Decoding poxvirus genome

Deciphering the information encoded in genomic sequences is a key step in modern biomedical research. Recent findings indicate that this endeavor can be far more complex than anticipated, even for relatively small viral genomes. Vaccinia virus, the prototypic member of the poxvirus family, was initially annotated to have approximately 200 open reading frames (ORFs) of 65 or more amino acids within its 200 kbp double-stranded DNA genome. This annotation has framed the molecular biological studies of vaccinia virus since then. To further decode information in the vaccinia virus genome, we carried out systematic genome-wide ribosome profiling recently published in the Journal of Virology [1]. In ribosome profiling, only the mRNA fragments bound and protected by ribosomes are analyzed by next generation sequencing, which can quantify active protein translation with superb sensitivity, resolution and clarity [2]. We confirmed that the majority mRNAs of previously annotated ORFs are actively translated, although at greatly different frequencies. In addition, even though the long transcripts made during the late stages of infection read through adjacent ORFs, only the first is translated. The most intriguing finding of this study was the existence of numerous unsuspected translation initiation sites, which were revealed by ribosome profiling of infected cells treated with the translation inhibitor Harringtonine or Lactimydomycin. Both inhibitors arrest the ribosomes at or near the translation initiation site on mRNA with single nucleotide resolution. In addition to the start sites of most annotated ORFs, approximately 600 additional putative translational initiation sites using AUG or alternative near-cognate codons were discovered. Most of these ORFs are embedded in previously annotated ones including truncated non-frameshifting ORFs and downstream frameshifting ORFs. Others are located in non-coding regions including a few upstream ORFs located in the 5′ untranslated region of mRNA, ORFs in intergenic regions and antisense-strand of the annotated ORFs. These findings suggest multifaceted translation of the vaccinia virus genome. In concert with our earlier findings of pervasive transcription [3,4], these studies demonstrate that vaccinia virus encodes many more products than previously recognized, although their biological relevance is yet to be determined. Most of the newly identified ORFs are small in size, as they were below the previous arbitrary cutoff of 65-amino-acids. In fact, several recent studies have suggested that small ORFs encoded by vaccinia virus can perform essential functions in viral replication. The smallest protein characterized has only 35 amino acids and is required for virus entry into cells [5]. …