. Two of the basal phosphorylation sites were identified to be serine-53 and serine-116. The phosphorylation of the core protein could be enhanced by the PKC activator phorbol 12-myristic 13-acetate (PMA), and the PKA activator forskolin, and these enhancements could be abolished by the respective inhibitors of PKC and PKA, indicating that the core protein is a substrate of these two kinases. While both serine-53 and serine-116 served as the PKC phosphorylation sites, serine-116 appeared to be the major PKA phosphorylation site. Further analyses using serine-to-alanine mutation to mimic dephosphorylation and serine-to-aspartic acid mutation to mimic phosphorylation revealed that the conversion of serine-116 to aspartic acid led to an enhanced nuclear localization of the core protein. This observation indicates that one function of phosphorylation may be to regulate the nuclear localization of the core protein
Transcriptional Analysis and Preliminary Characterization of ORF Bm42 from Bombyx mori Nucleopolyhedrovirus

AbstractClusters of closely spaced genes are frequently present in baculovirus genomes. We report here the detailed transcriptional mapping of one such gene cluster from Bombyx mori nucleopolyhedrovirus (BmNPV) comprising five ORFs, viz., Bm42, Lef-10, VP1054, Bm44, and Bm45, which are closely spaced and transcribed in the same orientation. This region is homologous to ORFs 53, 53a, 54, 55, and 56, respectively, on the AcMNPV genome. Multiple, overlapping sets of polycistronic transcripts from this region, that initiate independently but terminate at a common 3′ end, were detected following BmNPV infection. Expression of these genes followed a temporal pattern where the shorter transcripts initiating downstream appeared early, and with the progress of infection the longer transcripts initiated from upstream. The first ORF in this cluster, Bm42, is conserved in almost all baculoviruses, but has not been characterized so far. The protein was localized in the cytosol, predominantly near the nucleus-cytoplasm boundary in BmNPV-infected BmN cells. Bm42 was present in the budded virions and was not associated with occluded virus particles or the polyhedral matrix
The Hepatitis B Virus X Protein Binds to and Activates the NH2-Terminal trans-Activation Domain of Nuclear Factor of Activated T Cells-1

AbstractWe have previously reported that the hepatitis B virus X protein (HBx) activates nuclear factor of activated T cells (NF-AT), a key regulator of the immune system, by a calcium/calcineurin-dependent pathway, involving dephosphorylation and nuclear translocation of this transcription factor. In addition, we showed that HBx synergizes with potent calcium-mobilizing stimuli to activate NF-AT-dependent transcription, suggesting that additional mechanisms might also be operative in the activation of NF-AT by HBx. Here we demonstrate that HBx activates the NH2-terminal transcription activation domain (TAD) of NF-AT1 by a mechanism involving protein–protein interaction. Targeting of HBx to the nucleus did not affect its ability to induce the transcriptional activity of NF-AT1. In contrast, mutations of HBx affecting its functional interaction with general transcription factors abrogated the HBx-induced activity of NF-AT1. Together these results indicate that HBx may exert its function by acting as a nuclear coactivator of NF-AT1
Expression of HCV Structural Proteins Impairs IFN-Mediated Antiviral Response

AbstractHepatitis C virus (HCV), especially the genotype 1, is naturally resistant to the antiviral effects of interferon-alpha (IFN-α). Expression of the whole HCV genome and the NS5A protein has been suggested to interfere with the antiviral activity of IFN-α. Here we have analyzed the effect of individual or various combinations of HCV proteins on IFN-α-mediated antiviral effect against vesicular stomatitis virus (VSV). When the structural proteins (core-E1-E2) of HCV genotype 1 were expressed in human osteosarcoma cells in a tetracycline-regulated manner, partial VSV resistance to IFN-α was established. This was seen as an enhancement of both viral protein synthesis and production of infectious virus. Priming of core-E1-E2-expressing cells with low doses of IFN-γ (10 IU/ml) partially restored the antiviral activity of IFN-α. The core (high-level expression) and NS4B protein expression also showed some rescue of VSV replication. In this model cell system NS3A–NS4A complex and NS5A showed no inhibition of IFN-α-induced antiviral activity. Our results indicate that the expression of structural proteins of HCV may impair the antiviral activity of IFNs
Genetic Basis of Attenuation of Dengue Virus Type 4 Small Plaque Mutants with Restricted Replication in Suckling Mice and in SCID Mice Transplanted with Human Liver Cells

AbstractMutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. Dengue virus type 4 (DEN4) was previously grown in Vero cells in the presence of 5-fluorouracil, and the characterization of 1248 mutagenized, Vero cell passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain (J. E. Blaney, Jr., D. H. Johnson, C. Y. Firestone, C. T. Hanson, B. R. Murphy, and S. S. Whitehead, 2001, J. Virol. 75(20), 9731–9740). The present investigation has extended these studies by identifying an additional 22 DEN4 mutant viruses which have a small plaque size (sp) phenotype in Vero cells and/or the liver cell line, HuH-7. Five mutant viruses have a sp phenotype in both Vero and HuH-7 cells, three of which are also ts. Seventeen mutant viruses have a sp phenotype in only HuH-7 cells, 13 of which are also ts. Each of the sp viruses was growth restricted in the suckling mouse brain, exhibiting a wide range of reduction in replication (9- to 100,000-fold). Complete nucleotide sequence was determined for the 22 DEN4 sp mutant viruses, and nucleotide substitutions were found in the 3′-untranslated region (UTR) as well as in all coding regions except NS4A. Identical mutations have been identified in multiple virus clones, suggesting that they may be involved in the adaptation of DEN4 virus to efficient growth in Vero cells. Six of the 22 sp 5-FU mutant viruses lacked coding mutations in the structural genes, and 17 recombinant DEN4 viruses were generated which separately encoded each of the mutations observed in these six sp viruses. Analysis of the recombinant DEN4 viruses defined the genetic basis of the sp, ts, and att phenotypes observed in the six sp viruses. Mutations in NS1, NS3, and the 3′-UTR were found to confer a greater than 100-fold, 10,000-fold, and 1000-fold reduction in replication of rDEN4 virus in SCID mice transplanted with HuH-7 cells, respectively, which serves as a novel small animal model for DEN4 infection
HIV-1 Gag Polyprotein Rescues HLA-DR Intracellular Transport in a Human CD4+ Cell Line

AbstractMajor histocompatibility complex class II HLA-DR molecules are plasma-membrane integral heterodimers, constitutively expressed in antigen-presenting cells. Their expression is known to be upregulated in peripheral T lymphocytes upon cell activation and to be constitutive in T cell lines. In H78-C10.0, a subclone of the human CD4+ T cell line HUT-78, the transport of MHC class II HLA-DR molecules is blocked, resulting in their localization within internal vesicular compartments rather than at the cell surface. In this article, we show that HIV-1HX10 infection of H78-C10.0 cells induces HLA-DR surface expression. Moreover, the produced infectious viruses harbor the heterodimer molecules in their envelopes. To define which of the viral proteins was involved in this phenomenon, we infected H78-C10.0 cells with recombinant vaccinia vectors containing either the gag-pro coding sequence or the entire env gene. Only gag expression was able to induce HLA-DR cell-surface localization in H78-C10.0 cells. RT-PCR analysis of the infected cells revealed no significant alteration in the amount of HLA-DRα-specific RNA compared to untreated cells. This implies that Gag acts on downstream events. When the env viral gene, coding for the precursor glycoprotein gp160, was expressed in H78-C10.0, the Env protein targeted to the cell surface was poorly processed to its final mature forms gp120 and gp41. However, coexpression of the env and gag genes led to restoration of this phenotype. Although the mechanism is unknown, the data compiled in this study strongly suggest that the viral Gag protein can interact with the cellular trafficking apparatus. Moreover, in a specific cell type as H78-C10.0 this interaction can even reverse intracellular transport defects
Interaction of HIV-1 Gag and Membranes in a Cell-Free System

AbstractA coupled transcription-translation (TNT) reticulocyte lysate system was used to examine posttranslational alterations in HIV-1 Gag upon addition of Jurkat T cell membranes. Incubation of the Gag precursor protein, Pr55gag, with membranes resulted in a time-dependent alteration in Gag resulting in partial resistance to trypsin treatment. Treatment of membranes and TNT extract with apyrase or pretreatment of membranes with trypsin prevented this posttranslational alteration of Gag. In contrast, this activity was not disrupted by pretreatment of membranes with Triton X-100 at 4°C, under conditions which do not solubilize raft-associated proteins. Flotation studies revealed that acquisition of trypsin-resistance was accompanied by Gag binding to membranes. The myristylation signal and nucleocapsid domain were found to mediate Gag binding to membranes. The posttranslational alteration of Gag accompanying membrane interaction may represent a conformational change, oligomerization, and/or association with or envelopment by membranes. These findings provide new clues to the stepwise process of HIV-1 assembly
Detection of Antibodies against the Four Subtypes of Ebola Virus in Sera from Any Species Using a Novel Antibody-Phage Indicator Assay

AbstractThe natural host for Ebola virus, presumed to be an animal, has not yet been identified despite an extensive search following several major outbreaks in Africa. A straightforward approach used to determine animal contact with Ebola virus is by assessing the presence of specific antibodies in serum. This approach however has been made very difficult by the absence of specific reagents required for the detection of antibodies from the majority of wild animal species. In this study, we isolated a human monoclonal antibody Fab fragment, KZ51, that reacts with an immunodominant epitope on Ebola virus nucleoprotein (NP) that is conserved on all four Ebola virus subtypes. The antibody KZ51 represents a major specificity as sera from all convalescent patients tested (10/10) and sera from guinea pigs infected with each of the four Ebola virus subtypes competed strongly with KZ51 for binding to radiation-inactivated Ebola virus. These features allowed us to develop a novel assay for the detection of seroconversion irrespective of Ebola virus subtype or animal species. In this assay, the binding of KZ51 Fab-phage particles is used as an indicator assay and the presence of specific antibodies against Ebola virus in sera is indicated by binding competition. A prominent feature of the assay is that the Fab-phage particles may be prestained with a dye so that detection of binding can be directly determined by visual inspection. The assay is designed to be both simple and economical to enable its use in the field
Azathioprine Inhibits Vaccinia Virus Replication in Both BSC-40 and Rag Cell Lines Acting on Different Stages of Virus Cycle

AbstractIn the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage
Evidence for a Conserved Polydnavirus Gene Family: Ichnovirus Homologs of the CsIV Repeat Element Genes

AbstractIn Campoletis sonorensis Ichnovirus (CsIV), the repeat element genes constitute a gene family of 28 members. In the present work, we document the presence of members of this gene family in two additional ichnoviruses, Hyposoter didymator Ichnovirus (HdIV) and Tranosema rostrale Ichnovirus (TrIV). Two repeat element genes, representing at least one functional gene, were identified in TrIV, whereas HdIV was found to contain at least three such genes. In both HdIV and TrIV, the known repeat element genes are encoded on single genome segments, with hybridization studies suggesting the presence of other, related but as yet uncharacterized genes. The HdIV and TrIV repeat element genes are all transcribed in infected caterpillars, although differences exist among genes in levels and in tissue specificity of expression. A heuristic tree was generated indicating that the repeat element genes are more similar within a species of wasp than between species, with TrIV genes being more closely related to the CsIV than to the HdIV genes. These results suggest that the most significant duplication, divergence, and expansion of the repeat element genes occurred after speciation. The finding that repeat element genes form an interspecific family within the genus Ichnovirus supports the view that the proteins they encode play an important role in ichnovirus biology
Transcriptional Analysis of the DNA Polymerase Gene of Shrimp White Spot Syndrome Virus

AbstractThe white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5′-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2–4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized
Compartmentalization of HIV-1 between Breast Milk and Blood of HIV-Infected Mothers

AbstractHIV-1 variants in breast milk and peripheral blood have been compared in three HIV-1 infected mothers. Analysis of DNA and RNA env C2-V3 sequences showed a differential distribution of HIV variants between the two compartments. The major provirus variant found in breast milk corresponds to a minor variant in the blood of two mothers. In the third mother, the predominant proviral variant detected in breast milk was not represented in the HIV-1 blood population. The major RNA variant in breast milk was not represented in the blood of two mothers. The predominant RNA variant in breast milk and blood was however the same for the third mother. Unexpectedly, the pattern of free virus variants in breast milk of three mothers did not correspond to that of the proviral form, suggesting that free viruses do not derive from infected cells in breast milk. The observation of a compartmentalization of HIV-1 between peripheral blood and breast milk emphasizes that postnatal transmission of HIV occurs with variants that may not be predicted from the analysis of circulating viral populations