Cyclin T1 has been identi®ed recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is su cient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-speci®c signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the ®rst time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.

A substantial proportion of HIV-1-infected individuals are intravenous drug users (i.v.DUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the mu-opioid receptor. Our results show that DAMGO, a selective mu-opioid agonist, increases CXCR4 and CCR5 expression in both CD3(+) lymphoblasts and CD14(+) monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the mu-opioid receptor based on the ability of a mu-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of mu-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.

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The opiates are well-established immunomodulatory factors, and recent evidence suggests that mu- and delta-opioid receptor ligands alter chemokine-driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of mu- and delta-opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell-derived factor-1alpha/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the micro-opioid receptor to determine whether cross-desensitization of CCR5 was a result of receptor internalization. Using radiolabeled-binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross-desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV-1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV-1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor-dependent HIV-1 infection. The interference with HIV-1 uptake by heterologous desensitization of CCR5 suggests that HIV-1 interaction with this receptor is not passive but involves a signal transduction process.

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