Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages

Abstract

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Molecular Mechanisms of Poxvirus Evolution

“…In myxoma virus, these genes have evolved to have distinct protein interaction partners after duplication ( 66 – 68 ). For example, only M062 has been shown to interact with sterile alpha motif domain-containing protein 9 (SAMD9) and thereby inhibit a cGAS-dependent interferon response ( 67 , 69 ). A recent study identified a similar example of duplication in cetacean poxvirus in which the virus genome was predicted to encode two tandem copies of the E3L ortholog, a full-length copy (CePV-TA-20) and a truncated copy (CePV-TA-21), which lacks the amino-terminal Zα domain ( 70 , 71 ).…”

Antagonizing the SAMD9 pathway is key to myxoma virus host shut-off and immune evasion

“…Although early gene expression of this virus did not show significant difference from that of the wildtype MYXV, 10,20 ΔM062R infection failed to lead to host shutoff (Figure 1B).…”

“…HeLa cells were transfected with the plasmid containing the luciferase expression cassette driven by one of following VACV intermediate gene promoters, pA2L, 24 pI1L, 24 and VACV intermediate consensus promoter sequence (pINT) 25 (M. Wiebe, University of Nebraska-Lincoln) using ViaFect (Cat# E4982, Promega, Madison, WI) at 0.8 µg/mL with the transfection agent to DNA ratio of 3 µL to 1 µg as previously described. 20 Next Generation RNA sequencing THP-1 cells (10 6 cells per 3.5 cm dish) were differentiated in PMA at 50 ng/mL for up to 48 hours before mock treated, transfection with interferon stimulated DNA (ISD) 47 Dual-RNAseq bioinformatic data processing and GO:BP analyses A dual-RNAseq bioinformatic data processing pipeline was utilized for the study as previously described. 20 Exploratory data analysis (EDA) and differential expression analysis was performed using R and DESeQ2 v1.32.0, where p-value and adjusted pvalue thresholds were set to 0.05 and 0.1, respectively.…”

“…21 The replication defective MYXV with targeted M062R gene induces a unique inflammatory state through the activation of both cGAS/STING/IRF3 DNA sensing axis and SAMD9 pathway. 20 In this study, we further delineated the infection defect of M062R-null MYXV (ΔM062R), characterized ΔM062R infection caused outcome on viral and host protein translation, and reported the effect of ΔM062R in potentiating macrophages in response to new danger signals. We also summarized the effect on the transcriptomic landscape in monocytes/macrophages induced by ΔM062R and concluded that these characteristics of ΔM062R may be key to its immunotherapeutic potential in reversing the immunosuppressive environment such as the tumor environment.…”

Role of cytokines in poxvirus host tropism and adaptation

“…Similarly, C7L-like host range proteins from other poxviruses contribute to their host and cellular tropism [10] . Functional and structural studies of these C7-like proteins demonstrated that some of them bind and antagonize host sterile alpha-motif domain-containing 9 protein to overcome type-I IFN-mediated host restriction 11 , 12 , 13• . Thus, poxvirus-encoded proteins known as host range factors can dictate which cells, tissues, or hosts they can productively infect.…”