In the presence of Mg(2+) and a specific dinucleotide primer (ApG or
GpG), the
influenza virion
transcriptase synthesizes the eight discrete segments of
complementary RNA (
cRNA) containing polyadenylic
acid (Plotch and Krug, J. Virol. 21:24-34, 1977). Virions were examined for their ability to cap and methylate
cRNA containing di- or triphosphorylated 5' termini. By using the primers ppApG, pppApG, or ppGpG, viral
cRNA was synthesized in vitro with [alpha-(32)P]-
GTP and S-[methyl-(3)H]adenosylmethionine as labeled precursors.
DEAE-Sephadex chromatography of the
RNase T2 digest of the
cRNA product demonstrated no (3)H incorporation at all and the absence of a (32)P-labeled cap structure. The 5' terminus of ppApG-primed
cRNA could be capped and methylated by
enzymes from vaccinia virus, indicating that the two 5'-terminal
phosphates derived from the primer were preserved in the product
cRNA. The cap structure formed by the
vaccinia enzymes and released by
RNase T2 digestion as
m(7)GpppA(m)pGp was radioactively labeled at its 3'-terminal
phosphate only when [alpha-(32)P]
CTP was used as the labeled precursor during transcription. This indicates that the 5'-terminal sequence of the
cRNA is ppApGpC and that, therefore, ppApG most probably initiates transcription exactly at the 3' GpCpU(
OH) terminus of the virion
RNA templates. Virions were also tested for their ability to cap and methylate ppApG in the absence of transcription. No such activities were detected, whereas under the same conditions the vaccinia virus
enzymes successfully capped and methylated this compound. Consequently, these experiments, together with those reported earlier, have not detected in
influenza virions any capping and methylating
enzymes active on the 5'-initiated termini of viral
cRNA chains synthesized in vitro, whether these termini possess one, two, or three
phosphates. Some mechanism for capping and methylation of viral
cRNA must, however, exist, because the viral
mRNA (
cRNA) synthesized in the infected cell contains 5'-terminal methylated cap structures (Krug et al., J. Virol. 20:45-53, 1976). Possible mechanisms are discussed.