In addition to studies of coreceptor active sites, we have previously focused on the roles of CD4 and CCR5 cell surface concentrations in controlling HIV-1 infections.  These investigations have implied that a critical complex containing 4-6 CCR5s may be essential, and that infections of cells with suboptimal receptor/coreceptor concentrations and/or mutant coreceptors therefore becomes slow, which results in inefficiency due to a poorly understood competing innate viral death pathway.  Consistent with this kinetic interpretation, entry inhibitors also function more efficiently when assembly of critical viral-receptor complexes becomes slow.  These results raise the possibility that the innate viral decay pathway may be a common final mechanism for viral inactivation by entry inhibitors and neutralizing antibodies.  We propose to extend our mechanistic analyses of HIV-1 infections by: (1) We have isolated variants of the R5 HIV-1 strain JRCSF that efficiently utilize CCR5(delta18), a coreceptor mutant that lacks the amino terminal sulfated region previously believed to be essential for HIV-1 infections.  Analyze the adaptive gp120 mutations, including a surprisingly potent N-glycan loss mutation in the V4 domain. ( 2) Similarly, isolate HIV-1 variants that require only the amino terminal domain of CCR5, using in part an N-glycan addition approach. ( 3) Using our target cells that have specific concentrations of CD4 and coreceptors, analyze the kinetics of viral entry and of the competing viral death pathway.  Learn whether entry inhibitors and neutralizing monoclonal antibodies function by competitive, noncompetitive, or suicide inhibitor mechanisms, a distinction likely to be critical for optimizing drug and vaccine developments.  Thereby, learn whether these agents inactivate HIV-1 or whether they merely slow entry and thereby enable the virus death pathway to predominate. ( 4) Using this kinetic and mechanistic information, investigate the hypothesis that the innate viral death pathway implied by the above studies involves endocytosis.  Determine whether inhibition of endocytosis can in some circumstances enhance HIV-1 titers and reduce efficacies of entry inhibitors and neutralizing antibodies.  This program will provide important cell lines and viruses, methods and information for improving the development of HIV-1 entry inhibitors and vaccine strategies for AIDS.