Requirements for minus-strand transfer catalyzed by Rous sarcoma virus reverse transcriptase

We have examined the specific minus-strand transfer reactions that occur af ter the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma v irus (RSV) reverse transcriptases. Three different types of reverse transcr iptases can be isolated from virions of RSV: heterodimeric alpha beta and h omodimeric alpha and beta. The mechanism of minus-strand transfer was exami ned using a model primer-template substrate corresponding to the 5'- and 3' -terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand trans fer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the alpha homodimer lack ing the integrase domain can perform strand transfer almost as efficiently as the alpha beta and alpha Pol heterodimers. In contrast, the activities o f beta and Pol for minus-strand transfer are reduced. furthermore, a two- t o fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.