1. 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
- Author
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Rahman, Masmudur M, Liu, Jia, Chan, Winnie M, Rothenburg, Stefan, and McFadden, Grant
- Subjects
Medical Microbiology ,Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Small Pox ,Emerging Infectious Diseases ,Prevention ,Rare Diseases ,Infectious Diseases ,Biodefense ,Vaccine Related ,2.2 Factors relating to the physical environment ,2.1 Biological and endogenous factors ,Aetiology ,Infection ,Animals ,Antiviral Agents ,Cell Line ,Cells ,Cultured ,DEAD-box RNA Helicases ,Disease Susceptibility ,Female ,Gene Knockout Techniques ,Humans ,Interferon Type I ,Monocytes ,Mutation ,Myxoma virus ,Myxomatosis ,Infectious ,Neoplasm Proteins ,RNA-Binding Proteins ,Rabbits ,Recombinant Fusion Proteins ,Recombinant Proteins ,Viral Proteins ,Viral Tropism ,Virus Replication ,eIF-2 Kinase ,Microbiology ,Immunology ,Virology ,Medical microbiology - 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.
- Published
- 2013