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M Bukrinsky

Publications and source records attributed to M Bukrinsky.

34 records · Page 2Linked to original sources
PubMed1998

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.

Biological Transport↗
PubMed1998

Relative contribution of transcription and translation to the induction of tumor necrosis factor-alpha by lipopolysaccharide.

The synthesis of tumor necrosis factor-alpha has been suggested to be regulated at both the transcriptional and translational levels in response to stimulation by bacterial lipopolysaccharide, although the relative contribution of these two mechanisms has not been quantitatively evaluated. Here, using the murine monocytic cell line RAW 264.7 as a model system, we show that steady-state TNF-alpha mRNA levels increase approximately 77-fold following treatment with lipopolysaccharide for 2 h and to a maximum of 164-fold after 8 h as measured by an RNase protection assay. The TNF-alpha gene transcription rate increases approximately 5-fold following exposure to lipopolysaccharide for 2 h as measured by a nuclear run-on assay. TNF-alpha mRNA stability did not change in the presence of lipopolysaccharide. A ribosomal sedimentation assay and an RNA transfection assay revealed that the translation rate of endogenous as well as transiently transfected TNF-alpha mRNAs increases only approximately 2-3-fold after stimulation with lipopolysaccharide for 2 h. Taken together, these results suggest that the large increase in the level of secreted TNF-alpha protein in RAW 264.7 cells is due primarily to activation of TNF-alpha gene transcription.

Animals↗
PubMed1998

Human immunodeficiency virus type 1 T-lymphotropic strains enter macrophages via a CD4- and CXCR4-mediated pathway: replication is restricted at a postentry level.

The human immunodeficiency virus type 1 (HIV-1) laboratory strains adapted to T-cell lines, as well as most syncytium-inducing primary isolates, replicate poorly in macrophages, which, beside CD4(+) T lymphocytes, are major targets of HIV-1. In the present work, we used a semiquantitative PCR-based technique to study viral entry into cells, kinetics of reverse transcription, and translocation of the viral DNA into the nucleus of macrophages infected with different HIV-1 strains. Our results demonstrate that T-lymphotropic strains efficiently enter macrophages. Entry was inhibited by a monoclonal antibody against CD4 and by stromal cell-derived factor 1alpha, a natural ligand of CXCR4, suggesting that both CD4 and CXCR4 act as receptors on macrophages for HIV-1 T-lymphotropic strains. Analysis of the kinetics of reverse transcription and nuclear import revealed that the most pronounced differences between T-lymphotropic and macrophagetropic strains occurred at the level of nuclear translocation of viral DNA, although a delay in reverse transcription was also observed. These results suggest that postentry steps are critical for restricted replication of T-lymphotropic HIV-1 strains in macrophages.

CD4 Antigens↗
PubMed1997

Cell proliferation is not required for productive HIV-1 infection of macrophages.

Lentiviruses, including HIV-1, are considered a rare example of retroviruses which do not require cell proliferation for their replication. However, this notion was questioned in several publications where productive HIV-1 infection was found to be restricted to a small fraction of macrophages with proliferative capacity. Since the mode of HIV-1 replication in macrophages is of great clinical relevance, we performed a single-cell analysis of HIV-1 replication and [3H]thymidine incorporation. Our results indicate that while 17% macrophages were detected as HIV-1 DNA-positive 12 hr after infection, only 2% of those cells had incorporated tritium, about the same percentage as in the uninfected cell population. Forty-eight hours after infection, 38% macrophages were HIV-1 DNA-positive and 47% of those had incorporated tritium, while the percentage of tritium-positive uninfected cells did not change (1%). These results demonstrate directly that HIV-1 DNA does not colocalize with [3H]thymidine and support the notion that cell proliferation is not required for HIV-1 infection of macrophages.

Cell Division↗
PubMed1997

The critical role of p38 MAP kinase in T cell HIV-1 replication.

BACKGROUND: Replication of HIV-1 in human T lymphocytes requires the activation of host cellular proteins. This study identifies p38 mitogen-activated protein kinase (MAPK) as one such kinase necessary for HIV-1 replication in T cells. MATERIALS AND METHODS: Primary human T lymphocytes were infected with the LAI strain of HIV-1 and Jurkat cells were infected with the RF strain of HIV-1. HIV replication was measured by reverse transcriptase activity. Cellular expression of endogenous p38 MAPK protein was analyzed using immunoprecipitation. Blockade of p38 MAPK expression was achieved using antisense oligonucleotides to p38 MAPK and the guanylhydrazone compound CNI-1493, an inhibitor of p38 MAPK activation. RESULTS: HIV-1 infection of both primary human T lymphocytes and a T cell line rapidly activated the cellular p38 MAPK pathway, which remained activated for the duration of the culture. Addition of phosphothioated antisense oligonucleotides to p38 MAPK specifically inhibited viral replication. Blockade of p38 MAPK activation by addition of CNI-1493 also inhibited HIV-1 viral replication of primary T lymphocytes in a dose- and time-dependent manner. Stimulation of p38 MAPK activation did not occur with the addition of heat-inactivated virus, suggesting that viral internalization, and not just membrane binding, is necessary for p38 MAPK activation. CONCLUSIONS: These results indicate that activation of the p38 MAPK cascade is critical for HIV-1 replication in primary T lymphocytes, and that blockade of this signal transduction pathway may be a novel therapeutic approach to the treatment of HIV-1 infection.

Blotting, Western↗
PubMed1996

Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation.

HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to karyopherin alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.

Antiviral Agents↗
PubMed1996

Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.

An overproduction of proinflammatory cytokines by activated macrophages/monocytes mediates the injurious sequelae of inflammation, septic shock, tissue injury, and cachexia. We recently synthesized a tetravalent guanylhydrazone compound (CNI-1493) that inhibits cytokine-inducible arginine transport and nitric oxide (NO) production in macrophages, and protects mice against lethal endotoxemia and carrageenan-induced inflammation. During these investigations we noticed that CNI-1493 effectively prevented lipopolysaccharide (LPS)-induced NO production, even when added in concentrations 10-fold less than required to competitively inhibit L-arginine uptake, suggesting that the suppressive effects of this guanylhydrazone compound might extend to other LPS-induced responses. Here, we report that CNI-1493 suppressed the LPS-stimulated production of proinflammatory cytokines (tumor necrosis factor [TNF], interleukins 1beta and 6, macrophage inflammatory proteins 1alpha and 1beta) from human peripheral blood mononuclear cells. Cytokine suppression was specific, in that CNI-1493 did not inhibit either the constitutive synthesis of transforming growth factor beta or the upregulation of major histocompatibility complex class II by interferon gamma (IFN-gamma). In contrast to the macrophage suppressive actions of dexamethasone, which are overridden in the presence of IFN-gamma, CNI-1493 retained its suppressive effects even in the presence of IFN-gamma. The mechanism of cytokine-suppressive action by CNI-1493 was independent of extracellular L-arginine content and NO production and is not restricted to induction by LPS. As a selective inhibitor of macrophage activation that prevents TNF production, this tetravalent guanylhydrazone could be useful in the development of cytokine-suppressive agents for the treatment of diseases mediated by overproduction of cytokines.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.

AIDS Dementia Complex↗
PubMed1996

A critical role of nitric oxide in human immunodeficiency virus type 1-induced hyperresponsiveness of cultured monocytes.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection leads to a general exhaustion of the immune system. Prior to this widespread decline of immune functions, however, there is an evident hyperactivation of the monocyte/macrophage arm. Increased levels of cytokines and other biologically active molecules produced by activated monocytes may contribute to the pathogenesis of HIV disease both by activating expression of HIV-1 provirus and by direct effects on cytokine-sensitive tissues, such as lung or brain. In this article, we investigate mechanisms of hyperresponsiveness of HIV-infected monocytes. MATERIALS AND METHODS: The study was performed on monocyte cultures infected in vitro with a monocytetropic strain HIV-1ADA. Cytokine production was induced by stimulation of cultures with lipopolysaccharides (LPS) and measured by ELISA. To study involvement of nitric oxide (NO) in the regulation of cytokine expression, inhibitors of nitric oxide synthase (NOS) or chemical donors of NO were used. RESULTS: We demonstrate that infection with HIV-1 in vitro primes human monocytes for subsequent activation with LPS, resulting in increased production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). This priming effect can be blocked by Ca(2+)-chelating agents and by the NOS inhibitor L-NMMA, but not by hemoglobin. It could be reproduced on uninfected monocyte cultures by using donors of NO, but not cGMP, together with LPS. CONCLUSIONS: NO, which is expressed in HIV-1-infected monocyte cultures, induces hyperresponsiveness of monocytes by synergizing with calcium signals activated in response to LPS stimulation. This activation is cGMP independent. Our findings demonstrate the critical role of NO in HIV-1-specific hyperactivation of monocytes.

Calcium↗
PubMed1995

Nuclear localization signal of HIV-1 as a novel target for therapeutic intervention.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is a lentivirus and shares with other members of this retroviral subfamily the ability to replicate in nondividing cells, in particular, cells of the monocyte/macrophage lineage. This feature relies on the presence of a specific nuclear localization signal (NLS) within the viral matrix protein (MA p17), which to some degree can be complemented by the activity of the viral vpr gene product. The MA p17 NLS ensures efficient transportation of the viral preintegration complex into the nucleus of an infected macrophage and confers persistence of HIV-1 in quiescent T cells, and therefore presents an attractive target for therapeutic intervention. MATERIALS AND METHODS: Nuclear localization signals (NLS) in general and the HIV-1 MA p17 NLS in particular are characterized by a stretch of positively charged amino acids including one or more lysine residues. A series of compounds potentially capable of binding and reacting with lysine by forming Schiff base adducts was synthesized. Our special consideration was to make compounds that would preferentially bind to two closely contiguous amino functions, as opposed to isolated single lysine residues. We assumed that this approach might specifically target the compound to NLS while affecting other regions less, thus reducing nonspecific cytotoxicity. Antiviral activity was assessed in primary monocytes and in peripheral blood lymphocytes (PBL) infected with HIV-1ADA strain. Viral replication was monitored by reverse transcriptase (RT) activity in the supernatant. Efficiency of nuclear importation of the viral preintegration complex was estimated by the formation of 2-LTR circle forms of HIV-1 DNA and also by in situ PCR techniques. RESULTS: Arylene bis(methyl ketone) compounds with a nitrogenous third subsituent, especially a pyrimidinic side-chain, inhibited HIV-1 replication in human monocytes at an IC50 as low as 1 nM. These compounds did not block HIV-1 replication in peripheral blood lymphocyte cultures. The inhibitory effect observed in monocyte cultures appeared in the context of markedly reduced nuclear importation of viral DNA in the presence of the drug. No cytotoxic effects of the compounds was observed in vitro at concentrations as high as 10 microM. An amidinohydrazone derivative of the most active compound was about 100 times less active than the parent, indicating that carbonyl groups were instrumental in the antiviral effect. CONCLUSIONS: These early results suggest that retroviral replication in nondividing cells is susceptible to pharmaceutical intervention targeted against the NLS activity of HIV-1 proteins in the viral preintegration complex. The compounds described efficiently block translocation of viral DNA to the nuclei of infected primary monocytes, and inhibit viral replication. This inhibition is effective only in nondividing cells and is not seen in proliferating cultures, such as activated PBLs. Thus, drugs that target HIV-1 NLS may be useful to specifically block the macrophage arm of HIV infection and could thereby be of value in treating macrophage-specific manifestations of HIV disease, such as HIV-1 dementia. In combination with other drugs, potential therapeutics exploiting this target may also help to control the progression of HIV-1 infection and disease.

Antiviral Agents↗
PubMed1995

An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality.

BACKGROUND: Nitric oxide (NO), a small effector molecule produced enzymatically from L-arginine by nitric oxide synthase (NOS), is a mediator not only of important homeostatic mechanisms (e.g., blood vessel tone and tissue perfusion), but also of key aspects of local and systemic inflammatory responses. Previous efforts to develop inhibitors of NOS to protect against NO-mediated tissue damage in endotoxin shock have been unsuccessful, largely because such competitive NOS antagonists interfere with critical vasoregulatory NO production in blood vessels and decrease survival in endotoxemic animals. Accordingly, we sought to develop a pharmaceutical approach to selectively inhibit NO production in macrophages while sparing NO responses in blood vessels. MATERIALS AND METHODS: The process of cytokine-inducible L-arginine transport and NO production were studied in the murine macrophage-like cell line (RAW 264.7). A series of multivalent guanylhydrazones were synthesized to inhibit cytokine-inducible L-arginine transport. One such compound (CNI-1493) was studied further in animal models of endothelial-derived relaxing factor (EDRF) activity, carrageenan inflammation, and lethal lipopolysaccharide (LPS) challenge. RESULTS: Upon activation with cytokines, macrophages increase transport of L-arginine to support the production of NO by NOS. Since endothelial cells do not require this additional arginine transport to produce NO, we reasoned that a competitive inhibitor of cytokine-inducible L-arginine transport would not inhibit EDRF activity in blood vessels, and thus might be effectively employed against endotoxic shock. CNI-1493, a tetravalent guanylhydrazone, proved to be a selective inhibitor of cytokine-inducible arginine transport and NO production, but did not inhibit EDRF activity. In mice, CNI-1493 prevented the development of carrageenan-induced footpad inflammation, and conferred protection against lethal LPS challenge. CONCLUSIONS: A selective inhibitor of cytokine-inducible L-arginine transport that does not inhibit vascular EDRF responses is effective against endotoxin lethality and significantly reduces inflammatory responses.

Animals↗
PubMed1993

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↗
PubMed1992

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↗
PubMedDate not supplied

Viral protein R of HIV-1.

Viral protein R (Vpr) of HIV-1 belongs to a class of so called 'accessory' proteins, originally thought to be dispensable for virus replication, at least in vitro. Indeed, viruses with mutated or deleted Vpr replicate well in transformed T cell lines. However, recently published results reveal several important functions performed by Vpr, which are critical for HIV-1 replication in vivo. Vpr plays an important role in regulating nuclear import of the HIV-1 pre-integration complex, and is required for virus replication in non-dividing cells. Vpr also induces cell cycle arrest in proliferating cells, stimulates virus transcription, and regulates activation and apoptosis of infected cells. These diverse functions are mediated by the interaction of Vpr with different cellular proteins, many of which carry the WxxF amino acid motif. The molecular events underlying the activity of Vpr are reviewed in this article.

Cell Cycle↗