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

Abstract

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.

Keywords: ADP-ribosylation; macrodomain; mass spectrometry; viral replication; virulence.

Fig. 1.

CHIKV nsP3^MD removes ADP-ribose from MARylated PARP10^CD. (A and B) Total protein stain, autoradiography, and quantification of ³²P-MARylated PARP10^CD demodification reactions by MacroD2 or CHIKV nsP3^MD at 1 h time-point (A) and over a time course of 2 h (B). (C) Quantification of LC-MS extracted ion chromatograms for phosphoribosylated PARP10^CD peptides, where modified residues are denoted in red.

Fig. 2.

Characterization of CHIKV nsP3^MD mutants that are defective in ADP-ribose–binding and/or MAR hydrolase activity. (A) Active site of CHIKV nsP3^MD bound to ADP-ribose (37) highlighting residues targeted in this study. (B) Quantitative representation of MAR hydrolase activity of nsP3^MD mutants relative to nsP3^MD WT. Assays were performed in triplicate, buffer control was subtracted, and obtained values were normalized to activity levels of nsP3^MD WT. Representative raw data shown in SI Appendix, Fig. S5. (C) Quantitative representation of ADP-ribose affinity of nsP3^MD mutants normalized to nsP3^MD WT. Raw data shown in Table 1 and SI Appendix, Fig. S7.

Fig. 3.

Characterization of CHIKV 181/25 nsP3 G32E V113R Y114N mutant revertants. (A) Sequencing chromatogram of nsP3 G32E V113R Y114N-containing plasmid clones and virus particles produced at P0 from BHK-21 cells. (B) Quantitative representation of MAR hydrolase activity of nsP3^MD mutants relative to nsP3^MD WT. Assays were performed in triplicate, buffer control was subtracted, and obtained values were normalized to activity levels of nsP3^MD WT. Representative raw data shown in SI Appendix, Fig. S9. (C) Quantitative representation of ADP-ribose affinity of nsP3^MD mutants normalized to nsP3^MD WT. Raw data shown in Table 1 and SI Appendix, Fig. S9.

Fig. 4.

Macrodomain mutations in CHIKV nsP3 impair replication kinetics in cell culture and attenuate virulence in vivo. (A) Replication of CHIKV 181/25 WT and nsP3 mutants in NSC-34 cells infected at an MOI of 10. Average data of three independent experiments are presented. Error bars indicate SEMs. **P < 0.01; ***P < 0.001; ****P < 0.0001; 181/25 vs. nsP3 mutants. Dunnett’s multiple comparisons test. (B) Mortality of 2-d-old neonatal mice (n = 24–28 per virus strain) after intracranial infection with CHIKV 181/25^{E2 I12T R82G} or nsP3 mutants. Percentage of survival was plotted by using Kaplan–Meyer analysis and compared with log rank test. Data were pooled from two independent experiments, ****P < 0.0001; WT vs. nsP3 mutants.