Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase ab: Localization of the Polymerase and RNase H Active Sites in the a Subunit

The genes encoding the a (63-kDa) and b (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV ab and aPol RTs within which one subunit was selectively mutated. When ab heterodimers contained the Asp 1813Asn mutation in their b subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their a subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in ab heterodimers mutated in the b subunit but was lost when the mutation was present in the a subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the a subunit of the heterodimeric RSV RT ab.