Identification of the 5′ terminal structure of influenza virus genome RNA by a newly developed enzymatic method

A combination of T4 polynucleotide kinase, Escherichia coli alkaline phosphatase, yeast Saccharomyces cerevisiae capping enzyme consisting of α (RNA guanylyltransferase) and β (RNA 5′-triphosphatase) subunits, and its α subunit without RNA 5′-phosphatase activity was used to establish a simple enzymatic method for determination of RNA species with 5′-hydroxyl, 5′-monophosphate, 5′-diphosphate or 5′-triphosphate termini. Using this method, we found that viral genome RNA (vRNA) segments of both A-type and C-type influenza viruses carry tri- or diphosphates at their 5′ termini. The conclusion was based on the observations that: (i) 5′ phosphorylation of vRNAs by T4 polynucleotide kinase takes place only after phosphatase treatment; and (ii) capping of vRNAs can be observed with both the intact yeast capping enzyme and its α subunit alone devoid of RNA 5′-triphosphatase activity; but (iii) the level of capping is higher for the αβ holoenzyme than the α subunit though the relative level varies depending on RNA preparations. The results support the de novo initiation for the RNA replication although transcription of influenza vRNAs is initiated by host cell capped RNAs as primers.