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Background:  HIV-1 and other primate lentiviruses selectively incorporate cellular membrane proteins that shuttle between endosomes and the plasma membrane. Also, recent biochemical and genetic studies implicate numerous components of the mammalian vacuolar protein sorting pathway in retroviral budding. The endosomal system thus plays an important role during HIV-1 assembly and release. The tetraspanin CD63 is abundantly present in late endosomes and lysosomes. Despite its predominant association with intracellular membranes, this protein is known to be specifically incorporated into HIV-1 not only in macrophages, where this virus buds into late endosomes, but also in cells were HIV-1 buds primarily from the plasma membrane.

Methods:  We have now analyzed the distribution of CD63 at the plasma membrane of such cells and found that this protein is present in discrete micro-domains.

Results:  Combined biochemical and fluorescent microscopy data demonstrate that Gag, the major viral structural component, and the viral envelope glycoprotein localize to these CD63-enriched microdomains. TSG101, a component of the mammalian ESCORT complex I that plays an important role in HIV-1 budding, also localizes to these microdomains, suggesting that viral egress is gated through these membrane segments.

Conclusions:  Utilizing evanescent wave fluorescent microscopy, we are currently investigating the dynamics of formation and turnover of the CD63-enriched microdomains. Because CD63 is a resident of late endosomes/multivesicular bodies, we are also analyzing whether these domains are derivatives of the limiting membrane of that compartment which have been recruited to the cell cortex by focal exocytosis or, alternatively, whether the domains accumulate nascent CD63 that trafficks to late endosomes via the plasma membrane.

Keywords: HIV-1 egress; CD63-enriched membrane segments; late endosomal membranes