AbstractThe role of the chikungunya virus (CHIKV) non-structural protein 3 (nsP3) in the virus lifecycle is poorly understood. The protein comprises 3 domains. The N-terminus is a macro domain, biochemically characterised to bind both RNA and ADP-ribose, and to possess ADP-ribosyl hydrolase activity – an enzymatic activity that removes ADP-ribose from mono-ADP-ribosylated proteins. As ADP-ribosylation is important in the signalling pathway leading to activation of the transcription factor NF-κB, we sought to determine if the macro domain might perturb NF-κB signalling. We first show that CHIKV infection did not induce NF-κB activation, and could not block exogenous activation of the pathway via TNFα, although TNFα treatment did reduce virus titres. Ectopic expression of nsP3 was able to block TNFα-mediated NF-κB activation and this was dependent on the macro domain, as mutations previously shown to disrupt either ADP-ribose binding or hydrolase activity lost the ability to inhibit NF-κB activation. Lastly, we determined the phenotype of the macro domain mutants in the context of virus infection in a range of cell types. Our data are consistent with cell- and species-dependent roles of the macro domain, however, these phenotypes do not correlate with the ability to inhibit NF-κB activation suggesting that the macro domain plays multiple independent roles in the virus lifecycle.