Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA

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Abstract
The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 .times. 105 to 106 daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5'' end, as also appears to be the case with in vivo cRNA. The in vitro transcripts contain covalently linked poly(A) sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinucleoside monophosphate (DNMP), ApG or GpG. These DNMP stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5''-phosphorylated derivatives of these DNMP, 32pApG or 32pGpG, are incorporated at the 5'' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts was detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA. [Canine kidney MDCK and bovine kidney MDBK cells were used.].