Multiple RNase H activities in mammalian type C retravirus lysates

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• Retroviruses like Moloney murine sarcoma-leukemia virus (M-MSV(MLV)) possess RNAse H activity crucial for replication.
• Previous studies identified RNase H II in M-MSV(MLV) as an exohybridase.
• The characterization of distinct RNase H species and their origins is essential for understanding retroviral mechanisms.

Purpose of the Study:

• To identify and characterize the different RNase H species present in M-MSV(MLV) lysates.
• To elucidate the enzymatic properties and potential origins of these RNase H activities.
• To investigate the relationship between RNase H and DNA polymerase in M-MSV(MLV).

Main Methods:

• Polycytidylic acid [poly(C)]-agarose chromatography was used to separate RNase H species.
• Enzymatic assays were performed to determine substrate specificity, cofactor requirements, and mode of action.
• Gel filtration (Sephadex G-100) was used for molecular weight determination.
• Antisera against reverse transcriptase and protease inhibitors were employed to investigate enzyme origins.

Main Results:

• Three RNase H species (RNase H I, II, and III) were separated from M-MSV(MLV) lysates.
• RNase H I co-eluted with RNA-directed DNA polymerase, while RNase H II was independent of polymerase activity.
• RNase H III, purified and characterized, exhibited endoribonucleolytic activity, required Mn2+, and was inhibited by salt.
• RNase H III was identified as a proteolytic cleavage product of DNA polymerase-RNase H, as indicated by inhibition studies with protease inhibitors and antisera.

Conclusions:

• M-MSV(MLV) contains at least three distinct RNase H species with different biochemical properties.
• RNase H III is a distinct enzyme, likely derived from proteolytic processing of a larger DNA polymerase-associated protein.
• The characterization of these RNase H species provides insights into the enzymatic machinery of retroviruses.