PROJECT SUMMARY/ABSTRACT Human Immunodeficiency Virus 1 (HIV-1) is the causative agent behind acquired immunodeficiency syndrome (AIDS) and the resulting pandemic. Antiretroviral therapies are effective in controlling infections, however patients require lifelong treatment and can have severe side effects, resulting in the constant need for novel therapeutics. Therefore, a better understanding of the interaction between host cells and HIV-1 can unmask novel targets for therapeutics. Recent reports identified that MARCH1, 2 and 8, members of a family of host E3 ubiquitin ligases known as Membrane Associated RING-CH (MARCH) proteins, whose physiological functions involve immune receptor homeostasis, restrict HIV-1 by blocking the incorporation of viral envelope glycoproteins in nascent virions. Another member of the MARCH protein family, MARCH9, reduces HIV-1 infectivity, however, its mechanism of restriction is unknown and different from that of MARCH1, 2 and 8. MARCH9 targets immune cell receptors for ubiquitin and subsequential lysosomal degradation as a means of maintaining homeostasis including MHC-1 and ICAM-1. Preliminary data shown in this proposal demonstrate that MARCH9 reduces the cellular levels of all HIV-1 structural proteins thereby resulting in reduced viral particle production, which is quite different from what has been shown with MARCH1, 2 and 8. Additional data show that MARCH9 does not affect cell viability. The goal of this proposal is to determine the mechanism by which MARCH9 restricts HIV-1 infection. The experiments outlined here will characterize the step in the HIV-1 life cycle that MARCH9 acts on to inhibit HIV-1 particle production and the mechanism it utilizes to do so. Additionally, we will determine the role of endogenous MARCH9 through shRNA knockdown on CD4+ T cells and Monocyte derived Macrophages (MDMs) to better elucidate its antiviral mechanism. The proposed work will determine the role of MARCH9 in HIV-1 infection. In doing so, this proposal will provide new insights into the role of the MARCH protein family during retrovirus infection. Finally, the findings of this proposal have the potential to provide new therapeutic targets for the development of novel antiretrovirals.