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Influenza viral mRNA contains internal N6-methyladenosine and 5'-terminal 7-methylguanosine in cap structures
Abstract:
Influenza viral complementary RNA (cRNA), i.e., viral mRNA was radioactive when purified from the cytoplasmic fraction of cordycepin-treated canine kidney cells that were incubated with [methyl-3H]methionine during infection. Approximately 55 to 60% of the methyl-3H radioactivity was in internal N6-methyladenosine, a feature distinguishing this mRNA from those viral mRNA's that are known to be synthesized in the cytoplasm. The remaining methyl-3H radioactivity was in 5'-terminal cap structures that consisted of 7-methylguanosine in pyrophosphate linkage to 2'-o-methyladenosine, N6, 2'-O-dimethyladenosine, or 2'-O-methylguanosine. Methylated adenosine was the predominant penultimate nucleoside in caps, suggesting that cRNA synthesis in infected cells initiates preferentially with adenosine at the 5' end. In contrast to cRNA, influenza virion RNA segments extracted from purified virus contained mainly 5'-terminal ppA and no detectable cap structures.
Insights
Influenza virus mRNA synthesized in the cytoplasm is methylated internally and at the 5' end. This differs from virion RNA, which lacks these methylation patterns.
Area of Science:
- Molecular Virology
- RNA Biology
- Biochemistry
Background:
- Influenza virus replication involves the synthesis of viral RNA (vRNA) and complementary RNA (cRNA), which also functions as viral messenger RNA (mRNA).
- Understanding the post-transcriptional modifications of viral mRNA is crucial for elucidating viral replication strategies and host-pathogen interactions.
Purpose of the Study:
- To characterize the methylation status of influenza viral complementary RNA (cRNA) synthesized in infected cells.
- To compare the 5' end structures of cytoplasmic viral mRNA with those of virion RNA.
Main Methods:
- Canine kidney cells infected with influenza virus were treated with cordycepin to inhibit cellular RNA synthesis.
- Cells were incubated with [methyl-3H]methionine to radiolabel methylated nucleotides.
- Purification of cytoplasmic viral cRNA and analysis of its radioactive methylation patterns.
- Analysis of 5'-terminal structures of purified influenza virion RNA.
Main Results:
- Radioactive methyl-3H labeling revealed that 55-60% of the cRNA's methylation was internal, specifically N6-methyladenosine.
- The remaining methyl-3H radioactivity was associated with 5'-terminal cap structures (m7GpppNmpNmp), with methylated adenosine being the predominant penultimate nucleoside.
- Influenza virion RNA segments primarily contained 5'-terminal ppA and lacked detectable cap structures.
Conclusions:
- Influenza viral cRNA synthesized in the cytoplasm possesses internal N6-methyladenosine modifications and distinct 5'-terminal cap structures.
- The preferential use of adenosine at the 5' end suggests a specific initiation mechanism for cRNA synthesis.
- The absence of cap structures in virion RNA highlights differences between cytoplasmic mRNA and packaged viral genomes.