Subproject for Institution # SP0058262: Hultquist Project for "Functionally Defining HIV-Host Interactions During the Early HIV-1 Lifecycle"

The early phase of the HIV lifecycle encompasses the steps from virus fusion to provirus integration representing a critical therapeutic target. Small molecule inhibitors of HIV encoded reverse transcriptase and integrase are central components of many therapeutic treatment regimens and pre-exposure prophylaxis. Despite the therapeutic importance of these steps, the field still lacks consensus on several outstanding questions including how trafficking and uncoating are linked to reverse transcription, how and in what state the provirus transits through the nuclear pore, what host factors are involved in these processes, and why do so many genomes fail to integrate. Due to the inefficient and relatively stochastic nature of early phase replication, only a small percentage (~15%) of particles that enter the cytoplasm after fusion will result in successful provirus integration. As a result, population-based assays that measure what most viruses do may or may not actually capture what successful viruses do. Nevertheless, technical limitations have historically mandated a reliance on population-based assays, immortalized cell line models, and indirect measurements of biological processes whose underlying assumptions don’t necessarily reflect the biological priors. Only recently have innovations in single-particle tracking, molecular imaging, gene editing, and structural determination allowed for researchers to overcome these limitations, but these specialized technologies have not yet been brought together to answer these critical questions in HIV biology. Here, we assemble a team of HIV researchers with complementary expertise in these powerful approaches to overcome these barriers and to dissect and define the interactions, kinetics, and dynamics between fusion and integration that result in productive infection. We propose to leverage a newly optimized toolbox of molecular labeling methods, a technique collectively termed Infectious Virion Tracking (IVT), to image and track the behavior of individual viral components from fusion to integration, ultimately separating individual virions that result in successful infection from those that enter the cell non-productively. Additional advances in specialized technologies including primary cell CRISPR-Cas9 gene editing to modify host cell genetics and cryogenic electron microscopy (cryo-EM) to analyze complex structure in vivo. Wielding this novel and innovative series of tools, approaches, and equipment, we aim to: 1) Define the infectious pathway of HIV from fusion to integration in optimized cell culture models and primary human target cells; 2) Determine the role of host permissivity factors and viral components in the processes of the early phase of the HIV life-cycle; and 3) Visualize and define the structure of the viral based machines associated with the HIV genome as it progresses through reverse transcription, traffics through the cytoplasm, and ultimately localizes in the nucleus to integrate in the host chromosomal DNA. As a collaborative team with unique specialties that address critical limitations in the field, we are in a unique position to make significant contributions to our current understanding of the early phases of HIV replication.