Cell-Type-Dependent Role for nsP3 Macrodomain ADP-Ribose Binding and Hydrolase Activity during Chikungunya Virus Infection

Abstract

Chikungunya virus (CHIKV) causes outbreaks of rash, arthritis, and fever associated with neurologic complications, where astrocytes are preferentially infected. A determinant of virulence is the macrodomain (MD) of nonstructural protein 3 (nsP3), which binds and removes ADP-ribose (ADPr) from ADP-ribosylated substrates and regulates stress-granule disruption. We compared the replication of CHIKV 181/25 (WT) and MD mutants with decreased ADPr binding and hydrolase (G32S) or increased ADPr binding and decreased hydrolase (Y114A) activities in C8-D1A astrocytic cells and NSC-34 neuronal cells. WT CHIKV replication was initiated more rapidly with earlier nsP synthesis in C8-D1A than in NSC-34 cells. G32S established infection, amplified replication complexes, and induced host-protein synthesis shut-off less efficiently than WT and produced less infectious virus, while Y114A replication was close to WT. However, G32S mutation effects on structural protein synthesis were cell-type-dependent. In NSC-34 cells, E2 synthesis was decreased compared to WT, while in C8-D1A cells synthesis was increased. Excess E2 produced by G32S-infected C8-D1A cells was assembled into virus particles that were less infectious than those from WT or Y114A-infected cells. Because nsP3 recruits ADP-ribosylated RNA-binding proteins in stress granules away from translation-initiation factors into nsP3 granules where the MD hydrolase can remove ADPr, we postulate that suboptimal translation-factor release decreased structural protein synthesis in NSC-34 cells while failure to de-ADP-ribosylate regulatory RNA-binding proteins increased synthesis in C8-D1A cells.

In silico identification of chikungunya virus replication inhibitor validated using biochemical and cell‐based approaches

“…3A). A previous study has shown that mutations in the CHIKV nsP3 MD Asp10, Gly32, Val33 Thr111, Gly112, Tyr114 and Arg144 residues impair both ADPr binding and hydrolysis activity, indicating their critical role in enzyme activity [13–15,18,41].…”

“…Baicalin, a compound that interacts with nsP3 and demonstrates anti-CHIKV effects in cell culture, has shown promise [28]. Other small-molecule inhibitors specifically targeting CHIKV nsP3 MD including harringtonine, atovaquone, RYL 634, 6-azauridine, favipiravir and sofosbuvir have been identified but provide limited long-term relief to patients [29][30][31][32][33][34][35][36][37][38][39][40][41]. Given the lack of specific and effective inhibitors, the present study aimed to identify a potent inhibitor capable of halting viral replication.…”

Special Issue “Chikungunya Virus and Emerging Alphaviruses”

“…The study compared the replication kinetics of the wild-type CHIKV with that of CHIKV containing a mutant MD site. This mutation led to either decreased ADPr binding and hydrolase activity (G32S) or increased ADPr binding with decreased hydrolase activity (Y114A) [ 15 ]. Finally, the ultrastructural morphology and mechanical properties of CHIKV were examined using atomic force microscopy and Raman spectroscopy.…”