This is the second re-submission of the PI's first independent support request in which we propose to study the molecular mechanisms involved in beta-chemokine regulation of HIV-1 infection in primary macrophages (versus lymphocytes). The members of the chemokine receptor family, CXCR4 and CCR5, have been shown to act as fusion cofactors for HIV-1 T-tropic (X4) and M-tropic (R5) strains, respectively. The discovery in humans of the association of a homozygous defective CCR5 allele (CCR5 delta32) with resistance to HIV-1 infection demonstrated the importance of CCR5 in HIV-1 transmission. Furthermore, this observation suggested that targeting the HIV-1-CCR5 interaction may provide a means of interfering with HIV-1 infection. However, while a large body of work has demonstrated that the natural ligands of CCR5 (beta-chemokines; MIP-1alpha, MIP-1beta, and RANTES) inhibit HIV-1 replication in lymphocytes, our results demonstrated an enhancing rather than an inhibitory effect of beta-chemokines on HIV-1 replication in primary macrophages. We have begun to address the mechanisms which underlie this phenomenon and our preliminary data suggest that in macrophages beta-chemokine-treatment induces only transient inhibition of HIV-1 entry followed by upregulation of virus replication (inhibition of HIV-1 replication in lymphocytes combines sustained inhibition at the entry and post-entry level). Since macrophages are an important target of HIV-1 during virus transmission, a major goal of this proposal is to define the mechanisms which facilitate upregulation of HIV-1 replication in beta-chemokine-treated macrophages. We propose to characterize the regulation of CCR5 expression by beta-chemokines, and to establish whether altered expression of CCR5 in macrophages (as compared to lymphocytes) contributes to the observed phenomenon. Further, we propose to identify G proteins and downstream effectors activated by beta-chemokine binding to CCR5 receptors expressed in macrophages, and to characterize their effects on the signaling pathways involved in HIV-1 replication in macrophages (lymphocytes will be assayed in parallel for comparison). Together, these proposed studies should provide new insights into the mechanisms by which beta-chemokines regulate HIV-1 infection in primary macrophages, an important target of the virus during transmission of HIV-1 infection, and thus contribute to a better understanding of the unique aspects of AIDS pathogenesis.
