The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses. To mount an immune response to an invading bacterium or virus (pathogens), the host must detect foreign molecules from the pathogen. Pathogens have conserved features called pathogen associated molecular patterns (PAMPs) that are distinct from host cells and which are recognised by the host using specific sensors (called pattern recognition receptors, PRRs). One example of a PAMP is DNA in the cytoplasm. Cytoplasmic DNA activates the innate immune system, but the PRRs responsible remain incompletely understood. One such PRR, DNA-PK, was identified recently. Here we demonstrate that vaccinia virus (VACV), the vaccine used to eradicate smallpox, encodes a protein called C16 which binds to the DNA-PK complex and prevents it from sensing foreign DNA and activating the immune response. A VACV strain lacking C16 showed reduced virulence and, consistent with this, the host mounted a stronger innate immune response to infection. This illustrates the importance of DNA-PK as a sensor for foreign DNA, and increases understanding of the interaction between VACV and the host. It also illustrates how the study of virulence factors of pathogens can lead to the identification of novel components of the immune system.

The vaccinia virus (VACV) strain Western Reserve C16 protein has been characterized and its effects on virus replication and virulence have been determined. TheC16Lgene is present in the inverted terminal repeat and so is one of the few VACV genes that are diploid. The C16 protein is highly conserved between different VACV strains, and also in the orthopoxviruses variola virus, ectromelia virus, horsepox virus and cowpox virus. C16 is a 37.5 kDa protein, which is expressed early during infection and localizes to the cell nucleus and cytoplasm of infected and transfected cells. The loss of theC16Lgene had no effect on virus growth kinetics but did reduce plaque size slightly. Furthermore, the virulence of a virus lackingC16L(vΔC16) was reduced in a murine intranasal model compared with control viruses and there were reduced virus titres from 4 days post-infection. In the absence of C16, the recruitment of inflammatory cells in the lung and bronchoalveolar lavage was increased early after infection (day 3) and more CD4⁺and CD8⁺T cells expressed the CD69 activation marker. Conversely, late after infection with vΔC16 (day 10) there were fewer T cells remaining, indicating more rapid clearance of infection. Collectively, these data indicate that C16 diminishes the immune response and is an intracellular immunomodulator.