The vaccinia virus (VACV) is a prototypical poxvirus that was originally used to eradicate smallpox. Half a century ago, investigation into VACV mRNA substantially contributed to the fundamental discovery of the 5' mRNA cap, a hallmark of all eukaryotic and many viral mRNAs. VACV research also facilitated the identification and understanding of the general mechanism of 5' mRNA cap synthesis. We analyzed VACV transcripts at the level of individual mRNA molecules using a modified 5' RACE method. Our results demonstrate that VACV mRNAs containing long nontemplated 5' poly(A) leaders lack the 5' cap structure in vivo. The probability of the m7G cap occurrence decreases with the increasing number of nontemplated adenosines in the 5' poly(A) leader. Although half of VACV mRNAs with a single nontemplated adenosine still contain the m7G cap, only about 4% of viral mRNAs with leaders consisting of six or more nontemplated adenosines retain the cap. Uncapped mRNA can be transcribed from all genes containing adenosine-rich initiator sequences (INR) within their promoters. Early genes with INR still produce mostly capped transcripts (40%-59%, depending on the gene). However, intermediate mRNAs are less capped (11%-56%) and late mRNAs are mostly uncapped (0%-10% of capped mRNAs, depending on the gene).

Biotechnology and Biomedical Center in Vestec 1st Faculty of Medicine Charles University Vestec Czech Republic

Institute of Medical Microbiology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Czech Republic

Laboratory of RNA Biochemistry Department of Genetics and Microbiology Faculty of Science Charles University Prague Czech Republic

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