EXCEEDTHE SPACE PROVIDED. HIV infection of primary human cells typically requires interaction with both the primary receptor CD4 and a chemokinecoreceptor either CCRS or CXCR4. We have discovered a potential role for CD63 in HIV infection of M0, as monoclonal antibodies (mAb specific for CD63 can prevent HIV entry into MO, but not T-cells. CD63 is a tetraspan membrane glycoprotein, best known as a1 activation marker on platelets, that is nearly ubiquitous on human cells and is closely associated with integrins. Our experimentswil investigate the role of CD63 in HIV entry into M0. These studies may lead to developmentof novel antiretroviraltherapies targetini HIV entry. We will pursue these Aims over five years:
Specific Aim 1. To test the hypothesis that infection of M0by diverse primary HIV-1 strains can be inhibited by anti-CD63 mAb This will include both subtype B and non-subtypeB strains, includingsome that use CCRS as a coreceptor (R5), as well as dual-tropic strains that use either CXCR4 or CCRS (RSX4), and X4 strains able to replicate in M0. Amphotropic MLV-Env-pseudotyped virus which enters independent of CD4 and CCRYCXCR4 interactions, will be assessed to determine if anti-CD63 irhbition acts at post binding retroviral entry events.
Specific Aim 2. To test the hypothesis that cell-specific differences between HIV target cells affect the role of CD63 in facilitatini HIV entry. We will control for and test effects of various levels of receptor/coreceptor/CD63 expression and activation markers 01 susceptibility to anti-CD63 inhibition. We will also test cell line models of anti-CD63 inhibition, which would facilitate cel manipulation and the consistencycif lnhibitoryeffects in these studies,and in the mechanistic studiesin Aim 3.
Specific Aim 3. To test the hypothesis that CD63 has a specific role in facilitating HIV entry into macrophages, we will identify potential mechanisms of CD63-mediatedHIV entry, including (a) receptor/coreceptor interactions or colocalization with CD63, (b) CD63 binding to gp120, (c) the role of CD63 in CD4 turnover or virus/receptor endocytosis and (d) signal transduction. These experiments will be prioritized based on anti-CD63 susceptibility of MLV-Env pseudotyped virus (and X4 HIV-1 strains), which would direct us to focus on late events if inhibition is not dependent on CD4 and CCRS-associated events. Achieving these Aims will provide important new insights into the mechanism of HIV-1 entry, and may well provide important new targets for antiretroviraltherapy. PERFORMANCESITE( ========================================Section End===========================================
