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"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). We now find that the competitive inhibitor of methylation, S-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNAs containing 5'-terminal GpppA and ppA, rather than m(7)GpppAm as observed with Adomet. In addition to AdoHcy, other AdoMet analogues, including S-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. In order 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 analogues, 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 prerequisite to mRNA synthesis-i.e., a "pretranscriptional" event.
Insights
Cytoplasmic polyhedrosis virus (CPV) mRNA synthesis is stimulated by methylation inhibitors like S-adenosylhomocysteine (AdoHcy). Cap formation is a prerequisite for CPV mRNA synthesis, indicating a pretranscriptional event.
Area of Science:
- Molecular Virology
- Biochemistry
- RNA Synthesis
Background:
- Cytoplasmic polyhedrosis virus (CPV) mRNA synthesis requires the methyl donor S-adenosylmethionine (AdoMet).
- The role of methylation in CPV mRNA synthesis and cap formation requires further elucidation.
Purpose of the Study:
- To investigate the effect of methylation inhibitors and analogues on CPV mRNA synthesis.
- To determine the relationship between mRNA cap formation and the synthesis process in CPV.
Main Methods:
- Assessing CPV mRNA synthesis in vitro using S-adenosylhomocysteine (AdoHcy) and other S-adenosylmethionine (AdoMet) analogues.
- Utilizing beta,gamma-imido nucleoside triphosphate analogues to probe the role of nucleotide phosphohydrolase in cap formation and mRNA synthesis.
Main Results:
- S-adenosylhomocysteine (AdoHcy) efficiently stimulates CPV mRNA synthesis, producing viral mRNAs with 5 -terminal GpppA and ppA.
- Other AdoMet analogues like S-adenosylethionine and adenosine also stimulate synthesis, though less effectively.
- mRNA synthesis occurred with UMP-pNHp and GMP-pNHp but not with AMP-pNHp, suggesting ATP cleavage is essential for cap formation.
Conclusions:
- Cap formation is a prerequisite for cytoplasmic polyhedrosis virus mRNA synthesis.
- This suggests that cap formation is a pretranscriptional event in this viral system.