We have demonstrated that M029 is both a critical host range factor for MYXV replication in a wide spectrum of diverse mammalian cell types as well as a critical virulence factor for myxomatosis in the European rabbits. Our inability to isolate pure recombinant M029-defective MYXV constructs using any of the standard mammalian cell lines where the parental MYXV can robustly replicate indicated that M029 plays essential function(s) in MYXV replication for most mammalian cells. The M029-minus viruses were only successfully isolated away from complementing parental MYXV and propagated as pure clones in cell lines that stably express VACV E3, a complementing comparable poxvirus protein. The purified vMyx-M029KO and vMyx-M029ID viruses that were grown in these E3-complementing RK13 cells exhibited severe defects in replication in essentially all established mammalian cell lines originated from diverse species, where the parental wildtype-MYXV replicates permissively. The most profound tropism defect of the M029-mutant viruses was their complete inability to replicate in any of the tested cells derived from humans (eg HeLa, A549, THP-1, various cancer cell lines, primary fibroblasts, etc), non-human primates (eg BSC40) and mouse (eg NIH3T3) ( and ). In these cell lines, no expression of viral late proteins was detected in the absence of M029 expression. On the other hand, abundant expression of early viral proteins was detected from M029-minus viruses at a comparable level with wildtype-MYXV infection (), suggesting that there was no defect in virus binding, entry, or early viral gene expression. Both vMyx-M029KO and vMyx-M029ID viruses were also unable to replicate in rabbit RL5 T lymphocytes, and although they were capable of at least some detectable progeny virus synthesis in RK13 cells, replication kinetics were reduced and cell-cell transmission was compromised. Slower replication kinetics of M029-defective viruses was also observed in hamster BHK21 cell lines. In both RK13 and BHK21 cells, the synthesis of viral late protein was significantly delayed, which resulted in a reduced progeny virus production at 12 hpi when tested by single step growth curve analysis. It is possible that the level of dsRNA produced by the viruses in these semi-permissive cell lines is lower than the completely nonpermissive cell lines, as has been suggested for VACV 
The deletion or insertional disruption of M029 also resulted in the elimination of essentially all disease symptoms associated with myxomatosis in susceptible rabbits. This extreme attenuation was associated with the absence of obvious viral spread and extreme reductions in the replication of virus at the primary site of infection. Following full recovery of the M029-mutant infected animals, very variable levels of protection was generated against subsequent challenge by wildtype MYXV, suggesting that virus replication at the primary inoculation site was likely aborted quickly before the elements of acquired immune responses were significantly engaged.
Analyzing the distribution of V5-tagged MYXV M029 protein revealed that M029 was expressed at an early time points of infection and was packaged into infectious virions (), which suggests a critical role of M029 in the modulation of host innate sensing/signaling pathways during early stages of infection. This observation is particularly important because VACV E3 has not been reported in virions and we were also unable to detect E3 in purified VACV particles using comparable strategy used for vMyx-M029V5N (
, and data not shown). Localization studies using immunofluorescence microscopy indicated that M029 protein is localized to both the nuclear and cytosolic compartments of MYXV-infected cells. This same cellular localization of M029 was also observed when V5-tagged M029 was transiently expressed in uninfected cells using plasmids under CMV promoter (data not shown), suggesting that nuclear-cytoplasmic dual localization is not dependent on viral infection. Like M029, VACV E3 protein is also localized in both the nucleus and cytoplasm, however, the nuclear localization of E3 is dependent on the N-terminal 83 amino acid residues, which are missing in the M029 protein 
. This suggests that M029 may have acquired a different mechanism for nuclear localization.
The major role of VACV E3 is thought to antagonize the PKR-mediated anti-viral functions of the infected host cell, which are upregulated by IFN and activated by dsRNA 
. eIF2α is a primary cellular substrate that becomes phosphorylated in cells infected with E3-minus VACV, which then leads to the global inhibition of host and viral protein synthesis, the induction of apoptosis and eventual inhibition of virus replication. Infection with VACVΔE3 resulted in the phosphorylation of both PKR and eIF2a, however the defect of VACVΔE3 can be rescued in PKR-deficient HeLa cells 
. In the PKR-deficient cells, late viral protein synthesis was restored and virus-induced apoptosis was also abolished, which resulted in productive virus replication even in the absence of E3 expression. PKR is one of the host innate immune proteins that undergo rapid evolution in order to escape being targeted by viruses capable of anti-PKR strategies, such as poxviruses 
Based on the profound host range phenotype of M029-defective viruses in a wide spectrum of mammalian cells, we created MYXV that expressed V5-tagged M029 and performed co-IP and mass-spectrometry to identify putative host binding partner proteins that might be functionally targeted by M029. Based on the previous report that PKR interacts with VACV E3, we first identified cellular PKR as an M029 interacting protein in various human cells. The interaction of PKR and M029 was validated by co-IP assay in virus-infected mammalian cells with either V5 or PKR antibody (). Significantly, this PKR-M029 protein interaction was eliminated by digestion with RNaseV1, which cleaves dsRNA, but not with RNaseA/T1 that targets only ssRNA, suggesting that this interaction requires dsRNA. Since both PKR and M029 possess dsRNA-binding domains, it is presumed that these proteins become linked via dsRNA bridging. VACV E3, on the other hand, has been reported to have direct protein-protein interaction with PKR; however, the samples were not treated with an RNase that specifically cleaves dsRNA 
. Although M029 interacts with PKR indirectly via dsRNA, this interaction is still biologically relevant, since the replication of M029-minus MYXV viruses were specifically rescued in PKR knockdown cells. In stable PKR knockdown human cells, the synthesis of viral late proteins was fully restored and allowed complete replication and progeny virus formation by vMyx-M029KO and vMyx-M029ID (), which demonstrates that PKR is a major functional target for M029 host range functions, as it is for VACV E3.
In addition to PKR, we also identified several other cellular RNA binding proteins that were consistently present in the co-IP samples for V5-tagged M029 in virus-infected human cells. Among these, we were particularly interested in investigating the potential interaction of M029 with RHA, a cellular RNA helicase, also known as DHX9. RHA/DHX9 is a 130 kDa protein that belongs to the DEXD/H box family of proteins, which can unwind both double-stranded RNA and DNA, and can also regulate cellular processes such as pre-mRNA splicing, ribosome biogenesis, transcription, RNA nuclear export, and translation initiation 
. RHA/DHX9 is also associated with regulating the replication efficiencies of various RNA viruses, including HIV-1, HCV, FMDV and influenza A 
. These viral regulatory functions of RHA/DHX9 are mediated by interactions with unique viral proteins, for example, the Gag protein of HIV and NS1 protein of Influenza A virus 
. DHX9 also function as a dsRNA sensor in selected cell types 
. In one recent report, it was demonstrated that NS1 can rescue VACVΔE3 virus replication, at least in vitro
, and thus we postulated that RHA/DHX9 might also have potential role(s) in some aspect of poxvirus tropism. VACV E3 has also been reported to interact with a wide spectrum of host proteins, including RHA/DHX9, but the functional significance of this particular protein interaction has not been reported 
. The interaction of DHX9/RHA with V5-tagged M029 protein was validated in virus-infected cells by co-IP assay using either anti-RHA/DHX9 or V5 antibody. Interestingly, unlike the PKR-M029 interaction, the RHA-M029 protein interaction was not eliminated by digestion of dsRNA with RNaseV1 (), suggesting that interaction between RHA/DHX9 and M029 is through direct protein-protein interactions. RHA/DHX9 has also been reported to interact with PKR, and is in fact a substrate of activated PKR kinase 
. In the virus infected cells we have not observed any alteration in the nuclear localization of DHX9 (data not shown). We therefore assessed the potential biological significance of RHA/DHX9 interaction with M029, and discovered that this interaction regulated virus host range in a very cell-specific fashion. Knockdown of RHA/DHX9 gene expression in human THP-1 myeloid cells that had been depleted of PKR reduced the replication and viral protein synthesis for both the M029-expressing vMyx-GFP and significantly for the M029-defective viruses, indicating that RHA/DHX9 can play a required pro-viral regulatory role in the virus life cycle of MYXV in monocytic cell lines when PKR is absent or repressed.
It is well known that certain individual virus-encoded immunomodulators can inhibit multiple ligands or pathways. For example, the M-T7 protein of MYXV binds and inhibits both rabbit interferon-gamma and diverse chemokines, the SECRET family of orthopoxvirus proteins can bind and co-inhibit both TNF and chemokines, the M-T5 intracellular host range factor of MYXV targets both Akt and the Skp1 component of the cellular SCF complex, and the M13 protein of MYXV targets both ASC-1 of the inflammasome complex and NF-κB1 
. However, this is the first report of a single viral host range protein, M029, with two different functional cellular targets, one of which (PKR) is bound and inhibited to alleviate anti-viral signaling, while the other (RHA/DHX9) is bound and conscripted as a pro-viral effector to upregulate viral replication in a cell-specific fashion.