Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients.

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4(+) T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM-100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4(+) T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4(+) T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4(+) T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients.

Cocaine enhances HIV-1-induced CD4(+) T-cell apoptosis: implications in disease progression in cocaine-abusing HIV-1 patients.

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 µmol/L concentrations) did not induce apoptosis, but 200 to 1000 µmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.

Effect of dietary fat on metabolism and DNA adduct formation after acute oral exposure of F-344 rats to fluoranthene.

Adverse health effects such as cancer and toxicity may be attributed to consumption of chemically contaminated food rich in fat. This leads to a larger intake and retention of lipophilic toxic chemicals in the body with an increase in risks to human health. The objective of this study was to characterize the effect of dietary fat on disposition and metabolism of fluoranthene (FLA), a polycyclic aromatic hydrocarbon compound. FLA was administered to F-344 rats in monounsaturated (peanut oil), polyunsaturated (corn oil) and saturated (coconut oil) fats at doses of 50 and 100 microg/kg via oral gavage. Blood, small intestine, liver, lung, testis, adipose tissue, urine and feces were collected at various time points' post-FLA exposure. Samples were analyzed by reverse-phase high-performance liquid chromatography for FLA parent compound and metabolites. DNA was isolated from the tissues and subjected to (32)P-post labeling to measure FLA-DNA adducts. The concentrations of unchanged FLA (FLA parent compound) and its metabolites showed an increase for the saturated fat treatment group compared with mono- and polyunsaturated fat groups. The FLA-DNA adduct concentrations were high in tissues of rats that received FLA through saturated fat. The toxicokinetic parameters, concentrations of FLA metabolites and FLA-DNA adduct showed a dose-dependent increase, and this increase was statistically significant (P<.05) for saturated fat. These findings clearly demonstrate that the high residence time of FLA parent compound in saturated fat allows extensive metabolism, contributing reactive metabolites of FLA that bind with DNA and causing marked damage in a long-term exposure scenario.