This proposal is for continuation of long-standing research studies in our laboratory that focused on HIV-1 cell-specific tropism. This proposal addresses three related topics based on their innovative and unique approaches and promising recent preliminary findings that will provide a comprehensive view of HIV entry. The aims have been focused based on study section critiques and exciting new findings. Aim 1. Cell Biology Studies of Fusion- Role of the Actin Filament Network. We found that inhibitors of actin-based dynamics, as well as dominant-negative mutants of Rhofamily GTPase, Rac, blocked HIV-mediated cell-cell fusion. The current studies ask, a) Is Rac activation required for virus-cell fusion? b) Does SU ligation with CD4 and coreceptor activate Rac? c) Which signaling pathway, activated by Rac, is critical for HIV-1 fusion? Aim 2. Role of Tetraspan Proteins in HIV Entry We will extend preliminary findings suggesting a role of the tetraspan protein, CD9, in HIV entry, to examine its mechanism of action. The current studies ask, a) Is CD9 important in the producer or target cells? b) Does CD9 promote virus uncoating and/or targeting the viral core to the site of PIC formation? c) Do CD9 and CD63 function in the same way in promoting infection? d) What domains of CD9 are critical for this activity? Aim 3. Coreceptor-Envelope Interactions We will extend our previous studies of X4 and SU determinants to map the interactive domains of each. a) We will focus on the minimal domains of X4 extracellular loops (ECL) 2 and 3 that mediate SU binding, fusion, and virus entry, b) We will also examine the evolution of HIV-1 isolates to use X4 using the R5/X4 chimeras.