The chemokine receptors CCR5 & CXCR4 serve as co-receptors for HIV-1 entry. Since their normal function is to transduce signals in response to chemokines, HIV-1 gp12O could also activate intracellular signals, with consequences for pathogenesis by modifying viral entry, post-entry steps or cell functions apart from infection. Ca+2 elevations or protein phosphorylation responses to gp12O through CCR5 or CXCR4 have been reported in lymphoid cells in some but not all studies. While co-receptor signaling domains are dispensable for entry in transfected cell lines, recent studies suggest that signaling may affect entry or post-entry infection steps in some primary cells. Macrophages express CD4, CCR5 & CXCR4, and are important targets of HIV-1 in vivo but gpl2O/chemokine receptor signaling has not been addressed in them. Indeed, relatively little is known in general about mechanisms of chemokine receptor signaling in primary macrophages. In preliminary studies, we have found that gp12O initiates intracellular signals in primary human macrophages through CCR5 & CXCR4, activates K+, Cr, & non-selective cation channels, and elevates intracellular Ca +. R5 and X5 gp12O elicited qualitatively similar but quantitatively different responses and, unexpectedly, the patterns of ion channel signaling elicited by gp120 differed from those elicited by the receptors' natural chemokine ligands. Our hypothesis is that HIV-1 Env initiates intracellular signals in macrophages through the co-receptors that lead to critical alterations in cellular function, virus entry, and/or post-entry steps of infection. To better understand the mechanisms of chemokine receptor signaling & ion channel activation in macrophages by gp120 (as well as chemokines), and consequences of gp120-mediated signals for macrophage function & infection, we will (a) Define the ionic signaling pathways activated in primary macrophages by gp 120 using primary & prototype HIV- 1 & SIV strains and strains that differ in ability to utilize macrophage chemokine receptors, and chemokines; (b) Identify the mechanisms of chemokine receptor ionic signaling in macrophages by defining CCR5 & CXCR4 coupling to K+, Cl- & non-selective cation channels, role of CD4 co-engagement, source & coupling for Ca2 elevations, and structural elements in CCR5 involved; (c) Determine the role of Env signaling in entry, infection & replication in macrophages, including the formation & significance of gp12O-induced capping as well as post-entry events, and; (d) Determine the effects of gp120 signaling on macrophage function such as aberrant secretion of mediators, phagocytosis, and killing.
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