Ancestral reconstruction supports that loss of Nef-mediated T cell modulation coincided with the emergence of pathogenic lentiviruses.

Human immunodeficiency virus type 1 (HIV-1) originated following cross-species transmission of simian immunodeficiency virus (SIV) infecting chimpanzees (SIVcpz). While SIV infection of its natural primate hosts is typically non-pathogenic, SIVcpz/HIV-1 is highly pathogenic. SIV/HIV pathogenesis is influenced by the viral accessory protein Nef, which manipulates several immune factors to facilitate disease progression. Specifically, Nef can modulate T cell activation by downregulating the T cell receptor subunit CD3ζ and the co-stimulatory receptor CD28. Several studies have demonstrated that while Nef downregulates CD28 from the T cell surface, this ability is not completely conserved throughout the SIV/HIV phylogeny. In comparison, Nef-mediated CD3 downregulation is highly conserved in SIV derived from lower-order primates yet completely absent from SIVcpz/HIV-1. Here, we used Nef proteins previously generated via an ancestral reconstruction pipeline, corresponding to common ancestors in the SIV/HIV phylogeny leading to HIV-1 group M, and evaluated their ability to downregulate CD3ζ and CD28 in both expression and transduction systems. We observed that only the ancestral Nef of all primate lentiviruses efficiently downregulated both CD3ζ and CD28, while ancestors along the SIVcpz/HIV-1 lineage failed to downregulate CD3ζ and minimally recovered the ability to downregulate CD28 over time. Furthermore, we showed that the loss of CD3ζ and CD28 downregulation was associated with increased expression of cell surface T cell activation markers and inflammatory cytokines, consistent with a pathogenic infection. Altogether, this study illustrates the evolutionary dynamics of Nef modulation of T cell activation and its impact on pathogenesis in the SIV/HIV lineage.IMPORTANCEWe characterized ancestral Nef proteins derived from the lineage that gave rise to HIV-1 group M. We show that the loss of Nef-mediated CD3ζ and CD28 downregulation occurred in the common ancestor of the SIVcpz/HIV-1 lineage and coincided with increased T cell activation, a hallmark of pathogenicity. Moreover, this suggests that evolutionary changes to Nef-mediated immune modulation contributed to the emergence of pathogenic viruses like HIV-1. Together, these findings support the application of ancestral reconstruction to better understand the functional evolution of viral proteins, particularly in the context of cross-species transmission.