RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease

The influenza virus polymerase complex contains a metal ion-dependent endon uclease activity, which generates short capped RNA primer molecules from ca pped RNA precursors. Previous studies have provided evidence for a two-meta l ion mechanism of RNA cleavage, and the data are consistent with a direct interaction of a divalent metal ion with the catalytic water molecule. To r efine the model of this active site, we have generated a series of DNA, RNA , and DNA RNA chimeric molecules to study the role of the 2'-hydroxy groups on nucleic acid substrates of the endonuclease, We could observe specific cleavage of nucleic acid substrates devoid of any 2'-hydroxy groups if they contained a cap structure (m7GpppG) at the 5'-end. The capped DNA endonucl ease products were functional as primers for transcription initiation by th e influenza virus polymerase, The apparent cleavage rates were about 5 time s lower with capped DNA substrates as compared with capped RNA substrates. Cleavage rates with DNA substrates could be increased to RNA levels by subs tituting the deoxyribosyl moieties immediately 5' and 3' of the cleavage si te with ribosyl moieties. Similarly, cleavage rates of RNA substrates could be lowered to DNA levels by exchanging the same two ribosyl groups with de oxyribosyl groups at the cleavage site. These results demonstrate that the 2'-hydroy groups are not essential for binding and cleavage of nucleic acid s by the influenza virus endonuclease, but small differences of the nucleic acid conformation in the endonuclease active site can influence the overal l rate of hydrolysis, The observed relative cleavage rates with DNA and RNA substrates argue against a direct interaction of a catalytic metal ion wit h a 2'-hydroxy group in the endonuclease active site.