RNA helicase A/DHX9 forms unique cytoplasmic anti-viral granules that restrict oncolytic myxoma virus replication in human cancer cells.

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions, anti-viral responses and also hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the dsDNA poxvirus family that is being developed as an oncolytic virus, DHX9 forms unique granular cytoplasmic structures, which we named "DHX9 anti-viral granules". These DHX9 anti-viral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 anti-viral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene(s) transcription and translation was also significantly compromised, particularly in non-permissive or semi-permissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 anti-viral granules are induced by MYXV, and other poxviruses, in human cells, and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 anti-viral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel anti-viral function for DHX9 that is recruited from the nucleus into the cytoplasm and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV.ImportanceThe cellular DHX9 has both pro-viral and anti-viral roles against diverse RNA and DNA viruses. In this manuscript we demonstrate that DHX9 can form unique anti-viral granules in the cytoplasm during MYXV replication in human cancer cells. These anti-viral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9 the formation of DHX9 anti-viral granules can be inhibited which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 anti-viral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has anti-viral activity in human cancer cells and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.