Background:
Lenacapavir (LEN) is the first capsid inhibitor to be FDA-approved for HIV-1 treatment. Despite high potency and slow-release kinetics, a significant drawback of LEN is its low barrier to viral resistance. A mutation in the HIV-1 capsid, M66I, confers > 80,000-fold resistance to LEN and has been observed in cultured cells and in HIV-1-infected individuals treated with LEN. However, in the absence of LEN, M66I causes a substantial defect in viral fitness (<5% infectivity relative to WT). Given the high mutation rate of HIV-1, it is important to understand how HIV can adapt to circumvent the M66I-induced fitness defect before compensatory mutations are manifested in patients.
Methods:
We propagated LEN-resistant HIV-1 mutants (pNL4-3) in T-cell lines (SupT1 and MT4) to select for compensatory mutations. Replicating viruses were sequenced to identify compensatory mutations. Identified mutations were sub-cloned into pNL4-3 to examine their effects on virus infectivity and drug sensitivity.
Results:
M66I propagation in T-cells repeatedly led to WT reversion (I66M). We examined the effects of mutating M66 to other amino acids and determined whether these substitutions recapitulate the behavior of M66I, specifically its resistance to LEN and fitness defect. Of the M66 mutants examined, M66L, M66V, and M66F exhibited similar infectivity defects evident in M66I, but only M66V displayed high-level resistance to LEN. Propagation of these M66 mutants led to several second-site mutations. Of note, H12Y, in combination with A105T and other capsid substitutions, resulted in a >10-fold rescue of M66L infectivity.
Conclusions:
This study investigates viral escape strategies of M66I, a highly LEN-resistant but fitness-impaired HIV-1 mutant. As a clinically significant variant, this work will reveal important insights into how HIV-1 may maintain LEN resistance while bypassing the fitness defect inherent to M66I.