Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including t he polypurine tracts, remain annealed to the new DNA. Several RTs are belie ved to bind after synthesis to degrade all RNA fragments except the polypur ine tracts by a polymerization-independent mode of RNase H activity. For th is latter process, we found that RT positions the RNase H active site appro ximately 18 nt from the 5' end of the RNA, making the Primary cut. The enzy me rebinds or slides toward the 5' end of the RNA to make a secondary cut c reating two products 8-9 nt long. RT then binds the new 5' end of the RNA c reated by the first primary,or the secondary cuts to make the next primary cut. In addition,, we observed another type of RNase H cleavage specificity . RT aligns the RNase H active site to the 3' end of the RNA, cutting 5 res idues in. We determined the relative rates of these cuts, defining their te mporal order. Results show that the first primary cut is fastest, and the s econdary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progre ssively cleaves RNA fragments.