HIV infection of target cells requires at least three different molecules, the HIV receptor DC4, a coreceptor such as CXCR4 or CCR5, and the viral envelope protein. Much is known about the structure, biochemistry, and molecular biology of these essential proteins. Unfortunately, our understanding of the cell biology of these molecules and HIV entry limits our ability to understand how these proteins interact over time to mediate fusion between the virial and cellular membranes. To gain insights into this process, we will utilize a system recently developed in my laboratory, which allows individual virions of HIV to be imaged using fluorescent microscopy. The studies outlined here will combine our ability to visualize HIV interaction with living cells with the methods of modern cell biology to gain new insights into the process of HIV entry. Our preliminary studies reveal that the HIV receptor and coreceptor(s) are specifically localized on the cell surface to regions of active membrane activity. This observation suggests that the localization of these molecules is regulated. Using fluorescent protein fusions to CD4 and coreceptor(s) we will determine how the presence of HIV effects the cell biology of these cell surface proteins during infection. Using methods which allow the fusion of viral and cellular membranes to be detected visually we will determine the spatial interactions between the viral envelope protein and receptor/coreceptor(s) required for virion entry. Finally, we will utilize fluorescent HIV to characterize the role of DC-SIGN. The studies proposed here are significant for completing our understanding of HIV entry into cells because they will provide an important'context for the wealth of details which are currently known about HIV entry. This understanding can in turn be useful for the current push to bring compounds which specifically target, and disrupt, HIV entry to the clinic.
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