Project Summary The HIV-1 Nef protein enhances the infectivity of virus particles (virions) by an incompletely described mechanism. When virions are produced in the presence of Nef, they are intrinsically more infectious. Recently, two laboratories reported members of the SERINC protein-family as cellular factors that inhibit the infectivity of retroviruses including HIV-1 and that are counteracted by Nef. This finding is consistent with previous data and has opened the field to new questions regarding how the SERINC proteins inhibit viral infectivity and how Nef counteracts them. Our laboratories have a long-standing collaboration to understand at the cell biologic and structural levels how Nef and Vpu hijack the membrane trafficking machinery of the cell to create an environment optimal for viral replication and immune evasion. We plan to leverage this experience to determine how Nef counteracts the SERINC proteins and to determine the relationship between the SERINC proteins and the HIV-1 envelope glycoprotein (Env), the presumed target of SERINC. To accomplish these objectives, we have three specific aims: 1) Identify the determinants in the SERINC proteins and in Nef that are required for the infectivity phenotype. SERINC5 will be emphasized as the prototype family member. Two non- exclusive cell biologic models will be explored: Nef links SERINC5 to the clathrin adaptor AP2 to stimulate removal from the plasma membrane in a manner similar to its action on CD4, and Nef links SERINC5 to AP1 at the trans-Golgi network to stimulate degradation in a manner similar to its action on class I MHC. We will combine our biochemical, cell biologic, and virologic assays to validate and elaborate these mechanisms. 2) Determine the structural basis of SERINC antagonism by Nef. We aim to provide high-resolution structures of the interaction of Nef with SERINC proteins and clathrin adaptors. 3) Determine how SERINC proteins affect infectivity in an Env-dependent manner. To facilitate an understanding of SERINC function, we will characterize a full-length protein biophysically and aim to provide a high-resolution structure. We will test the hypothesis that SERINC proteins interact with Env to inhibit infectivity and map the interaction. We will determine whether SERINC proteins affect the conformation of Env trimers as displayed on the cell surface and on virions. Lastly, we will elaborate a model in which the cytoplasmic domain of Env contributes to counteraction of SERINC proteins. When the proposed experiments are complete, we should have a high- resolution mechanistic understanding of how Nef counteracts the inhibitory influence of the SERINC proteins on viral infectivity, and a model for how the activity of Env is inhibited by these proteins.