br Results br Discussion C is related

Results
Discussion C4 is related to another VACV protein, C16, and the C-terminal domain of C16 also inhibits DNA-PK-mediated DNA sensing via the same mechanism and to a similar level as C4. Moreover, mutagenesis of C16 at the CYC residues equivalent to those in C4 abrogated the C16-Ku interaction, indicating that C4 and C16 bind to Ku in a similar manner. A summary of the C4 and C16 functional domains is shown in Figure S6. In vivo, C4 and C16 are non-redundant in that AMD3100 of either caused reduced virulence after i.n. infection (Ember et al., 2012, Fahy et al., 2008), and a double deletion virus was attenuated further. In contrast, redundancy was observed in the i.d. model because deletion of either C16 or C4 alone did not affect virulence (Ember et al., 2012), but double deletion reduced virulence. This may be due to the common inhibition of DNA-PK, and the lack of redundancy in the i.n. model is likely due to other functions of these proteins (Figure S6). Indeed, C16 also induces a hypoxic response (Mazzon et al., 2013), reprogramming the central energy metabolism (Mazzon et al., 2015), and C4 inhibits NF-κB signaling (Ember et al., 2012), and other unknown functions may exist. The response to infection with vΔC16ΔC4 showed that C4 inhibits the recruitment of macrophages, neutrophils, and NK cells, and reduces the recruitment and activation of CD4+ and CD8+ T cells. C4 also inhibits the secretion of IL-6 and CXCL10 in vivo, and this may explain the changes in infiltrating leukocytes. Similar analyses with viruses expressing C4 and C16 with point mutations that abrogate each of their functions alone would be interesting. This was attempted for the C4 AAA mutant; however, expression of C4 AAA by the virus was too low to justify in vivo infection (data not shown). It is remarkable that VACV proteins C4 and C16 perform the same function by the same mechanism. This might be explained by C4 and C16 having other functions that are enhanced by binding Ku, or redundancy might enable each protein to carry out other functions better. Alternatively, one protein alone might be insufficient to inhibit DNA-PK, particularly early in infection, given that DNA-PK is a very abundant complex. Both C4 and C16 are made early p.i. with nucleocytoplasmic localizations (Ember et al., 2012, Fahy et al., 2008); however, slight differences in expression levels and localization might exist. Having two proteins may also be beneficial should the host act to prevent either protein from functioning. Finally, because VACV has a broad host range, variations in Ku proteins in different hosts may require slightly different viral proteins to ensure functional binding. Interestingly, VACV was shown recently to also inhibit STING activation in a C16-independent manner by an alternative mechanism, acting at or downstream of cGAMP (Georgana et al., 2018). The expression of multiple inhibitors of a single pathway is common in pathogen evasion. For example, >10 VACV proteins inhibit NF-κB activation (Bowie et al., 2000, Chen et al., 2008, Cooray et al., 2007, DiPerna et al., 2004, Ember et al., 2012, Gedey et al., 2006, Mansur et al., 2013, Myskiw et al., 2009, Schröder et al., 2008, Shisler and Jin, 2004, Stack et al., 2005), and these proteins are not redundant in vivo because, where tested, deletion of each one alone attenuates virulence (Bartlett et al., 2002, Benfield et al., 2013, Brandt and Jacobs, 2001, Chen et al., 2006, Ember et al., 2012, Harte et al., 2003, Mansur et al., 2013, Stack et al., 2005). C4 and C16 are also not the only example of VACV proteins that perform the same function via the same mechanism. The VACV enzymes D9 and D10 share ∼25% sequence identity and both decap host and viral mRNAs (Parrish et al., 2007, Parrish and Moss, 2007). This prevents an accumulation of dsRNAs to reduce the anti-viral response to this PAMP (Liu et al., 2015). Redundancy was found as WT virus and D9 or D10 catalytic site mutants retained virulence during i.n. infection, but a double mutant was attenuated (Liu et al., 2015). This is similar to viruses lacking C4, C16, or both proteins in the i.d. model.