The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleosidereverse transcriptase inhibitors alters the size distribution of RNase H cleavages

We investigated the effects of the nonnucleoside reverse transcriptase inhi bitor-resistant mutant Y181C on RNA 5'-end-directed RNase H cleavage by HIV -1 reverse transcriptase, using an RNA DNA hybrid in which a radiolabeled R NA 5' end was recessed. Y181C produced a higher ratio of secondary (9 nucle otide long) to primary (18 nucleotide long) products than wild type. When t he RNA was 3'-end-labeled, Y181C generated a long product, which results wh en secondary cleavage precedes the primary. When using an RNA DNA hybrid in which the labeled RNA 5' end and DNA 3' end were flush, formation of secon dary product by both enzymes was inhibited. Under these conditions, Y181C c leaved closer to the RNA 5' end than wild type. Studies with this substrate labeled at the RNA 3' end showed that Y181C is no more likely than wild ty pe to cleave toward the RNA 3' end. Thus, Y181C RT has a strong preference to cleave in the direction of the RNA 5' end even when secondary cleavage i s prevented, resulting in a disruption of the normal sequence of primary fo llowed by secondary cleavages.