Synthesis and functional characterization of a recombinant HIV-1 reverse transcriptase variant carrying twelve drug-resistance mutations as a platform for antiretroviral screening.

HIV-1 multidrug resistance remains a major barrier to effective long-term antiretroviral therapy and highlights the need for robust biochemical models for screening next-generation inhibitors. Reverse transcriptase (RT) is a central therapeutic target whose inhibition is compromised by the accumulation of resistance mutations that alter substrate usage and reduce drug susceptibility. Here, we engineered, purified, and functionally characterized a recombinant HIV-1 RT variant carrying twelve clinically relevant resistance-associated mutations (12MRT) representative of highly treatment-experienced patient genotypes. The 12MRT enzyme was expressed in E. coli and purified to >95% homogeneity. Kinetic analysis revealed reduced catalytic efficiency, with a 2.1- to 2.6-fold increase in Km and a 30-40% reduction in Vmax relative to wild-type RT (wtRT). In drug susceptibility assays, 12MRT exhibited markedly increased IC50 values for first-generation NNRTIs (8- to 15-fold) and moderate resistance to NRTIs (4- to 7-fold), while retaining measurable polymerase activity suitable for quantitative inhibition studies. Structural analysis suggests that mutation clustering perturbs inhibitor binding without disrupting global folding. Together, these findings demonstrate that 12MRT authentically reproduces multidrug-resistant RT phenotypes and provides a scalable, mechanistically informative platform for preclinical antiviral screening and evaluation of next-generation therapeutic candidates.