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

Abstract

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, SERINC5 incorporates into virions and inhibits viral fusion with target cells, decreasing infectivity. However, whether Nefs counteraction of 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 relatively robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1{Delta}Nef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc3 or serinc5 failed to restore the infectivity of HIV1{Delta}Nef virions produced from infected CEM cells. Taken together, our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 cannot fully explain the virology of HIV-1 lacking Nef.