A Deep Mutational Scanning Platform to Map the Landscape of HIV-1 Capsid Fitness and Resistance to Lenacapavir

About This Grant

PROJECT SUMMARY The HIV-1 capsid protein is a validated target for antiretroviral therapeutics, as demonstrated by the recent FDA approval of lenacapavir (LEN). Administered every six months, LEN effectively reduces viral loads and prevents HIV-1 infection. Despite its efficacy, resistance mutations have emerged in 19% of treated individuals enrolled in the CAPELLA clinical trial of this drug. Specific mutations and mutation combinations were repeatedly observed; however, the mechanisms underlying their recurrence remain unknown. Conventional single-variant mutagenesis approaches do not allow systematic evaluation of all mutations and their combinations to explain the basis for such preferences. This project will develop a new deep mutational scanning (DMS) platform to systematically evaluate how all amino changes in the capsid protein affect HIV-1 fitness and resistance to capsid inhibitors. Building on our prior work with DMS systems that apply replication-competent virus, we will establish a DMS system based on replication-defective virus to enable safe, accurate and high-throughput profiling of all capsid variants. We will first develop the single-site DMS system to examine the effects of all amino acid changes at individual sites of the protein. We will optimize the accuracy of DMS measurements, determine their reproducibility using three independent biological replicates for each site, and compare outputs using different cell types. Fitness and LEN-resistance profiles measured by DMS will be validated using standard single-variant assays with replication-competent and replication-defective HIV-1. To address the frequent emergence of specific mutation combinations in clinical and in vitro studies, we will extend this platform to perform dual-site DMS. This approach will resolve epistatic interactions between key capsid residues linked to LEN resistance and quantitatively determine how all combinations at two positions of the protein affect viral fitness and drug resistance. The proposed work will generate a powerful experimental framework to comprehensively map the mutational space of HIV-1 capsid. These insights will improve our understanding of resistance mechanisms to capsid inhibitors, inform prediction of clinically relevant escape pathways, and guide the design of next-generation capsid-targeting antivirals.