Molecular investigations of HIV-1 maturation pathways and inhibitor actions in situ

PROJECT SUMMARY: The HIV-1 life cycle critically depends on a maturation process that transforms the viral core from a spherical to a conical shape, a crucial step for productive infection of host cells. This process is driven by the proteolytic cleavage of Gag and Gag-Pol polyproteins, resulting in the formation of the mature capsid. Although significant progress has been made in understanding Gag assembly and capsid structures, the dynamic transitions and molecular mechanisms underlying HIV-1 maturation remain inadequately understood, particularly within native cellular environments. To address these gaps, the Xiong laboratory has developed an advanced in situ cryo- electron microscopy (cryo-EM) approach, enabling high-resolution visualization of these processes in their native context. The research is structured around two specific aims: Aim 1 focuses on determining the high- resolution structural pathway of HIV-1 Gag assemblies and capsid during maturation within their native environment. This will involve using in situ cryo-EM and cryo-electron tomography (cryo-ET) to visualize the complex structural transitions of HIV-1 Gag and capsid during maturation. These studies will be conducted on both purified virus-like particles (VLPs) and budding viruses from infected cells, including clinically derived HIV- 1 isolates and primary CD4+ T cells. Preliminary data identify a novel hybrid capsid structure that may represent a transitional state in HIV-1 maturation, supporting the hypothesis of a replacive maturation pathway. This will be further tested through biochemical, structural, and functional experiments. Aim 2 will define the mechanisms by which HIV-1 capsid cofactors and maturation inhibitors influence virus maturation during budding. This will involve investigating how capsid inhibitors impact the maturation process using a comprehensive experimental system, including in vitro assembled Gag constructs, purified VLPs, and budding viruses on the cell surface. Preliminary findings have revealed new binding sites for Lenacapavir and a novel cellular cofactor within the immature Gag lattice. The cellular cofactor will be thoroughly investigated to determine its identity and function. The insights gained from this research will advance the understanding of HIV-1 maturation, guide the development of new therapeutic strategies targeting the immature Gag structures and the capsid, and provide valuable tools for studying host-virus interactions within their native cellular environments.