Multiple RNase H activities in mammalian type C retravirus lysates

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Abstract
Lysates of Moloney murine sarcoma-leukemia virus [M-MSV(MLV)], a virus complex grown in the rat cell line 78A-1, contained 3 RNase H species separable by [poly(C)]-agarose chromatography. RNase H activity (RNase H I) associated with RNA-directed DNA polymerase eluted at 0.23 M KCl from poly(C)-agarose. RNase H II, which eluted from poly(C)-agarose at 0.12 M KCl and was not associated with DNA polymerase activity, was identical to an RNase H species (designated RNase H II) previously isolated from M-MSV(MLV) by a different procedure. M-MSV(MLV) RNase H II was established to be a random exohybridase that requires free-chain termini in its hybrid substrate for activity. Lysates of Rickard feline leukemia virus also contained RNase H activity not associated with DNA polymerase activity that eluted from poly(C)-agarose at 0.12 M KCl. A 3rd species of enzyme from M-MSV(MLV) lysates, called RNase H III, did not bind to poly(C)-agarose in 0.06 M KCl. RNase H III was purified from lysates of M-MSV(MLV) and M-MLV (grown in mouse cells) by sequential chromatography on poly(C)-agarose, DEAE-cellulose, phosphocellulose and poly(U)-Sepharose. Purified RNase H III was free of any associated DNA polymerase activity, had an apparent MW of 30,000 determined by Sephadex G-100 gel filtration, had an absolute requirement for Mn2+ (1 mM optimum) for the degradation of [3H](A)n.cntdot.(dT)n, was inhibited by the presence of any salt in reaction mixtures, and was endoribonucleolytic in its mode of action as indicated by the size distribution of limited degradation products of [3H](A)n.cntdot.(dT)n. RNase H III was inhibited by antisera prepared against Rauscher MLV and simian sarcoma virus reverse transcriptase, and the quantity of RNase H III and RNase H I present in lysates of M-MLV were reduced and increased proportionately if virus was lysed in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Apparently, RNase H III is a proteolytic cleavage product of DNA polymerase-RNase H. Substantial RNase H activity that did not bind to poly(C)-agarose in 0.06 M KCl was also found in lysates of Harvey MSV(MLV), Rauscher MLV and Rickard feline leukemia virus, but not in lysates of avian myeloblastosis virus.