Background:  HIV reverse transcriptase (RT) is a multifunctional enzyme possessing polymerase and ribonuclease (RNase) H activities. However, despite the absolute necessity of the RNase H function for viral replication, all clinically available RT inhibitors target the polymerase function of the enzyme. Although several compounds that block the cleavage reaction in cell-free assays have been described, the specific RNase H inhibitors among these compounds do not appear to exhibit antiviral activity. Here we asked whether the bound nucleic acid substrate in a polymerase-competent configuration may diminish binding of RNase H inhibitors.

Methods:  We have used the hydroxylated tropolone β-thujaplicinol in gel-based assays to determine cleavage positions in the absence and presence of β-thujaplicinol. b-thujaplicinol was tested on binary complexes as well as stalled ternary complexes that were generated with a chain-terminated primer. The next complementary nucleotide stabilizes a ternary complex in its post-translocational state, while the PPi-analogue foscarnet stabilizes the complex in its pre-translocational state. We have analyzed how the formation of these complexes may influence RNase H cleavage and its inhibition.

Results:  In the absence of nucleotide substrate or foscarnet, RNase H cleavage is seen at multiple positions on the template. These reactions are homogeneously blocked with β-thujaplicinol. In the presence of a ligand, RNase H cleavage is restricted to specific cuts at a fixed distance to the polymerase active site. In this case β-thujaplicinol is unable to block cleavage, which suggests that this compound may not bind to a stable ternary complex.

Conclusions:  Here we show mechanistic differences with regards to inhibition of the RT-associated RNase H activity in the context of binary and ternary complexes. Inhibition of RNase H cleavage is severely compromised with stable ternary complexes that freeze the substrate in the binding channel of RT, which is a possible factor that can compromise the antiviral effects of certain RNase H inhibitors.