Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.

Abstract

Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain.