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At least 811 records · Page 45Linked to original sources
PubMed1967

Primary virus-cell interactions in the immunofluorescence assay of Venezuelan equine encephalomyelitis virus.

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.

Animals↗
PubMed1982

Accurate transcription of simian virus 40 chromatin in a HeLa cell extract.

During simian virus 40 viral maturation, a series of modifications occur which alter the composition of viral nucleoprotein complexes. As a consequence, the chromatin that is extracted from extracellular simian virus 40 virions exhibits properties that are similar to those of transcriptionally active eucaryotic chromatin. The influence of this chromatin structure on specific RNA initiation by RNA polymerase II was examined by using the in vitro HeLa cell extract of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980). The 5' ends of RNA transcripts were positioned by the "run-off" assay, in which transcripts extend from the initiation site to termination sites created by restriction cleavage and by S1 nuclease analysis, using DNA probes labeled at their 5' termini. Two major early RNA transcripts, which originated at map positions 5,240 +/- 10 and 5,145 +/- 10, and two major late RNA transcripts, which originated at map positions 325 +/- 10 and 185 +/- 10, were identified. Transcripts were initiated with comparable relative efficiencies at the same 5' site when either purified DNA or chromatin was used as the template. Our results suggest that extracellular simian virus 40 virion chromatin modifications do not regulate simian virus 40 promoter selection but function to increase the accessibility of RNA promoter sequences to RNA polymerase II and allow efficient elongation of the RNA chain after the initiation event.

Chromatin↗
PubMed1995

Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.

Amino Acid Sequence↗
PubMed1995

Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract.

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and J. G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this site is due to a stem-loop structure in the RNA template, formed by interaction of a number of bases in the polypurine tract, including the six G's, and a 3' sequence which includes four C's. Addition of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein to reverse transcriptase reactions reduces pausing by approximately 8- to 10-fold and stimulates synthesis of full-length DNA. Thus, NC functions as an accessory protein during elongation of minus-strand DNA and increases the efficiency of DNA synthesis, in this case, by apparently destabilizing a region of secondary structure in the template. Since NC is associated with genomic RNA in the viral core and is likely to be part of a viral replication complex, these results suggest that NC may also promote efficient DNA synthesis during virus replication. Mutational analysis indicates that the features of HIV-1 NC which are important for reduction of pausing include the basic amino acids flanking the first zinc finger, the zinc fingers, and the cysteine and aromatic amino acids within the fingers. These findings suggest that reverse transcription might be targeted by drugs which inactivate the zinc fingers of HIV-1 NC.

Amino Acid Sequence↗
PubMed1996

The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a nuclear export signal.

The Rex protein of human T-cell leukemia virus type 1 is required for the nuclear export of unspliced viral mRNA and, therefore, for virus replication. In this manuscript, we demonstrate that Rex shuttles between the nucleus and the cytoplasm and that its activation domain constitutes a nuclear export signal that specifies efficient transport to the cytoplasm. These findings are consistent with a model for Rex-mediated trans-activation in which Rex-viral mRNA complexes are targeted for nuclear export by the direct action of the activation domain.

Amino Acid Sequence↗
PubMed1997

Virus-specific cytotoxic T-lymphocyte activity elicited by coimmunization with human immunodeficiency virus type 1 genes regulated by the bacteriophage T7 promoter and T7 RNA polymerase protein.

Cytotoxic T lymphocyte (CTL) activity was assessed in mice immunized with DNA plasmids containing the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (pTMIgp120) or p55gag (pTMIgag) gene regulated by the bacteriophage T7 promoter. Immunization with either plasmid resulted in CTL activity against class I major histocompatibility complex-restricted viral epitopes when coadministered with a recombinant vaccinia virus expressing the T7 RNA polymerase protein (T7 RNAP) but not a control vaccinia virus. Recombinant vaccinia-T7 RNAP virus (VTF7-3) could be replaced with a noninfectious source of T7 RNAP. A three-component vaccine consisting of pTMIgag, a recombinant subunit T7 RNAP protein, and a plasmid (pT7T7) encoding T7 RNAP under the control of its own promoter induced gag-specific CTL activity. Intramuscular immunization with the pTMIgag plasmid delivered with either the T7 RNAP protein or pT7T7 plasmid alone also induced HIV-1-specific CTL. Thus, there is adventitious expression of the pT7T7 plasmid in vivo, and enough T7 RNAP is produced to result in production of p24gag protein from the pTMIgag plasmid. The results demonstrate that regulated expression of genes in vivo is possible with this T7-based expression system, and may be useful in vaccine settings where short-term cytoplasmic expression of protein in antigen presenting cells is desired.

AIDS Vaccines↗
PubMed1999

Mosaic structure of the human immunodeficiency virus type 1 genome infecting lymphoid cells and the brain: evidence for frequent in vivo recombination events in the evolution of regional populations.

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17(gag), pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17(gag) and V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Phosphorylation of human immunodeficiency virus type 1 Vpr regulates cell cycle arrest.

Human immunodeficiency virus type 1 (HIV-1) Vpr regulates nuclear transport of the viral preintegration complex, G(2) cell cycle arrest, and transcriptional transactivation. We asked whether phosphorylation could affect Vpr activity. Vpr was found to be phosphorylated on serine residues in transiently transfected and infected cells. Residues 79, 94, and 96 were all found to be phosphorylated, as assessed by alanine mutations. Mutation of Ser-79 to Ala abrogated effects of Vpr on cell cycle progression, whereas mutation of Ser-94 and Ser-96 had no effect. Simultaneous mutation of all three Vpr serine residues attenuated HIV-1 replication in macrophages, whereas single and double Ser mutations had no effect.

Amino Acid Sequence↗
PubMed2000

Role of Vif in stability of the human immunodeficiency virus type 1 core.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription. vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.

Cells, Cultured↗
PubMed2001

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

Amino Acid Sequence↗
PubMed2003

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch.

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.

Amino Acid Sequence↗
PubMed2004

Murine T cells potently restrict human immunodeficiency virus infection.

Development of a mouse model for human immunodeficiency virus type 1 (HIV-1) infection has advanced through the progressive identification of host cell factors required for HIV-1 replication. Murine cells lack HIV-1 receptor molecules, do not support efficient viral gene expression, and lack factors necessary for the assembly and release of virions. Many of these blocks have been described using mouse fibroblast cell lines. Here we identify a postentry block to HIV-1 infection in mouse T-cell lines that has not been detected in mouse fibroblasts. While murine fibroblastic lines are comparable to human T-cell lines in permissivity to HIV-1 transduction, infection of murine T cells is 100-fold less efficient. Virus entry occurs efficiently in murine T cells. However, reduced efficiency of the completion of reverse transcription and nuclear transfer of the viral preintegration complex are observed. Although this block has similarities to the restriction of murine retroviruses by Fv1, there is no correlation of HIV-1 susceptibility with cellular Fv1 genotypes. In addition, the block to HIV-1 infection in murine T-cell lines cannot be saturated by a high virus dose. Further studies of this newly identified block may lend insight into the early events of retroviral replication and reveal new targets for antiretroviral interventions.

Animals↗
PubMed2005

Identification of an essential molecular contact point on the duck hepatitis B virus reverse transcriptase.

The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.

Amino Acid Sequence↗
PubMed1999

Sperm chromatin decondensation by template activating factor I through direct interaction with basic proteins.

Template activating factor I (TAF-I) was originally identified as a host factor required for DNA replication and transcription of adenovirus genome complexed with viral basic proteins. Purified TAF-I was shown to bind to core histones and stimulate transcription from nucleosomal templates. Human TAF-I consists of two acidic proteins, TAF-Ialpha and TAF-Ibeta, which differ from each other only in their amino-terminal regions. Here, we report that TAF-I decondenses demembraned Xenopus sperm chromatin. Human TAF-Ibeta has a chromatin decondensation activity comparable to that of NAP-I, another histone binding protein, whereas TAF-Ialpha has only a weak activity. Analysis of molecular mechanisms underlying the chromatin decondensation by TAF-I revealed that TAF-I interacts directly with sperm basic proteins. Deletion of the TAF-I carboxyl-terminal acidic region abolishes the decondensation activity. Interestingly, the acidic region itself is not sufficient for decondensation, since an amino acid substitution mutant in the dimerization domain of TAF-I which has the intact acidic region does not support chromatin decondensation. We detected the beta form of TAF-I in Xenopus oocytes and eggs by immunoblotting, and the cloning of its cDNA led us to conclude that Xenopus TAF-Ibeta also decondenses sperm chromatin. These results suggest that TAF-I plays a role in remodeling higher-order chromatin structure as well as nucleosomal structure through direct interaction with chromatin basic proteins.

Amino Acid Sequence↗
PubMed2004

Heat-shock protein 70 exerts opposing effects on Vpr-dependent and Vpr-independent HIV-1 replication in macrophages.

HIV-1 viral protein R (Vpr) shuttles between the nucleus and the cytoplasm and is believed to contribute to the process of nuclear translocation of the viral preintegration complex, thus facilitating HIV-1 replication in macrophages. In this report, we demonstrate that Hsp70, a heat-shock protein contributing to cellular stress responses, inhibits nuclear translocation of HIV-1 Vpr. In macrophages, Hsp70 is induced shortly after HIV-1 infection. Recombinant Hsp70 or a mild heat shock diminished replication of the wild-type HIV-1, suggesting that Hsp70 might function as an innate antiviral factor. Surprisingly, Hsp70 stimulated nuclear import and replication in macrophages of the Vpr-deficient HIV-1 construct. This finding suggests that Hsp70 and Vpr may function in a similar manner when expressed separately, but they neutralize each other's activity when present together. Consistent with this interpretation, Hsp70 coprecipitated with Vpr from HIV-1-infected cells.

Active Transport, Cell Nucleus↗