Poxvirus-encoded noncanonical open reading frames

Poxviruses are a large family of viruses including species that are highly pathogenic for humans and animals, including smallpox and monkeypox viruses. Some poxviruses are developed as vaccine vectors and oncolytic virotherapies to fight other infectious diseases and cancers. Vaccinia virus, the prototype poxvirus, has previously been annotated to encode over 200 open reading frames in its 200kbp genome. Our genome-wide analysis of vaccinia virus gene expression using RNA-sequencing uncovered that numerous unexpected mRNAs are transcribed. Furthermore, our previous study discovered a large number of (>500) novel translation initiation sites associated with previously unrecognized non-canonical open reading frames (ORFs) using ribosome profiling. Much remains unknown about the biological relevance of these non-canonical ORFs. The objective of this proposal is to characterize the peptides encoded by the non-canonical vaccinia virus ORFs and explore the biological relevance and functions. The central hypothesis is that peptides/proteins are produced from the non-canonical ORFs, which may play important roles in VACV replication. In Specific Aim 1, peptides/proteins encoded by vaccinia virus non-canonical ORFs will be identified. In Specific Aim 2, the roles of representative non-canonical ORFs in vaccinia virus replication will be determined. Completion of the project is expected to provide knowledge of the expression and functions of the non-canonical poxvirus ORFs that are previously unrecognized. It has profound implications in interpreting poxvirus genetic studies since the past studies have been focusing on previously annotated large ORFs. The outcomes may transform the understanding of poxvirus gene expression and break ground toward new directions in the field. Better understanding these newly identified ORFs may ultimately lead to novel intervening strategies and improving poxviruses as vaccine vectors and cancer therapeutics agents. Therefore, the outcomes are also expected to have a broad impact beyond the poxviruses, as the genomes of various organisms, including humans, also encoded many previously unrecognized ORFs.