Both the polymerase and RNase H of HIV-1 RT are essential for viral replication.  After the polymerase has copied the RNA genome, RNase H degrades the RNA, setting the stage for the second strand of viral DNA synthesis.  However, RNase H also generates (and removes) the polypurine tract (PPT) primer, which is used to initiate plus-strand DNA synthesis and removes the tRNA used to initiate minus-strand DNA synthesis.  These events define the ends of the HIV-1 genome; the generation and removal of the PPT primer define the left end of the linear viral DNA; the removal of the tRNA primer defines the right end of the linear viral DNA.  This means that the RNase H must be able to cleave with considerable precision; the ends of the linear DNA are the substrates for the integration reaction.

Since RNase H does not appear to recognize specific sequences, this raises a question of how the enzyme is able to cleave these substrates specifically.  Although we do not have a complete answer, we do have the structure of HIV-1 RT in a complex with an RNA/DNA duplex that contains the PPT.  This structure shows that there are unpaired and mispaired bases in the PPT.  The structure also shows where there are contacts between amino acids in the RT and the RNA/DNA substrate.  Based on the contacts seen in the structure of the RT/RNA/DNA complex, we made mutations in amino acids in the RNase H that contact either the RNA or the DNA strand.  Analysis of the cleavages made by RNase H, both in vitro and in vivo, makes it possible to ascertain which contacts are the most important in determining the specificity of RNase H cleavage.