Poxviruses have double stranded DNA
Poxviruses have double-stranded DNA (dsDNA) genomes, yet they replicate within the cytoplasm. Poxviruses that cause human disease include monkeypox virus (Reynolds et al., 2004), molluscum contagiosum virus (Hanson and Diven, 2003) and variola virus (VARV), the cause of smallpox, a disease eradicated by vaccination with vaccinia virus (VACV) (Fenner et al., 1988). VACV is a useful expression vector (Mackett et al., 1982, Panicali and Paoletti, 1982) and has applications as vaccines against other pathogens (Panicali et al., 1983, Smith et al., 1983a, Smith et al., 1983b) and as an oncolytic agent (Buller et al., 1985, Heo et al., 2013, Kirn and Thorne, 2009). In addition, interest in VACV endures because it is an excellent model to study host-pathogen interactions and cell biology (Smith et al., 2013). The detection of DNA by PRRs triggers the production of type I IFN, cytokines, and chemokines (Stetson and Medzhitov, 2006) via a pathway that requires stimulator of IFN genes (STING) (Ishikawa et al., 2009), TANK-binding kinase 1 (TBK1), and IFN regulatory factor 3 (IRF3) (Ishii et al., 2008, Ishii et al., 2006, Tanaka and Chen, 2012). For example, the detection of VACV DNA induced IFN-β independently of Toll-like receptors (TLRs) and RNA polymerase III but was dependent on STING, TBK1, and IRF3 (Unterholzner et al., 2010). DNA-PK is a heterotrimeric complex consisting of the catalytic subunit DNA-PKcs and a heterodimer of Ku70 and Ku80. DNA-PK binds dsDNA breaks and functions in non-homologous end joining (NHEJ) (Pannunzio et al., 2018) and also in DNA sensing, upregulating type I IFN and cytokines via the STING pathway (Ferguson et al., 2012). The kinase activity of DNA-PKcs is essential for DNA repair (Kurimasa et al., 1999) but not for innate immune signaling. Cells and mice lacking DNA-PK components show impaired response to infection with VACV and herpes simplex virus 1 (HSV-1) (Ferguson et al., 2012). Ku70 also induces the expression of type III IFN in response to DNA (Sui et al., 2017, Zhang et al., 2011). The autumn wind of pathogens with their hosts has produced intriguing strategies for both host detection and pathogen subversion. VACV is a good example of this, and between one-third and one-half of its 200 proteins modulate the immune response (Bowie and Unterholzner, 2008, Elde et al., 2012, Gubser et al., 2004). However, for dsDNA-binding PRRs, the only known direct inhibitor is protein VACV C16, an inhibitor of DNA-PK signaling (Peters et al., 2013). Mechanistically, the C-terminal region of C16 binds directly to the Ku heterodimer to block its binding to DNA. C16 also induces a hypoxic response by binding to PHD2 via its N-terminal domain (Mazzon et al., 2013) and, in doing so, reprograms cellular energy metabolism (Mazzon et al., 2015). In a murine intranasal (i.n.) model of infection, the VACV strain Western Reserve (WR) that lacks C16 caused an increase in infiltrating leukocytes, less weight loss with fewer signs of illness, and greater cytokine synthesis (Fahy et al., 2008, Peters et al., 2013). Here, another VACV protein, C4, is shown to target DNA-PK and inhibit DNA sensing. C4 shares sequence similarity to C16 and, like C16, binds to Ku to diminish DNA binding. This function mapped to the C-terminal domain of C4, and mutagenesis of three residues in both C4 and C16 abrogated binding to Ku. The infection of mice by viruses lacking C4 or C16 singly or together showed that these two proteins have both redundant and non-redundant functions. The loss of C4 increased recruitment and activation of cells involved in both innate and adaptive immunity. This phenotype is attributed to the suppression of cytokine production by C4 both in vitro and in vivo. Overall, this study identifies a second DNA-PK inhibitor encoded by a virus, adding to the evidence that DNA-PK is important to innate immunity. The complexity of host-pathogen interactions is highlighted by the finding that VACV encodes two multifunctional proteins that inhibit the same PRR to directly evade the detection of its genome, but, as yet, there are no reported inhibitors of other DNA sensors.