HIV Nef-mediated WAVE2-ARP2/3 inhibition underlies CD4+ T-cell lamellipodial abnormalities and immune dysfunction

CD4+ T cells are central regulators of adaptive immune responses, and their depletion following HIV infection leads to AIDS. HIV Nef and Gag weaken CD4+ T cells by disrupting the actin cytoskeleton, which leads to impaired cell migration and immune synapse formation. Several mechanisms of cortical actin disruption have been proposed; however, a unifying and detailed mechanism has remained elusive. This study investigates how HIV alters the actin cytoskeleton of primary CD4+ T cells, aiming to reconcile disparate mechanisms reported in the literature. Using a multi-modal approach combining ultrastructural microscopy, time-lapse imaging, small-molecule inhibitors, and proteomics, we identified identified potential actin regulators affected affectedaffected by HIV. Two distinct lamellipodial abnormalities were observed following infection, both of which are indicative of ARP2/3 inhibition. These morphologies were highly dependent on Nef in vitro and in vivo. Direct chemical inhibition of ARP2/3 recapitulated the lamellipodial defects in Nef-expressing cells. Proteomics of primary cells indicates ARP2/3 inhibition at the lamellipodia of infected T cells may occur through Nef-mediated inhibition of the WAVE2 complex, specifically via inhibitory phosphorylation of WASF2. Together with previous reports of WAVE2 regulation in HIV infection, these results support HIV Nef as a major disruptor of cortical actin through WAVE2-ARP2/3 inhibition at the lamellipodia, providing a novel mechanism of HIV-mediated CD4+ T-cell dysfunction and depletion. IMPORTANCE CD4+ T cells migrate throughout the body and form immune synapses to carry out their functions. Both of these actions require dynamic actin structures, which are disrupted by HIV proteins. Our study suggests that a key HIV protein, Nef, might disrupt a vital internal cellular machinery that helps immune cells move and function properly. Our microscopic and proteomics studies suggest a new model in which Nef inhibits a large protein complex at the front of migrating T cells. Restoring this cytoskeletal dysfunction may be key to restoring CD4+ T-cell survival and function, which may improve adaptive immune responses during HIV infection.