Highly active antiretroviral therapy and beta-chemokines.
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Anti-HIV Agents↗
Biomedical subjects
M Bukrinsky
Publications and source records attributed to M Bukrinsky.
CD147 is a signaling receptor for cyclophilin B.
Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins.
Animals↗
CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A.
Cyclophilin A (CyPA) is specifically incorporated into the virions of HIV-1 and has been shown to enhance significantly an early step of cellular HIV-1 infection. Our preliminary studies implicated CD147 as a receptor for extracellular CyPA. Here, we demonstrate a role for CyPA-CD147 interaction during the early steps of HIV-1 infection. Expression of human CD147 increased infection by HIV-1 under one-cycle conditions. However, susceptibility to infection by viruses lacking CyPA (simian immunodeficiency virus or HIV-1 produced in the presence of cyclosporin A) was unaffected by CD147. Virus-associated CyPA coimmunoprecipitated with CD147 from infected cells. Antibody to CD147 inhibited HIV-1 entry as evidenced by the delay in translocation of the HIV-1 core proteins from the membrane and inhibition of viral reverse transcription. Viruses whose replication did not require CyPA (SIV or mutant HIV-1) were resistant to the inhibitory effect of anti-CD147 antibody. These results suggest that HIV-1 entry depends on an interaction between virus-associated CyPA and CD147 on a target cell.
Animals↗
The binding subunit of pertussis toxin inhibits HIV replication in human macrophages and virus expression in chronically infected promonocytic U1 cells.
We have recently shown that the binding subunit of pertussis toxin (PTX-B) inhibits the entry and replication of macrophage-tropic (R5) HIV-1 strains in activated primary T lymphocytes. Furthermore, PTX-B suppressed the replication of T cell-tropic (X4) viruses at a postentry level in the same cells. In this study we demonstrate that PTX-B profoundly impairs entry and replication of the HIV-1(ADA) (R5), as well as of HIV pseudotyped with either murine leukemia virus or vesicular stomatitis virus envelopes, in primary monocyte-derived macrophages. In addition, PTX-B strongly inhibited X4 HIV-1 replication in U937 promonocytic cells and virus expression in the U937-derived chronically infected U1 cell line stimulated with cytokines such as TNF-alpha and IL-6. Of interest, TNF-alpha-mediated activation of the cellular transcription factor NF-kappaB was unaffected by PTX-B. Therefore, PTX-B may represent a novel and potent inhibitor of HIV-1 replication to be tested for efficacy in infected individuals. In support of this proposition, a genetically modified mutant of PTX (PT-9K/129G), which is safely administered for prevention of Bordetella pertussis infection, showed an in vitro anti-HIV profile superimposable to that of PTX-B.
Antiviral Agents↗
Chemokine receptors on brain endothelia--keys to HIV-1 neuroinvasion?
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AIDS Dementia Complex↗
HIV-1 penetrates coronary artery endothelial cells by transcytosis.
BACKGROUND: The pathogenesis of HIV-1-related cardiomyopathy is poorly understood, but HIV-1 has been detected in cardiomyocytes. Whether HIV-1 penetrates into the myocardium by infection of coronary artery endothelial cells (CAEC) or using transcellular or paracellular routes across CAEC has not been resolved. MATERIALS AND METHODS: A model of the CAEC barrier was constructed with primary CAEC (derived from human coronary vessels). Polymerase chain reaction (PCR) assay, infectious assay, and immunofluorescence were employed to show abortive nature of HIV-1 infection of CAEC. Tight junction (TJ) and cell adhesion proteins were visualized by immunofluorescence. The time course of HIV-1 invasion was measured by HIV-1 RNA assay. Inulin permeability assay determined paracellular leakage. Transmission electron microscopy demonstrated virus-induced endothelial vacuolization. RESULTS: Despite a strong display on CAEC of CXCR4 and a lesser expression of CCR3 and CCR5, HIV-1 did not productively replicate in CAEC, as shown by infectious assay, immunofluorescence, and electron microscopy. HIV-1 infection of CAEC was abortive with minimal reverse transcription of strong stop DNA and pol but not full-length or two LTR DNA circles. Upon infection of the model with 1 million RNA copies of HIV-1JR-FL, virus penetration 2 hr postinfection (PI) was negligible but increased by 1,750% 24 hr PI. The paracellular permeability increased during this period by only 25%. Neither AOP-RANTES nor v-MIPII significantly reduced HIV-1JR-FL invasion. Virus infection did not alter the integral TJ protein occludin and the TJ-associated protein ZO-1. HIV-1 exposed CAEC and brain microvascular endothelial cells (BMVEC) developed extensive cytoplasmic vacuolization with retroviral-like particles in the vacuoles. CONCLUSIONS: The endothelium is not an impenetrable barrier to HIV-1. The virus opens a transcellular route across coronary and brain endothelia in cytoplasmic vacuoles.
Arteries↗
Lipopolysaccharide stimulates HIV-1 entry and degradation in human macrophages.
The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and has been shown to protect differentiated macrophages from de novo infection by HIV-1. However, the mechanisms of this inhibitory activity of LPS are not fully understood. We investigated the effect of LPS on the early post-binding steps of HIV-1 replication in primary macrophages. Paradoxically, when applied together with the virus, LPS stimulated entry of HIV-1 into macrophages, as judged by the amount of internalized HIV-1 RNA and p24. This stimulatory activity did not depend on receptors used for entry and did not require new protein synthesis. However, internalized viral RNA and p24 were rapidly degraded in LPS-stimulated macrophages. Surprisingly, while degradation of HIV-1 p24 in LPS-treated cells was inhibited by bafilomycin A1, HIV-1 RNA was not protected by this agent, suggesting that viral RNA is degraded by a pH-independent mechanism. These results indicate that LPS stimulates both virus uptake and virus degradation in macrophages.
Cell Line↗
Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex.
Heat-shock proteins (Hsp's) are a family of molecular chaperones that contribute to protection from environmental stress. In this report, we demonstrate that a member of this family, Hsp70, facilitates nuclear import of HIV-1 preintegration complexes (PICs). The mechanism of this activity appears to be similar to the one used by Vpr, an HIV-1 protein regulating viral nuclear import and replication in macrophages. Indeed Hsp70 stimulated binding of HIV-1 matrix antigen to GST-karyopherin alpha fusion protein and rescued nuclear import of a Vpr-defective HIV-1 strain in vitro. Binding studies with truncated forms of GST-karyopherin alpha demonstrated that both Vpr and Hsp70 bind to a region in the amino-terminal part of the karyopherin alpha molecule. This region appears to be distinct from the binding sites for two other karyopherin alpha cargoes, basic-type NLS-containing proteins and transcription factor STAT-1. Vpr competed with Hsp70 for binding to karyopherin alpha. These results suggest the presence of a novel regulatory site on karyopherin alpha which is used by Hsp70 and Vpr to stimulate interaction between the HIV-1 PIC and karyopherin alpha and thus promote viral nuclear import.
Antigens, Polyomavirus Transforming↗
Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.
Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.
Amino Acid Sequence↗
Lipopolysaccharide inhibits HIV-1 infection of monocyte- derived macrophages through direct and sustained down-regulation of CC chemokine receptor 5.
It is now well established that HIV-1 requires interactions with both CD4 and a chemokine receptor on the host cell surface for efficient infection. The expression of the CCR5 chemokine receptor in human macrophages facilitates HIV-1 entry into these cells, which are considered important in HIV pathogenesis not only as viral reservoirs but also as modulators of altered inflammatory function in HIV disease and AIDS. LPS, a principal constituent of Gram-negative bacterial cell walls, is a potent stimulator of macrophages and has been shown to inhibit HIV infection in this population. We now present evidence that one mechanism by which LPS mediates its inhibitory effect on HIV-1 infection is through a direct and unusually sustained down-regulation of cell-surface CCR5 expression. This LPS-mediated down-regulation of CCR5 expression was independent of de novo protein synthesis and differed from the rapid turnover of these chemokine receptors observed in response to two natural ligands, macrophage-inflammatory protein-1alpha and -1beta. LPS did not act by down-regulating CCR5 mRNA (mRNA levels actually increased slightly after LPS treatment) or by enhancing the degradation of internalized receptor. Rather, the observed failure of LPS-treated macrophages to rapidly restore CCR5 expression at the cell-surface appeared to result from altered recycling of chemokine receptors. Taken together, our results suggest a novel pathway of CCR5 recycling in LPS-stimulated human macrophages that might be targeted to control HIV-1 infection.
CCR5 Receptor Antagonists↗
Small molecule inhibitor of HIV-1 nuclear import suppresses HIV-1 replication in human lymphoid tissue ex vivo: a potential addition to current anti-HIV drug repertoire.
Despite recent progress in anti-HIV therapy, which has to do mainly with introduction of protease inhibitors into clinical practice, drug toxicity and emergence of drug-resistant isolates during the long-term treatment of the patients necessitates search for new drugs that can be added to currently used components of a multi-drug cocktail in highly active anti-retroviral therapy (HAART). Recently, we described a class of arylene bis(methylketone) compounds that inhibit nuclear import of HIV-1 pre-integration complexes and suppress viral replication in macrophages and PBMC in vitro. In this report, we demonstrate that one of these compounds, CNI-H1194, inhibited HIV-1 replication in primary lymphoid tissue ex vivo. The compound did not antagonize the activity of currently used anti-HIV drugs that inhibit viral reverse transcriptase or protease. These results suggest that arylene bis(methylketone) compounds might be a valuable addition to HAART.
Aniline Compounds↗
The B-oligomer of pertussis toxin inhibits human immunodeficiency virus type 1 replication at multiple stages.
We have recently demonstrated that the binding subunit (B-oligomer) of pertussis toxin (PTX-B) deactivates CCR5 and inhibits entry of R5 human immunodeficiency virus type 1 (HIV-1) strains in activated primary T lymphocytes (M. Alfano et al., J. Exp. Med. 190:597-605, 1999). We now present evidence that PTX-B also affects a post-entry step of HIV-1 replication. While PTX-B inhibited fusion induced by R5 but not that induced by X4 envelopes, it blocked infection of T cells with recombinant HIV-1 particles pseudotyped with R5, X4, and even murine leukemia virus or vesicular stomatitis virus envelopes. It also suppressed HIV-1 RNA synthesis in cultures of infected peripheral blood mononuclear cells when new infections had been inhibited by zidovudine, and it reduced Tat-dependent expression of the luciferase reporter gene controlled by the HIV-1 long terminal repeat (LTR). Surprisingly, PTX-B did not affect expression from the cytomegalovirus promoter, nor did it reduce the basal (Tat-independent) expression from the LTR promoter. These results indicate that PTX-B inhibits HIV-1 infection at both the entry and the post-entry stages of viral replication, with the post-entry activity specifically affecting transcription or stability of Tat-stimulated HIV-1 mRNAs.
Cells, Cultured↗
Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response.
BACKGROUND: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. MATERIAL AND METHODS: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. RESULTS: LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. CONCLUSIONS: These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.
Base Sequence↗
The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.
Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein-coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line-adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G(i) proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G(i)-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.
CCR5 Receptor Antagonists↗
Activation-induced resistance of human macrophages to HIV-1 infection in vitro.
Cells of the monocyte/macrophage lineage are the first targets of HIV-1 in patients and also serve as reservoirs for the virus during the course of infection. We investigated the effects of cell activation on early events of HIV-1 infection of monocyte-derived macrophages. Addition of LPS, a potent stimulator of macrophages, at the time of infection stimulated entry of HIV-1 into monocyte-derived macrophages, as judged by accumulation of early products of RT, but inhibited the synthesis of late RT products and strongly repressed nuclear import of the viral DNA, resulting in protection from infection. This effect was mediated by the CD14 receptor and involved activation of the p38 mitogen-activated protein kinase pathway. Disruption of this signaling pathway using a specific inhibitor of the p38 mitogen-activated protein kinase (SB203580) restored HIV-1 infection in the presence of LPS. These results suggest a novel view of the role of macrophage activation in anti-HIV responses of the immune system.
Calcium-Calmodulin-Dependent Protein Kinases↗
Bacterial lipopolysaccharide is a potent inhibitor of HIV-1 replication in T lymphocytes and macrophages.
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Cell Separation↗
Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex.
Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.
Cell Line↗
Role of cyclophilin A in the uptake of HIV-1 by macrophages and T lymphocytes.
Cyclophilins are a family of proteins that bind cyclosporin A (CsA) and possess peptidyl-prolyl cis-trans isomerase activity. In addition, they are secreted by activated cells and act in a cytokine-like manner, presumably via signaling through a cell surface cyclophilin receptor. More recently, host-derived cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 virions and its incorporation essential for viral infectivity. Here we present evidence supporting a role for viral-associated CyPA in the early events of HIV-1 infection. We report that HIV-1 infection of primary peripheral blood mononuclear cells can be inhibited by: (i) an excess of exogenously added CyPA; (ii) a CsA analogue unable to enter the cells; (iii) neutralizing antibodies to CyPA. Taken together with our observations that recombinant CyPA-induced mobilization of calcium in immortalized, as well as primary, CD4+ T lymphocytes, and that incubation of T cells with iodinated CyPA, followed by chemical cross-linking, resulted in the formation of a high molecular mass complex on the cell surface, these results suggest that HIV-1-associated CyPA mediates an early event in viral infection via interaction with a cellular receptor. This interaction may present a target for anti-HIV therapies and vaccines.
Animals↗