Identification of factors that govern HIV-1 replication in nondividing host cells.
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Amino Acid Sequence↗
Biomedical subjects
M Stevenson
Publications and source records attributed to M Stevenson.
A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells.
Permissiveness of the host cell to productive infection by oncoretroviruses is cell-cycle dependent, and nuclear localization of viral nucleoprotein preintegration complexes will occur only after cells have passed through mitosis. In contrast, establishment of an integrated provirus after infection by the lentivirus HIV-1 is independent of host cell proliferation. The ability of HIV-1 to replicate in non-dividing cells is partly accounted for by the karyophilic properties of the viral preintegration complex which, after virus infection, is actively transported to the host cell nucleus. Here we report that the gag matrix protein of HIV-1 contains a nuclear localization sequence which, when conjugated to a heterologous protein, directs its nuclear import. In addition, HIV-1 mutants containing amino-acid substitutions in this nuclear localization signal integrate and replicate within dividing but not growth-arrested cells, and thus display a phenotype more representative of an oncoretrovirus.
Amino Acid Sequence↗
Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection.
We have examined components of the preintegration complex of human immunodeficiency virus type 1 (HIV-1) and have analyzed features which govern the association of these components. HIV-1 nucleoprotein complexes, isolated from nuclear and cytoplasmic extracts of CD4+ cells after acute virus infection, contained viral RNA and DNA in association with viral matrix (MA), integrase (IN), and reverse transcriptase (RT) antigens but not capsid (CA) antigens and possessed integration activity in vitro. Association of IN but not RT or MA antigens with viral DNA was detergent-stable. Analysis of viral DNA synthesis and nuclear import of viral nucleoprotein complexes in the presence of a reversible RT inhibitor demonstrated that reverse transcription of viral RNA could be completed entirely in the host cell nucleus. Our studies demonstrate structural and functional features of the nucleoprotein (preintegration) complex of HIV-1 which are pertinent to the understanding of early events in the lentiviral life cycle.
Base Sequence↗
A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors.
Several nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been described, including Nevirapine, thiobenzimidazolone (TIBO) derivatives, pyridinone derivatives such as L-697,661, and the bis(heteroaryl)piperazines (BHAPs). HIV-1 resistant to L-697,661 or Nevirapine emerges rapidly in infected patients treated with these drugs, and the resistance is caused primarily by substitutions at amino acids 181 and 103 of RT that also confer cross resistance to the other nonnucleoside inhibitors. We describe derivation and characterization of two BHAP-resistant HIV-1 variants that differ from this pattern of cross resistance. With both variants, HIV-1 resistance to BHAP RT inhibitors was caused by a RT mutation that results in a proline-to-leucine substitution at amino acid 236 (P236L). Rather than conferring cross resistance to other RT inhibitors, this substitution sensitized RT 7- to 10-fold to Nevirapine, TIBO R82913, and L-697,661 without influencing sensitivity to nucleoside analogue RT inhibitors. This sensitization caused by P236L was also observed in cell culture with BHAP-resistant HIV-1. The effects of the P236L RT substitution suggest that emergence of BHAP-resistant virus in vivo could produce a viral population sensitized to inhibition by these other nonnucleoside RT inhibitors.
Antiviral Agents↗
The nuts and bolts of behavioral therapy for insomnia.
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Adaptation, Psychological↗
Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex.
The preintegration complex of human immunodeficiency virus type 1 (HIV-1) is a large nucleoprotein complex containing viral nucleic acids in association with products of the viral gag and pol genes. One of these proteins, integrase, is absolutely required for the integration and formation of the provirus. Although HIV-1-specific 2-LTR circles from nuclei of HIV-1-infected cells were found to be associated within a high-molecular-weight nucleoprotein complex, antibodies to HIV-1 integrase failed to precipitate this form of viral DNA. This result indicates that circular forms of HIV-1 DNA are not associated with integrase. These viral DNA forms seem to exist in a context of a nucleoprotein complex that is different from a preintegration complex of HIV-1.
Cell Line↗
Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.
After cell infection by the human immunodeficiency virus type 1 (HIV-1), nascent viral DNA in the form of a high molecular weight nucleoprotein preintegration complex must be transported to the nucleus of the host cell prior to integration of viral DNA with the host genome. The mechanism used by retroviruses for nuclear targeting of preintegration complexes and dependence on cell division has not been established. Our studies show that, after infection, the preintegration complex of HIV-1 was rapidly transported to the nucleus of the host cell by a process that required ATP but was independent of cell division. Functional HIV-1 integrase, an essential component of the preintegration complex, was not required for nuclear import of these complexes. The ability of HIV-1 to use host cell active transport processes for nuclear import of the viral preintegration complex obviates the requirement for host cell division in establishment of the integrated provirus. These findings are pertinent to our understanding of early events in the life cycle of HIV-1 and to the mode of HIV-1 replication in terminally differentiated nondividing cells such as macrophages (monocytes, tissue macrophages, follicular dendritic cells, and microglial cells).
Adenosine Triphosphate↗
Metabolic and biochemical status of articular cartilage following cryopreservation and transplantation: a rabbit model.
To determine the fate of transplanted cryopreserved articular cartilage, an animal model employing the proximal humerus in the rabbit has been developed. Previous studies have been hindered by problems of postoperative joint instability, secondary injury due to immobilization, and paucity of cartilage for analysis. This experiment demonstrates the survival and function of transplanted cartilage by quantitative assessment of metabolic and biochemical parameters. Forty-five New Zealand white rabbits underwent transplantation of the right proximal humerus. In 29 animals, the proximal half of the humerus was resected and replaced by a cryopreserved osteoarticular allograft. Autograft procedures were carried out in the remaining animals. Following sacrifice at 3, 6, 9, and 12 months postoperatively, articular cartilage was analyzed for gross appearance, collagen synthesis, proteoglycan synthesis, and water, hydroxyproline, hexosamine, and hexuronic acid contents. The results indicate that the cryopreserved osteoarticular allografts retained their metabolic and biochemical integrity and behaved as viable and biologically functional units 1 year postoperatively.
Animals↗
HIV-1 replication and potential targets for intervention.
Intense research into fundamental processes of human immunodeficiency syndrome type 1 (HIV-1) replication has yielded knowledge that in many aspects equals or exceeds that of the oncogenic retroviruses. The availability of sensitive virus detection methods has allowed a more thorough characterization of the biology of virus persistence and latency in vivo and removed the dependence on in vitro models. As a clearer picture of the pattern of HIV-1 replication in vivo evolves, it becomes apparent that HIV-1 biology is distinct from that of the prototypic oncogenic retroviruses in several key aspects, particularly with regard to host cell range and determinants of viral permissiveness. In this respect it may be appropriate to examine the lentivirus, rather than the oncovirus model system to better understand the biology and pathogenesis of HIV-1 infection. This synopsis of recent and ongoing research developments in HIV-1 replication and pathogenesis emphasizes the determinants of host cell permissiveness, early events in virus replication, and underlying features in HIV-1 cytopathogenesis. In addition, basic viral replication processes which can be exploited for therapeutic intervention are discussed.
Animals↗
Effect of a diet high in monounsaturated fat from almonds on plasma cholesterol and lipoproteins.
The effect of almonds as part of a low saturated fat, low cholesterol, high-fiber diet was studied in 26 adults (13 men, 13 women). The baseline diet was modified in a similar way for all subjects by limiting meat, fatty fish, high-fat milk products, eggs, and saturated fat. Grains, beans, vegetables, fruit, and low-fat milk products were the foundation of the diet. During the almond diet period, raw almonds (100 mg/day) supplied 34 g/day of monounsaturated fatty acid (MUFA), 12 g/day of polyunsaturated fatty acid, and 6 g/day of saturated fatty acid. Almond oil was the only oil allowed for food preparation. There was a rapid and sustained reduction in low-density lipoprotein cholesterol without changes in high-density lipoprotein cholesterol. This was reflected in a total plasma cholesterol decrease from (means +/- SEM) 235 +/- 5.0 at baseline to 215 +/- 5.0 at 3 weeks, and to 214 +/- 5.0 mg/dl at 9 weeks (p less than 0.001). When the consumption of nuts high in MUFA increases the fat content of the diet, reduction rather than elevation of plasma cholesterol has to be expected, possibly due to the MUFA content of these nuts.
Adult↗
Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection.
To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.
DNA, Viral↗
Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection.
Alterations in two highly conserved N-linked glycosylation sites within the gp120 envelope glycoprotein of human immunodeficiency virus type I (HIV-1) implicated in the phenotype of a noncytopathic HIV-1 variant were introduced independently and in combination into a cytopathic, infectious HIV-1 clone by site-specific mutagenesis. Neither mutation affected the synthesis of HIV-1 envelope glycoproteins. However, one of the mutations restricted the ability of HIV-1 envelope to localize on the cell membrane and thus markedly impaired virus assembly. The HIV-1 assembly defect could be overcome in trans if site-specific mutants were packaged in HeLa cells constitutively producing wild-type HIV-1 envelope glycoprotein. In addition to inefficient virus assembly, this mutation impaired the ability of the virus to infect CD4+ T cells, but did not affect CD4-independent infection of muscle cells. These results suggest additional functions of posttranslational modification in virus replication (i.e., envelope glycoprotein transport). Given that such modifications can restrict CD4-mediated uptake without affecting CD4-independent uptake, variations in posttranslational env processing between different HIV-1 genotypes may affect virus tropism in vivo.
Amino Acid Sequence↗
Predominance of distinct viral genotypes in brain and lymph node compartments of HIV-1-infected individuals.
A modified polymerase chain reaction protocol was used to amplify the entire envelope-coding region of HIV-1 directly from brain and lymph node tissue obtained at autopsy from three HIV-1-infected individuals. Molecular analysis of amplified DNA by digestion with 18 restriction endonucleases, singly and in combination, revealed different HIV-1 genotypes in the brain and lymph node compartments in each of the three individuals. This anatomic compartmentalization of HIV-1 populations may reflect different viral genomic sequences that determine tropism or differences in host immune selection pressures in the brain and lymphoid compartments that drive the emergence of distinct viral populations.
Acquired Immunodeficiency Syndrome↗
Lymphotropic viruses as etiologic agents of lymphoma.
DNA and RNA virus with immortalized lymphocytes are described including Epstein-Barr virus and HTLV-I. The roles of immune deficiency and genetic alterations occurring in a proliferating cell are also considered.
Animals↗
Transformation of rat bladder epithelial cells by introduction of a single oncogene.
Malignant transformation of cells in vitro requires the action of cooperating oncogenes. However, the action of a single activated oncogene, if placed under a strong constitutive promoter, is sufficient to transform in vitro established cells. We have investigated the role of cooperating oncogenes in the transformation of normal rat bladder epithelial cells. Effects of polyoma middle T, v-Ha-ras or activated rat c-Ha-ras, independently or in combination with v-myc on in vitro and in vivo behavior of rat bladder epithelial cells were studied. Introduction of polyoma middle T alone, c-Ha-ras or in some cases v-myc into "normal" rat bladder epithelial cells was sufficient for full malignant transformation of these cells. In addition, introduction of polyoma middle T, activated c-Ha-ras or v-myc transformed cells into nude mice, resulted in development of highly invasive adenocarcinomas. These results indicate that full malignant transformation of normal rat bladder epithelial cells did not require the action of introduced cooperating oncogenes.
Adenocarcinoma↗
HIV-1 replication is controlled at the level of T cell activation and proviral integration.
During progression of the Acquired Immune Deficiency Syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1) is harbored in CD4+ T cells, which act as the primary reservoir for the virus. In vitro, HIV-1 requires activated T cells for a productive infection; however, in vivo, the number of circulating T cells in the activated state that are potential targets for HIV-1 infection is low. We have investigated the ability of HIV-1 to infect resting T cells, and the consequences of such an infection. T cell activation was not required for HIV-1 infection; however, viral DNA was unable to integrate in resting T cells and was maintained extrachromosomally. Subsequent T cell activation allowed integration of extrachromosomal forms and led to a productive viral life cycle. Extrachromosomal forms of viral DNA were found to persist for several weeks after infection of resting T cells and, following T cell activation, these forms maintained their ability to integrate and act as a template for infectious virus. Several lines of evidence, including temporal analysis of HIV-1 replication and analysis of an HIV-1 integrase deletion mutant, indicated that extra-chromosomal HIV-1 DNA genomes were transcriptionally active. These results are compatible with a model whereby HIV-1 can persist in a non-productive extra-chromosomal state in resting T cells until subsequent antigen-induced or mitogen-induced T cell activation, virus integration and release. Thus agents that induce T cell activation may control the rate of HIV-1 replication and spread during AIDS progression.
Acquired Immunodeficiency Syndrome↗
Autonomous growth of lymphoid cells following IL-2 expression from retrovirus vectors containing HIV-1 trans-acting elements.
The human immunodeficiency virus type I (HIV-1) possesses powerful regulatory elements that control the rate of replication of HIV-1 and subsequent processing of HIV-1 genes. We have used this regulatory mechanism to drive expression of foreign genes inserted in retrovirus vectors. This approach was used to express the human IL-2 gene in IL-2-dependent mouse CTLL-2 cells to determine the role of autonomous growth in maintaining proliferation of virus-infected T lymphocytes during HTLV-1-induced adult T-cell leukemia (ATL). Expression of IL-2 sequences in IL-2-dependent mouse CTLL-2 cells resulted in autonomous growth of IL-2-independent CTLL-2 clones. Endogenous expression of IL-2 appeared to interrupt normal constraints of growth in that these IL-2-independent clones showed reduced cell-density-dependent inhibition but not a tumorigenic phenotype. IL-2-independent CTLL-2 clones did not secrete detectable quantities of IL-2 into culture supernatant and exhibited reduced sensitivity to the inhibitory effects of both IL-2 and IL-2 receptor antibody. These results suggest that the IL-2 autocrine loop within these cells involves intracellular IL-2/IL-2 receptor binding. The apparent lack of IL-2 production and poor responsiveness to IL-2 or IL-2 antibodies displayed by cell lines from ATL patients may be explained by an intracellular IL-2/IL-2 receptor autocrine loop.
Animals↗
Rearrangement and expression of T-cell receptor genes in marmoset lymphoid cell lines with natural killer activity.
To study the role of the T-3/T-cell receptor complex in mediating natural killer activity, we have examined T-cell receptor gene configuration and expression in five transformed marmoset cell lines with natural killer activity. All lines showed rearrangement of the beta chain of the T-cell receptor and expressed full size beta chain transcripts as well as full size delta chain transcripts. Only three lines contained full size T-cell receptor alpha chain transcripts. Of the three lines which had potential for encoding functional T-3/T-cell receptor complex, those which possessed natural killer activity also expressed T-gamma transcripts. In addition, one clone with high natural killer activity which did express high levels of T-gamma mRNA was unable to produce functional T-3/T-cell receptor complex due to truncated T-cell receptor alpha chain message. The results in this paper provide strong evidence that natural killer activity is dependent upon expression of the T-cell receptor gamma chain.
Animals↗