Conformational Dynamics in HIV-1 Gag during Viral Maturation

About This Grant

Project Summary Human immunodeficiency virus type 1 (HIV-1) remains a major global health challenge. While viral maturation has long been a promising target for inhibitor development, the emergence of resistance to protease inhibitors continues to limit their effectiveness. As such, the Group-Specific Antigen (Gag) polyprotein presents an alternative avenue for therapeutic intervention, marked by the recent development of novel antiretrovirals such as bevirimat and lenacapavir, which target the immature Gag and mature capsid (CA). Gag encodes the core structural proteins that form the viral particle and undergoes cleavage by the viral protease during maturation. Among the domains in Gag, CA-SP1 cleavage is the key determinant for CA maturation, a process marked by CA undergoing drastic conformational changes. In Aim 1, I will elucidate how the stability of SP1 helices evolves during proteolysis. SP1 helices are arranged mostly in relatively stable six-helix bundles in the immature lattice but are bound to the protease extended and unfolded. It remains unclear how SP1 loses its secondary structure as it binds to the protease. I hypothesize that the initial cleavage of a single SP1 helix destabilizes the six-helix bundle, promoting the unfolding of neighboring helices and allowing for easier access to the protease. Using MD simulations, I will elucidate and compare the unfolding timescale and mechanisms of SP1 helices within fully intact and partially cleaved hexamers. I predict that SP1 helices in the cleaved hexamers will unfold more readily, whereas the intact hexamer will remain predominantly stable. These simulations will provide mechanistic insights into how SP1 proteolysis propagates structural changes through the hexamer and contributes to how the crucial last step of Gag proteolytic processing occurs. The expertise from the Schiffer lab provides me with the necessary training needed to perform robust structural modeling and molecular dynamics simulations. In Aim 2, I will investigate how SP1 cleavage mediates CA conformational changes that facilitate maturation. The destabilization of the six-helix bundle has been identified as the key structural switch for CA maturation. Using smFRET, I will visualize the conformational changes of single CA molecules within oligomeric assemblies. I predict that destabilization of the six-helix bundle following CA-SP1 cleavage induces conformational changes to the CA domain that are relevant for virion maturation. These findings will provide mechanistic insights into the allosteric communication between the CA-SP1 cleavage site and CA domains, revealing how initial cleavage events prime the hexamer for further structural transitions. The Munro lab has a proven track record and expertise in visualizing dynamic events in virions using single molecule fluorescence- based techniques. This study will significantly advance our understanding of the molecular mechanisms underlying HIV-1 maturation, potentially shedding light on the maturation processes of other retroviruses.