Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

The lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 incorporate into virions and inhibit viral fusion with target cells, decreasing infectivity. However, whether Nef’s counteraction of SERINC3 and SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was severely attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether or not the coding regions of the serinc3 and serinc5 genes were intact. Moreover, knockout of serinc3 and serinc5 failed to restore the infectivity of HIV-1ΔNef virions produced from infected CEM cells in single-cycle replication experiments using CD4-positive HeLa cells as targets. Taken together, our results corroborate a recent study using another T-lymphoid cell line (MOLT-3) and suggest that Nef modulates a still unidentified host protein(s) to enhance viral growth rate and infectivity in CD4-positive T cells.