“Pretranscriptional capping” in the biosynthesis of cytoplasmic polyhedrosis virus mRNA

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Abstract
The in vitro synthesis of cytoplasmic polyhedrosis virus (CPV) mRNA was previously shown to be dependent upon the presence of the methyl donor S-adenosylmethionine (AdoMet). The competitive inhibitor of methylation, S-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNA containing 5''-terminal GpppA and ppA, rather than m7GpppAm as observed with AdoMet. Besides AdoHcy, other AdoMet analogs, including S-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. To study the relationship between cap formation and mRNA synthesis, nucleoside triphosphates were replaced in the RNA-synthesizing reaction mixture (containing AdoMet) by the corresponding .beta.,.gamma.-imido analogs, which are resistant to nucleotide phosphohydrolase, an enzyme involved in cap formation. Although mRNA synthesis occurred in the presence of UMP-pNHp or GMP-pNHp, none was observed when AMP-pNHp was substituted for ATP. Because the ATP molecule that becomes the 5''-terminal nucleotide of CPV mRNA must be cleaved at the .beta.-.gamma. position during cap formation, the results suggest that, in this viral transcription system, cap formation is a prerequisite to mRNA synthesis, i.e., a pretranscriptional event.
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