ABSTRACT The accessory HIV-1 protein Nef is a major pathogenicity determinant that is crucial for high virus loads and for progression to AIDS. Nef increases the specific infectivity of HIV-1 virions, but the mechanism has long remained enigmatic. We have recently identified the multipass transmembrane proteins SERINC3 and SERINC5 as novel anti-viral factors whose down- regulation by Nef accounts for its effect on HIV-1 infectivity. SERINC5 in particular strongly inhibits the infectivity of Nef-deficient HIV-1 progeny virions. HIV-1 Nef induces the removal of SERINC3 and SERINC5 from the surface of virus-producing cells, which prevents their incorporation into HIV-1 virions and consequently counteracts their inhibitory effects on HIV-1 infectivity. However, it remains unclear how SERINCs affect HIV-1 virions. We propose to investigate what determines the susceptibility of HIV-1 envelope glycoproteins to SERINCs, whether SERINCs account for the ability of Nef to protect a membrane-proximal region of gp41, and whether SERINCs affect the viral lipid envelope. It also remains unclear what step during the early phase of HIV-1 replication is inhibited by virion-associated SERINCs. We propose to investigate the hypothesis that SERINCs inhibit the translocation of the mature viral core into target cells. Finally, we propose to investigate what step of SERINC trafficking is affected by Nef, and what determines the ability of Nef to selectively downregulate those SERINC family members that inhibit HIV-1. These studies are significant, because SERINC3 and SERINC5 are highly expressed in primary HIV-1 target cells. Inhibiting their downregulation by Nef is thus a potential strategy to combat HIV/AIDS.