Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.

We show that SAMD9 is an innate host antiviral stress response element that participates in the formation of antiviral granules. Poxviruses, myxoma virus and vaccinia virus specifically, utilize a virus-encoded host range factor(s), such as a member of the C7L superfamily, to antagonize SAMD9 to prevent granule formation in a eukaryotic initiation factor 2␣ (eIF2␣)-independent manner. When SAMD9 is stimulated due to failure of the viral antagonism during infection, the resulting antiviral granules exhibit properties different from those of the canonical stress granules. Poxviruses are enveloped large double-stranded DNA (dsDNA) viruses that replicate exclusively in the cytoplasm. Numbers of immunoregulatory factors are encoded in the poxvirus genome to antagonize multiple host defense pathways at all levels. We previously showed that a targeted knockout of M062R, an essential host range factor of the C7L superfamily in the myxoma virus (MYXV) genome, led to a profound defect of viral replication in cells from all species tested, including human, rabbit, and primate cells (1). Viral M062 antagonizes cellular SAMD9 during viral infection through direct protein-protein interactions, and knocking down the expression of SAMD9 in human cells lifts the block of late gene expression from MYXV-M062R-null infection (1). To investigate the antiviral functions of SAMD9, we examined the cellular localization of this host protein during permissive wild-type and nonpermissive MYXV-M062R-null infection by immunofluorescence (IF) staining. We observed that, during MYXV-M062R-null infection, SAMD9 formed a granule structure in the cytoplasm. The accumulation of concentrated SAMD9 was detected as early as 7 h postinfection (p.i.). By 18 h p.i., 91.0% (Ϯ6.2% standard deviation [SD]) of infected cells showed SAMD9 granules which were then mostly concentrated near the viral factories (Fig. 1A). During permissive infection by wild-type MYXV that expressed M062 protein, SAMD9 was observed to become distributed throughout the cells, which was also distinct from the exclusive cytoplasmic localization of SAMD9 in uninfected cells. We further investigated the SAMD9 granules and found that they are not conventional stress granules (SGs), as they cannot be dispersed by cycloheximide treatment (not shown) in a manner similar to that seen with the antiviral granules previously described, which form after infection with E3L-knockout vaccinia virus (VACV) (2). On the other hand, several hallmark markers of SGs, such as rasGTPase-activating protein-binding protein 1 (G3BP1) (3) (Fig. 1), T-cell intracellular antigen 1-related protein (TIAR) (4), USP10 (5), and key translation initiation factors such as eukaryotic initiation factor 4G (eIF4G) (6) (Fig. 1B), were readily detected surrounding the viral factories colocalized with the SAMD9 granules in infection by MYXV-M062-null. However, in addition to the SAMD9 granules that overlap G3BP1 staining, SAMD9 staining can be detected outside the viral factories. The polyclonal SAMD9 antibody used in thi...

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.

SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.