ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence

Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.