The work in my laboratory is focused on understanding a variety of aspects of HIV-1 assembly, release, and maturation. The major areas of investigation are described briefly below. 1. The subcellular targeting of HIV-1 assembly. The production of retrovirus particles from infected cells is mediated by the Gag precursor protein. Following its synthesis in the cytosol, Gag is rapidly and specifically transported to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant HIV-1 Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. We recently determined (Ono & Freed, J. Virol., 2004) that, in HeLa and T cells, the MA-mutant Gags that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that, in primary human macrophages, both wild type (WT) and MA-mutant Gag proteins are targeted specifically to the MVB. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages, and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6Gag and MVB sorting machinery (e.g., Tsg101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and Tsg101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB. Recent studies in the lab have provided evidence of a role for specific phosphoinositides in HIV-1 Gag targeting (Ono et al., PNAS, 2004). 2. Role of plasma membrane rafts in HIV-1 replication. We and others have observed that retroviral Gag proteins, rather than being uniformly distributed at the plasma membrane, concentrate in discrete regions at the cell surface. Since these presumed centers of assembly are likely to be enriched for host proteins that play an active role in virus assembly, Env incorporation, and particle release, it will be very important to determine the composition of these regions. Much excitement and controversy have been generated by the realization that the plasma membrane, rather than being a uniform sea of lipid, contains a variety of microdomains with specific lipid and protein compositions. Of particular interest has been the cholesterol/glycosphingolipid-enriched "rafts." We have demonstrated (Ono & Freed, PNAS, 2001) that HIV-1 Gag associates with rafts, and that disruption of these lipid domains with cholesterol-depleting agents markedly and specifically suppresses virus particle release. We also observe that virions produced from cholesterol-depleted cells display impaired infectivity. A top priority will be to develop a full understanding of the role rafts play throughout the virus replication cycle. 3. HIV-1 Env glycoprotein incorporation and Env/Gag interactions. A critical step in HIV-1 assembly involves the incorporation of the Env glycoproteins into budding particles. For a number of years, we have been investigating the mechanism by which this incorporation takes place. We demonstrated the importance of the long cytoplasmic tail of the transmembrane glycoprotein in Env incorporation, and described the cell-type-dependent nature of this function (Murakami & Freed, PNAS, 2000; Murakami & Freed, J. Virol., 2000).