Session 4Oral Abstract Presentations HIV Replication: Entry and Assembly Session Day and Time: Tuesday 10 am - 12:30 pm Presentation Time: 10:45 Room: Ballroom A
20 Recruitment of CD4, CCR5, Rafts, and Ezrin in Actin-dependent Cell Surface Structures: Implications for HIV Fusion and Entry Events C.M. Steffens*, T.J. Hope Univ of Illinois at Chicago
Background: The events leading to HIV fusion and entry require co-operative interactions between the receptor, co-receptor, and viral envelope proteins. These interactions are dependent on the concentration and distribution of receptor and co-receptor molecules on the cell surface. However, much of this process is still unexplained. Previous studies have demonstrated the importance of actin and lipid rafts in entry events, but the mechanism of their involvement is unclear. The extent to which these proteins co-localize in the cell membrane remains undetermined and the mechanism of fusion pore formation is still unknown.
Methods: To gain insight into the association of CD4 and CCR5, we conducted immunofluorescent analysis of CD4 and CCR5 using high-resolution deconvolution microscopy. Key for these experiments were fluorescent fusion constructs linking CD4 to yellow fluorescent protein (YFP) and CCR5 to cyan fluorescent protein (CFP). Both fusion constructs were functional as HIV receptor and co-receptor.
Results: Our studies demonstrate that CD4 and CCR5 accumulate in membrane protrusions. Although both proteins localize in the same cell-surface structures, the ability to induce the internalization of each protein independently suggests that these proteins do not exist in a complex. Interestingly, we find that ezrin is a marker for these actin-dependent structures, which also contain increased amounts of the lipid raft marker GM1.
Conclusions: Current models of HIV entry suggest that multiple receptor and co-receptor molecules are needed to interact with each envelope trimer, and that multiple trimers are necessary for fusion to occur. Our observation that CD4, CCR5, ezrin, and GM1 are all present in the same actin-dependent structures may unify previous reports that lipid rafts and actin are important in facilitating the interactions leading up to HIV fusion and entry. We find that all of these proteins accumulate in distinct structures which likely are important for entry. For example, crosslinking of receptor and co-receptor induces signaling through the formation of ezrin and lipid raft containing structures, and hence the observed recruitment. Thus, the preferential localization of both CD4 and CCR5 in actin-dependent structures may have broad implications for how HIV interacts with receptor and co-receptor to generate a potential site of fusion pore formation.
