Vaccinia virus mRNAs containing long 5'-poly(A)-leaders lack a canonical 5'-methylguanosine cap

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).