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

Abstract

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.

PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex

“…4), or resulted in a dramatic alteration in the formation of viral DNA ends (Tables 1 and 2). To test this hypothesis, we prepared cytoplasmic extracts containing PICs (Cy-PICs) from SupT1 cells acutely infected with a high concentration (ϳ1500 ng of p24 equivalent) of DNase I-treated HIV-1 particles (72,73,76). These PICs were subjected to integration assay in vitro, and viral DNA integration was quantified by nested qPCR (76,77).…”

“…SupT1 cells were inoculated with high-titer HIV-1 particles (1,500 ng/ml of p24), and PIC-containing cytoplasmic extracts (Cy-PICs) were isolated from these cells. In vitro integration assays using the Cy-PICs as the source of integration activity and quantification of viral DNA integration by nested qPCR were carried out as described previously(76,77).Several controls were included in parallel to ascertain the specificity of the PIC-associated integration activity. (B) Integration activity with different amounts of PICs.…”

Cocaine Enhances DC to T-cell HIV-1 Transmission by Activating DC-SIGN/LARG/LSP1 Complex and Facilitating Infectious Synapse Formation

“…Risky behavior associated with cocaine increases the probability of acquiring HIV and undermines adherence to treatment1234567. However, independent of behavioral factors, studies indicate enhanced replication and transmission of HIV-1 among cocaine using HIV-1 infected individuals489101112131415161718.…”

“…Cocaine is a commonly used illicit drug and prominently linked to HIV-1 infection and spread by both fostering high risk behaviors and facilitating the pathobiology of the virus1234567. Prior studies have shown that cocaine enhances viral replication in various cell types and alters the immune response by regulating the secretion of cytokines and expression of their receptors, accelerating the decline of CD4+ T-cells and disrupting the integrity of the blood-brain barrier489101112131415161718. However, the molecular mechanisms whereby cocaine may act as a cofactor for HIV-1 pathogenesis are not fully defined.…”

Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

“…This method utilizes a nested qPCR-based strategy for measuring PIC-associated IVI activity, and the procedures are adapted, with modifications, from protocols published by the Engelman laboratory 19,20,21 . In this method, the PIC-associated integration activity is initiated by providing a heterologous linear target DNA 21 , and the DNA products resulting from this integration reaction are purified and used as the starting material for subsequent PCR-based quantification. In the first-round conventional PCR, the viral-target DNA junctions are amplified using appropriate primers.…”