N24 mutations in the CHIKV macrodomain catalytic site rapidly select for second-site mutations at residue D31.

a, Schematic of the CHIKV genome highlighting the nsP3 gene. The nsP3 protein comprises three domains: an N-terminal macrodomain (first 160 amino acids), a central zinc-binding domain (ZBD), and a C-terminal hypervariable domain (HVD). Structure of the CHIKV macrodomain (PDB 3GPO) highlighting the catalytic asparagine 24 (N24) and bound ADP-ribose. b, Point mutations were introduced into the CHIKV Caribbean infectious clone to generate two mutant viruses: N24A (asparagine to alanine) and N24D (asparagine to aspartic acid). For viral stock production, in vitro transcribed RNAs for WT, N24A, and N24D viruses were transfected into Vero cells. Cell culture supernatant was collected at one day post-transfection (P0) and used to infect fresh Vero cells. Supernatants from this passage were collected at one and two days post-infection (P1) and subjected to RNA extraction, RT-PCR amplification of the nsP3 gene, and Sanger sequencing. c, Summary of amino acid and nucleotide identities at residues 24 and 31 in WT, N24A, and N24D viruses at one day post-transfection (P0) and one and two days post-infection (P1) in Vero cells. d, Representative Sanger sequencing chromatograms from P1 viral stocks. The WT sequence shows no changes at position 24. N24A and N24D viruses show novel second-site mutations at residue 31 (D31N and D31H/N respectively).

N24 catalytic mutations impair CHIKV replication in interferon-competent human cells but not in mosquito cells.

a, Schematic of the experimental design. In vitro transcribed N24A and N24D viral RNAs were transfected into A549 cells. Cell culture supernatants were collected at 0, 1, 2, and 3 days post-transfection (P0) and at 1 day post-infection (P1) and subjected to RNA extraction, RT-PCR amplification of the nsP3 gene, and Sanger sequencing. b, Amino acid and nucleotide identities at residues 24 and 31 in N24A and N24D viruses following transfection into A549 cells. Dashes indicate timepoints where insufficient viral RNA was recovered for sequencing. Double peaks indicating coexistence of multiple nucleotides at the same position are denoted by ‘/’. Amino acid abbreviations: N, asparagine; A, alanine; D, aspartic acid. c, Growth kinetics of WT, N24A-D31N, and N24D-D31H/N viruses in A549 and U4.4 cells. Cells were infected at a multiplicity of infection of 0.05. Data are plotted as mean ± SD for three independent biological experiments. Data were analyzed using a mixed-effects model with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test. Pink and teal asterisks indicate significant differences compared to WT.

Macrodomain catalytic mutations modulate CHIKV vector competence in Aedes mosquitoes.

a, Schematic of the experimental design. Laboratory colonies of Ae. albopictus and Ae. aegypti were exposed to an infectious blood meal containing WT, N24A-D31N, or N24D-D31H/N viruses. At 2, 5, and 7 days post-infection, individual mosquitoes were dissected into bodies and heads. Prevalence of infection and viral titers were determined by plaque assay. b, Prevalence of infection (top) and viral titers (bottom) in bodies and heads of Ae. albopictus mosquitoes. c, Prevalence of infection (top) and viral titers (bottom) in bodies and heads of Ae. aegypti mosquitoes. Numbers above prevalence bars indicate the number of infected individuals

Functional and structural characterization of CHIKV nsP3 macrodomain mutants.

a, Assays of the ADP-ribosylhydrolase activity of recombinantly expressed wild-type (WT) and mutant macrodomains. Macrodmains (200 nM) were incubated with auto-MARylated human PARP10 for 1 hour at room temperature and the production of ADP-ribose was measured using NUDT5 and an AMP-Glo luciferase assay4. Data are plotted mean ± SD for four technical replicates. b, Thermostability of CHIKV nsP3 macrodomain mutants measured by DSF using SYPRO orange. Data are plotted mean ± SD for three technical replicates. c, Change in CHIKV nsP3 macrodomain thermostability upon incubation with 1 mM ADP-ribose. d, Alignment of the crystallographic structures of WT, N24A, N24A-D31N, N24A-D31H, D31H and D31N reveals a peptide flip in P25 and a coupled shift in Y114 in structures containing the N24A mutation. For clarity, only chain A is shown (see Supplementary Fig. 4a for all chains). e, Difference electron density maps (FO-FC, contoured at 3 σ) calculated prior to modeling ADP-ribose. Maps for all chains are shown in Supplementary Fig. 5. f, Crystal structures of N24A, D31N, N24A-D31H and N24A-D31N bound to ADP-ribose. Chain A is shown for the P31 structures and chain D for the P41 structure. Although the ADP-ribose binding pose is conserved, there is a rotameric shift at residue 31 that accompanies ADP-ribose binding, and mutations at position 31 change the character of the exit path of the substrate suggesting a role for substrate-specific recognition.