Statin-induced inhibition of HIV-1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV-1 replication.

Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/gammac null mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy.     

Expanding the Clinical Indications for α1-Antitrypsin Therapy

α₁-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor. Production of AAT by the liver maintains 0.9–1.75 mg/mL circulating levels. During acute-phase responses, circulating AAT levels increase more than fourfold. In individuals with one of several inherited mutations in AAT, low circulating levels increase the risk for lung, liver and pancreatic destructive diseases, particularly emphysema. These individuals are treated with lifelong weekly infusions of human plasma–derived AAT. An increasing amount of evidence appears to suggest that AAT possesses not only the ability to inhibit serine proteases, such as elastase and proteinase-3 (PR-3), but also to exert antiinflammatory and tissue-protective effects independent of protease inhibition. AAT modifies dendritic cell maturation and promotes T regulatory cell differentiation, induces interleukin (IL)-1 receptor antagonist and IL-10 release, protects various cell types from cell death, inhibits caspases-1 and -3 activity and inhibits IL-1 production and activity. Importantly, unlike classic immunosuppressants, AAT allows undeterred isolated T-lymphocyte responses. On the basis of preclinical and clinical studies, AAT therapy for nondeficient individuals may interfere with disease progression in type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and human immunodeficiency virus (HIV), and is currently evaluated in clinical trials for type 1 diabetes, cystic fibrosis and graft versus host disease. Thus, AAT therapy appears to have advanced from replacement therapy, to a safe and potential treatment for a broad spectrum of inflammatory and immune-mediated diseases.     

Mechanisms for macrophage-mediated HIV-1 induction

Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.     

Picomolar dichotomous activity of gnidimacrin against HIV-1

Highly active antiretroviral therapy (HAART) has offered a promising approach for controlling HIV-1 replication in infected individuals. However, with HARRT, HIV-1 is suppressed rather than eradicated due to persistence of HIV-1 in latent viral reservoirs. Thus, purging the virus from latent reservoirs is an important strategy toward eradicating HIV-1 infection. In this study, we discovered that the daphnane diterpene gnidimacrin, which was previously reported to have potent anti-cancer cell activity, activated HIV-1 replication and killed persistently-infected cells at picomolar concentrations. In addition to its potential to purge HIV-1 from latently infected cells, gnidimacrin potently inhibited a panel of HIV-1 R5 virus infection of peripheral blood mononuclear cells (PBMCs) at an average concentration lower than 10 pM. In contrast, gnidimacrin only partially inhibited HIV-1 ×4 virus infection of PBMCs. The strong anti-HIV-1 R5 virus activity of gnidimacrin was correlated with its effect on down-regulation of the HIV-1 coreceptor CCR5. The anti-R5 virus activity of gnidimacrin was completely abrogated by a selective protein kinase C beta inhibitor enzastaurin, which suggests that protein kinase C beta plays a key role in the potent anti-HIV-1 activity of gnidimacrin in PBMCs. In summary, these results suggest that gnidimacrin could activate latent HIV-1, specifically kill HIV-1 persistently infected cells, and inhibit R5 viruses at picomolar concentrations.     

Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression.

We have designed a ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in U5 (at nucleotide +115). This ribozyme was tested in vitro and was found to give efficient and specific digestion of RNA containing the HIV-1 U5 sequence. When the U5 ribozyme was placed into the HIV-1 genome, virus replication was suppressed in tissue culture. Introduction of this ribozyme into cells by using an amphotropic retrovirus vector significantly reduced expression of U5-containing RNA in cells chronically infected with HIV-1. Naive T cells were cocultivated with packaging cells that produce defective amphotropic retroviruses containing the U5 ribozyme. These lymphocytes were found to be partially protected from HIV-1 infection.     

Macrophage colony-stimulating factor antagonists inhibit replication of HIV-1 in human macrophages

Macrophages infected with HIV-1 produce high levels of M-CSF and macrophage-inflammatory protein-1alpha (MIP-1alpha). M-CSF facilitates the growth and differentiation of macrophages, while the chemotactic properties of MIP-1alpha attract both T lymphocytes and macrophages to the site of HIV infection. Studies described in this work indicate M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and data suggest possible therapeutic strategies for decreasing viral load following HIV infection. We show that macrophage infection with measles virus or respiratory syncytial virus, in contrast to HIV-1, results in production of MIP-1alpha, but not M-CSF. Thus, M-CSF appears to be specifically produced upon infection of macrophages with HIV-1. Furthermore, addition of M-CSF antagonists to HIV-1-infected macrophages, including anti-M-CSF monoclonal or polyclonal Abs or soluble M-CSF receptors, dramatically inhibited HIV-1 replication and reduced production of MIP-1alpha. Our results suggest that biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-1 by 1) blocking virus replication in macrophages, 2) reducing recruitment of HIV-susceptible T cells and macrophages by MIP-1alpha, and 3) preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Chemokine receptor-usage of clinical HIV-1 isolates obtained from patients with HIV-1 infection in late clinical stages using PHA-blast

In the present sudy, chemokine receptor-usage of primary HIV-1 isolates was examined using U87-CD4 cells expressing chemokine receptors CCR3, CCR5 and CXCR4. HIV-1 was isolated from the peripheral blood mononuclear cells (PBMC) and/or plasma of eight HIV-1-infected individuals in late CDC-II and CDC-IV clinical stages using PHA-blast prepared from the PBMC of healthy blood donors. The primary HIV-1 isolates from patients in late CDC-II stage rarely infected monocyte-derived macrophages in the present study, whereas most isolates from patients in the CDC-IV stage infected the macrophages. In the experiments using U87-CD4 cells expressing chemokine receptors, the isolates from patients in the late CDC-II stage infected U87-CD4 cells expressing CXCR4, but not U87-CD4 cells expressing CCR5. In contrast, most isolates from patients in the CDC-IV stage infected both U87-CD4 cells expressing CXCR4 or CCR5. The isolates which infected both U87-CD4 cells were supposed to contain dual tropic HIV-1 or a mixture of CXCR4-tropic and CCR5-tropic HIV-1s. Analysis of the deduced amino acid sequence of the V3 region in proviral env gene showed that the V3 region in U87-CD4 cells infected with CXCR4-tropic HIV-1 isolates was largely different from that in the cells infected with CCR5-tropic isolates, but were highly similar to that in cells infected with dual tropic isolates. These results suggest that PHA-blasts may preferentially support the replication of the CXCR4-tropic and dual tropic HIV-1s, and that CXCR4-tropic and dual tropic HIV-1s are also present in peripheral blood from patients in the late stage of the asymptomatic phase.     

Expression of HIV-1 nef decreases basal phosphatidyl-inositol 3-kinase activity

CEM lymphoid cells expressing high levels of HIV-1 nef protein displayed a significant decrease in basal phosphatidyl-inositol 3-kinase (PI3-kinase) activity associated with immunoprecipitates with anti-p85 regulatory subunit. In contrast, chronically infected U937 monocytic cells displayed a significant increase in basal P13-kinase activity in cells infected with HIV-1 nef⁻ compared to those infected with isogenic HIV-1 nef⁺. These findings suggest that HIV-1-nef expression is accompanied by a decrease in basal intracellular phosphatidyl-inositol 3-kinase activity and suggest that PI3-kinase could be important for HIV-1 replication. Moreover, wortmannin, a potent in-vitro phosphatidyl-inositol 3-kinase inhibitor, can inhibit HIV-1 replication in U937 chronically infected cells. Together these results suggest a correlation between PI3-kinase activity and HIV-1 replication.     

Expanding the Clinical Indications for α(1)-Antitrypsin Therapy

α(1)-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor. Production of AAT by the liver maintains 0.9-1.75 mg/mL circulating levels. During acute-phase responses, circulating AAT levels increase more than fourfold. In individuals with one of several inherited mutations in AAT, low circulating levels increase the risk for lung, liver and pancreatic destructive diseases, particularly emphysema. These individuals are treated with lifelong weekly infusions of human plasma-derived AAT. An increasing amount of evidence appears to suggest that AAT possesses not only the ability to inhibit serine proteases, such as elastase and proteinase-3 (PR-3), but also to exert antiinflammatory and tissue-protective effects independent of protease inhibition. AAT modifies dendritic cell maturation and promotes T regulatory cell differentiation, induces interleukin (IL)-1 receptor antagonist and IL-10 release, protects various cell types from cell death, inhibits caspases-1 and -3 activity and inhibits IL-1 production and activity. Importantly, unlike classic immunosuppressants, AAT allows undeterred isolated T-lymphocyte responses. On the basis of preclinical and clinical studies, AAT therapy for nondeficient individuals may interfere with disease progression in type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and human immunodeficiency virus (HIV), and is currently evaluated in clinical trials for type 1 diabetes, cystic fibrosis and graft versus host disease. Thus, AAT therapy appears to have advanced from replacement therapy, to a safe and potential treatment for a broad spectrum of inflammatory and immune-mediated diseases.     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.     

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.