Structural Basis for HIV-1 Rev Nucleocytoplasmic Trafficking

Abstract Human immunodeficiency virus 1 (HIV-1) targets the human immune system and can lead to the development of acquired immunodeficiency syndrome (AIDS), increasing susceptibility to opportunistic infections and tumors. Current HIV-1/AIDS treatments involve the utilization of combination antiretroviral therapies (cART) that target and inhibit multiple steps of the HIV-1 replication cycle. People living with HIV must stay on treatment for the rest of their lives, which increases the risk of HIV-1 developing resistance to antiretrovirals. As a result, there is a need for the development of novel antiretrovirals. One target for possible antiretroviral treatment is the HIV-1 protein called Rev (regulator of expression of virion proteins). Rev functions as a nucleocytoplasmic shuttling protein for the nuclear export of viral mRNA necessary for virus assembly. To properly mediate the export of this mRNA, Rev must be able to translocate back and forth through the nuclear pore complex (NPC). Rev contains a nuclear localization signal and a nuclear export signal able to bind host importins and exportins, which are responsible for host cargo nucleocytoplasmic trafficking. This F31 proposal seeks to decipher how HIV-1 Rev is recognized, bound, and trafficked through the NPC by the host nuclear transport machinery. We hypothesize that Rev hijacks human importins and exportins, specifically importin-β and Crm1 (or exportin), for trafficking Rev through the NPC and preventing Rev aggregation and non-specific RNA binding. Aim 1 of this proposal explores binding determinants between HIV-1 Rev and importin-β. We will utilize cryogenic-electron microscopy (cryo-EM) and pull-down and turbidity assays to visualize and characterize the importin-β:Rev structure. Aim 2 explores the mechanisms of Crm1 nuclear export of Rev alone and bound to the HIV-1 genome. To better understand these mechanisms, we will use cryo-EM to determine the structures of Crm1 bound to Rev before and after adding viral mRNA.