Soluble Rous sarcoma virus reverse transcriptases alpha, alpha beta, and beta purified from insect cells are processive DNA polymerases that lack an RNase H 3 '-> 5 ' directed processing activity

Reverse transcriptase (RT) isolated from Rous sarcoma virus (RSV) consists of heterodimeric RT alpha beta, RT alpha, and RT beta. The alpha subunit (6 3 kDa) contains an N-terminal polymerase and a C-terminal RNase H domain. T he N terminus of beta (95 kDa) corresponds to alpha with the integrase doma in attached to the C terminus (32 kDa). We have constructed baculoviruses e xpressing the genes for alpha or beta or the entire pol (99 kDa). Infection of insect cells with recombinant virus yielded highly active and soluble R SV RT enzymes that could be purified to >90% homogeneity. HPLC gel filtrati on showed that a is a dimeric enzyme that can be partially monomerized upon the addition of 45% Me2SO, DNA synthesis on DNA-DNA and DNA-RNA primer-tem plates in the presence of competitor substrates revealed that alpha beta an d beta as well as a are processive polymerases. However, the affinity of be ta and alpha beta for primer-template substrates appears to be higher than that of alpha. All RSV enzymes investigated have the potential to displace RNA-RNA duplexes more efficiently than human immunodeficiency virus type 1 RT. Unlike human immunodeficiency virus type 1 RT, RSV RTs can catalyze an initial RNase H endonucleolytic cleavage of the RNA template but not a 3' - -> 5' directed processing activity.