title: Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.
abstract: Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Masmudur M
authors.initials: MM
authors.lastname: Rahman
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.forename: Winnie M
authors.initials: WM
authors.lastname: Chan
authors.forename: Stefan
authors.initials: S
authors.lastname: Rothenburg
authors.forename: Grant
authors.initials: G
authors.lastname: McFadden
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000627:therapeutic use; D002460:Cell Line; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004198:Disease Susceptibility; D005260:Female; D055786:Gene Knockout Techniques; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism / Q000627:therapeutic use; D009000:Monocytes / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D009154:Mutation; D009233:Myxoma virus / Q000502:physiology*; D009234:Myxomatosis, Infectious / Q000517:prevention & control / Q000821:virology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056189:Viral Tropism; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; C081374:E3L protein, Vaccinia virus; D007370:Interferon Type I; D009363:Neoplasm Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C583541:EIF2AK1 protein, human; D019892:eIF-2 Kinase; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases