Divalent Cation Modulation of the Ribonuclease Functions of Human Immunodeficiency Virus Reverse Transcriptase

The stimulatory effect of Mg²⁺ and Mn²⁺ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H* activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn²⁺. Enzyme altered at the p66 residue Glu478 (Glu⁴⁷⁸→Gln⁴⁷⁸), which participates in metal ion binding, is completely inactive in Mg²⁺. However, Mn²⁺ restores specifically its endoribonuclease activity. In the presence of Mn²⁺, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.