AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner.