64. Broad-Spectrum Anti-HIV RT RNA Aptamers

Despite recent therapeutic progress, HIV remains a significant problem, due in part to the propensity of the virus to develop resistance. New, broad-spectrum therapeutics targeting diverse HIV strains and drug-resistant mutants could curtail the emergence of viral escape mutants by increasing the genetic barrier to resistance. RNA aptamers that target HIV reverse transcriptase (RT) and inhibit RT activity through competition with primer/template for access to the RT active site. Some aptamer structural families inhibited RT from only certain subtypes of HIV in biochemical assays. For example, family 1 pseudoknots (F1pk) inhibited RT only from HIV-1 subtype B. More recently, we identified several classes of broad spectrum aptamers that biochemically inhibit diverse RT subtypes. However, neither aptamer class has been evaluated in cell culture for the ability to inhibit diverse HIV strains.To evaluate potentially broad-spectrum aptamers in cell culture, we engineered recombinant reporter virus plasmids encoding RT from phylogenetically diverse viruses (HIV-1 subtypes A and B, Group O, and HIV-2) in the HIV-1NL4-3 background, keeping all other viral components constant. Viability of the recombinant viruses was determined using single-cycle infectivity assays to compare each recombinant construct to the parent (HIV-1NL4-3 RT). The new constructs were then tested against aptamers known to inhibit the parent construct. Importantly, aptamer-mediated inhibition correlated with our previous biochemical results when these reporter viruses were produced in cells that expressed aptamers. Broad-spectrum inhibition was observed for one new class of aptamers using representative aptamer 88.1 (6/5 asymmetric loop motif). Notably, aptamer 70.05 (F1pk) inhibited RT from viral strain HXB2 (Group M, subtype B), but not RT from strain MVP5180 (Group O) in infectivity assays. This observation correlates with biochemical results and represents the first demonstration of aptamer-specific resistance in cell culture. In addition, a single point mutation (R277K) demonstrated to confer biochemical resistance also completely abolished inhibition of the provirus expressing HXB2 RT for aptamer 70.05. Thus, viruses expressing RTs from diverse HIV strains in a constant backbone served three functions: (1) to identify RT as the genetic locus responsible for aptamer-mediated inhibition, (2) to demonstrate aptamer-specific resistance for the first time in cell culture, and (3) to assay broad-spectrum activity of our new class of aptamers. Our current work is focused on further exploring aptamer-RT interactions to determine mechanisms governing antiviral specificity and viral encapsidation of anti-HIV RT aptamers.