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Query: UMLS:C0019693 (HIV)
170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.
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PMID:The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. 790 79

We have used a combination of genetic and immunological techniques to explore how amino acid substitutions in the second conserved (C2) domain of gp120 from human immunodeficiency virus type 1 (HIV-1) affect the conformation of the protein. It was reported previously (R. L. Willey, E. K. Ross, A. J. Buckler-White, T. S. Theodore, and M. A. Martin. J. Viol. 63:3595-3600, 1989) that an asparagine-glutamine (N/Q) substitution at C2 residue 267 of HIV-1 NL4/3 reduced virus infectivity, but that infectivity was restored by a compensatory amino acid change (serine-glutamine; S/N) at residue 128 in the C1 domain. Here we show that the 267 N/Q substitution causes the abnormal exposure of a segment of C1 spanning residues 80 to 120, which compromises the integrity of the CD4-binding site. The reversion substitution at residue 128 restores the normal conformation of the C1 domain and recreates a high-affinity CD4-binding site. The gp120 structural perturbation caused by changes in C2 extends also to the C5 domain, and we show by immunological analysis that there is a close association between areas of the C1 and C5 domains. This association might be important for forming a complex binding site for gp41 (E. Helseth, U. Olshevsky, C. Furman, and J. Sodroski. J. Virol. 65:2119-2123, 1991). Segments of the C1 and C2 domains are predicted to form amphipathic alpha helices. We suggest that these helices might be packed together in the core of the folded gp120 molecule, that the 267 N/Q substitution disrupts this interdomain association, and that the 128 S/N reversion substitution restores it.
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PMID:Immunological evidence for interactions between the first, second, and fifth conserved domains of the gp120 surface glycoprotein of human immunodeficiency virus type 1. 793 65

To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3. Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41). The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9). Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene. No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message. These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population. In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture. The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.
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PMID:Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells. 798 70

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a major determinant of phenotypic variability. The V3 domain of HIV-1 has many basic amino acid residues. Lymphocytotropic HIV-1 tends to have a V3 domain with a higher density of positive charge than does monocytotropic HIV-1. The importance of basic residues in the V3 domain for the HIV-1 infectivity, however, has not been well investigated. Here we show that mutation of basic amino acid residues at positions 303, 306, 309, 313, and 325 in the V3 domain of the lymphocytotropic isolate NL4-3 results in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Three basic amino acid substitutions (at position 306, 309, and 313) induced a decrease in the binding ability of two kinds of neutralizing antibodies (NEA9284 and 0.5 beta) that recognize a different site in the V3 domain. This suggests that the basic residues play an important role in maintaining the tertiary structure of the V3 domain. Monocytotropism was not simply dependent on either decreased positive charge in the V3 domain of NL4-3 or on mutation of lysine to glutamate at position 320, which is a characteristic amino acid of monocytotropic HIV-1. These findings contribute to our understanding of the significance of basic residues on the function of envelope glycoprotein.
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PMID:HIV type 1 infection of CD4+ T cells depends critically on basic amino acid residues in the V3 domain of envelope glycoprotein 120. 798 86

Molecular clones of three macrophage-tropic and three T-cell line-adapted strains of human immunodeficiency virus type 1 (HIV-1) were used to explore the mechanism of HIV-1 resistance to neutralization by soluble CD4 (sCD4). The three macrophage-tropic viruses, each possessing the V3 and flanking regions of JR-FL, were all resistant to sCD4 neutralization under the standard conditions of a short preincubation of the virus and sCD4 at 37 degrees C prior to inoculation of peripheral blood mononuclear cells. In contrast, the three T-cell line-adapted viruses, NL4-3 and two chimeras possessing the V3 and flanking regions of NL4-3 in the envelope background of JR-FL, were all sCD4 sensitive under these conditions. Sensitivity to sCD4 neutralization at 37 degrees C corresponded with rapid, sCD4-induced gp120 shedding from the viruses. However, when the incubation temperature of the sCD4 and virus was reduced to 4 degrees C, the three macrophage-tropic viruses shed gp120 and became more sensitive to sCD4 neutralization. In contrast, the rates of sCD4-induced gp120 shedding and virus neutralization were reduced for the three T-cell line-adapted viruses at 4 degrees C. Thus, HIV resistance to sCD4 is a conditional phenomenon; macrophage-tropic and T-cell line-adapted strains can be distinguished by the temperature dependencies of their neutralization by sCD4. The average density of gp120 molecules on the macrophage-tropic viruses exceeded by about fourfold that on the T-cell line-adapted viruses, suggesting that HIV growth in T-cell lines may select for a destabilized envelope glycoprotein complex. Further studies of early events in HIV-1 infection should focus on primary virus strains.
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PMID:Macrophage-tropic and T-cell line-adapted chimeric strains of human immunodeficiency virus type 1 differ in their susceptibilities to neutralization by soluble CD4 at different temperatures. 803 23

Auxiliary genes that are not essential for viral replication in cell culture are found in all known lentiviruses. The "nonessential" auxiliary genes of HIV-1 are vif, vpr, vpu, and nef. Sequences within the upstream region of U3 in the LTR are also not required for virus replication in cell culture. We constructed a panel of 23 mutants with single and combination deletions in these regions in the wild-type HIV-1 infectious molecular clone NL4-3. Deletion of the vpu, vpr, and nef genes and the U3 upstream sequence (US), individually or in combinations, did not appreciably alter virus replication in either chimpanzee PBMCs, human PBMCs, or in the B/T cell hybrid line CEMx174. In contrast, deletion of the vif gene dramatically delayed virus replication in all three cell types. This collection of HIV-1 deletion mutants will be useful for elucidating the functions of these genes and for investigating antiviral immunity in animal models.
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PMID:Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes. 806 14

Expression of reporter genes under the control of the HIV-1 long terminal repeat (LTR) was up-regulated in the murine macrophage cell line RAW264 by cotransfection of a plasmid coding for the viral regulatory protein Nef. To determine if a discrete section of the LTR was exclusively responsive to Nef, a series of promoters was produced by successive 5' deletions from the LTR up to the boundary of the enhancer region. These truncated promoters were as active as the full-length sequence in the RAW264 cells, but elimination of the direct repeats and one of the three Sp1 sites reduced promoter activity to minimal levels. Transcription driven by all constructs was equally susceptible to the trans-activating effect of Nef and could be increased further by the addition of a Tat-expressing plasmid to the cotransfection. Open reading frames of nef from NL4-3, from HXB2, which has a premature stop, and a fully functional hybrid of the two under the control of the SR alpha artificial promoter (SV40 early promoter plus HTLV-I R-U5') were able to transactivate the LTR in RAW264 cells to the same degree as HXB3 nef under the control of the cytomegalovirus (CMV) immediate-early promoter. A frameshift mutation of Nef at the XhoI site at position 8475 did not abrogate trans-activation of the LTR in macrophages. To further define the effective trans-activation region of Nef, internal deletions were made. Changes downstream of the XhoI site at amino acid 35 resulted in little or no reduction in trans-activation, whereas a deletion between the CMV promoter of the expression plasmid and the XhoI site largely abolished activity. Nef trans-activation of the LTR may be restricted to macrophages. Parallel cotransfection experiments in COS-1 simian fibroblast-like cells showed repression of reporter expression by Nef. Results suggested that the section of nef responsible for transactivation of the LTR in macrophages differed slightly from that sufficient for trans-repression in fibroblasts. Translation of the protein from the first translation start site (Met-1) rather than from the second in-frame ATG (Met-20) appears to be necessary for the trans-activating effect of Nef in RAW264 cells. Mutation of the initial ATG to ATA led to loss of trans-activating activity. Expression of Nef also has a cytostatic/cytotoxic effect on RAW264 cells indicated by a reduced rate of establishment of stably transfected clones. The cytostatic effect of Nef was not relieved by internal deletions in the coding sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The HIV-1 regulatory protein Nef has a specific function in viral expression in a murine macrophage cell line. 808 2

Two molecularly cloned viruses, human immunodeficiency virus type 1 (HIV-1)-NL4-3 (NL4-3) and HIV-1-HXB-2 (HXB-2), have been used to study the role of HIV-1 auxiliary genes in the establishment of chronic virus producers. NL4-3 encodes all known HIV-1 proteins, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. Studies were done in H9 cells, a T-cell line unusually permissive for the establishment of chronic virus producers. NL4-3 and HXB-2 undergo lytic phases of infection in H9 cultures with HXB-2, but not NL4-3, supporting the efficient establishment of chronic virus producers. Tests of mutant NL4-3 genomes containing various combinations of defective auxiliary genes revealed that both vpr and nef limited the ability of NL4-3 to establish chronic virus producers. Tests of a series of recombinants between NL4-3 and HXB-2 revealed that 5' internal sequences as well as fragments containing defective auxiliary genes affected the establishment of chronic virus producers. Viral envelope sequences and levels of virus production did not correlate with the ability to establish chronic virus producers. These results suggest that complex interactions of viral auxiliary and nonauxiliary gene functions with the host cell determine the ability to establish chronic virus producers.
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PMID:Context-dependent role of human immunodeficiency virus type 1 auxiliary genes in the establishment of chronic virus producers. 841 97

Aurothioglucose and aurothiomalate have anti-HIV-1 activity in vitro. Antiviral activity requires the formation of a reactive intermediate with a molar equivalent amount of a thiol ligand. This activates gold(I) ligand exchange between the reactive species bis(thiolato)gold(I) and acidic thiol groups exposed on the surface of proteins. Bis(thioglucose)gold(I) (bisAuTG) which is formed by the reaction of molar equivalent amounts of aurothioglucose and 1-thio-beta-D-glucose completely protected MT-4 and CEM cells against HIV-1NL4-3-induced cytopathogenicity. Although bisAuTG is an inhibitor of human immunodeficiency virus-1 (HIV-1) reverse transcriptase in a cell-free assay, its antiviral effect is due to modification of a surface component of the virion. The HIV-1 strain NL4-3 is 200-fold more sensitive to inhibition of infectivity by bisAuTG than are the strains MN, RF, and SF-2. HIV-1NL4-3 has a unique cysteine residue close to the amino terminus of its gp41 envelope glycoprotein (residue 532 of gp160) which we hypothesize is the target of bisAuTG binding. Mutation of that residue alters HIV-1NL4-3 infectivity and dominantly suppresses virus assembly when coexpressed with the wild-type NL4-3 genome. We show that bisAuTG treatment releases gp120 from the surface of cells expressing wild-type HIV-1NL4-3 envelope glycoprotein, but it does not release gp120 if Cys532 is mutationally altered to Ala. Thus, the antiviral effect of bisAuTG on HIV-1NL4-3 is due to an effect on the association of gp120 with gp41.
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PMID:Aurothiolates inhibit HIV-1 infectivity by gold(I) ligand exchange with a component of the virion surface. 842 3

Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclam-resistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791- and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.
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PMID:The molecular target of bicyclams, potent inhibitors of human immunodeficiency virus replication. 855 4

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