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

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.