Characterization of the RNase H activity of HIV-1 reverse transcriptase

The ribonuclease H (RNase H) of HIV-1 reverse transcriptase has been characterized with respect to endonuclease and 3$\sp\prime$-5$\sp\prime$ exonuclease activities by using synthetic homopolymers poly(rA)/poly(dT) and poly(rG)/poly(dC) as model substrates. The present study demonstrates that the RNase H of HIV-1 reverse transcriptase has a 3$\sp\prime$-5$\sp\prime$ exonuclease activity in addition to an endonuclease activity. Whether the RNase H functions as endonuclease or an exonuclease appears to be dependent on the base composition and/or sequence of the RNA/DNA hybrid substrate, as well as on the divalent cation activator. In the presence of Mg$\sp{2+},$ the initial cleavage of poly(rA)/poly(dT), as well as the subsequent generation of smaller oligonucleotides, is carried out by an endonuclease activity. However, when Mn$\sp{2+}$ is the divalent cation activator, it appears that, although the initial cleavage is catalyzed by an endonuclease activity, further hydrolysisis carried out by an exonuclease activity. In contrast to the hydrolysis of poly(rA)/poly(dT), it appears that both endonuclease and exonuclease activities are involved in the digestion of poly(rG)/poly(dC), in the presence of either Mg$\sp{2+}$ or Mn$\sp{2+}.$ With both substrates, the exonuclease activity is significantly more active in Mn$\sp{2+}$ than in Mg$\sp{2+}.$ These findings suggest that the RNase H of HIV-1 reverse transcriptase may exist in two forms: one in which both the endonuclease and exonuclease are active and another in which only the endonuclease is active.The mechanism of inhibition of RNase H by AZTMP has also been investigated by analysis of digestion products in the presence and absence of AZTMP and by kinetic analysis. The correlation of the sensitivity of the RNase H activity of HIV-1 reverse transcriptase to inhibition by AZTMP with the appearance of mononucleotides, and their disappearance in the presence of AZTMP, suggests that the inhibition of RNase H activity is primarily due to inhibition of the exonuclease activity. Analysis of the kinetics of inhibition indicates that AZTMP is a competitive inhibitor with respect to the substrate, suggesting that AZTMP binds at the active site of RNase H. This finding may allow the identification of the substrate contact site by co-crystallization of the p15 RNase H domain with AZTMP