A flexible Janus head: molecular determinants of a viral protein’s RNAi suppressor and capsid forming activities

Viral suppressors of RNA silencing (RNAi) expressed by plant viruses, VSRs, are exceptional proteins. Although not conserved, even within virus families, most VSRs bind small interfering RNAs, siRNAs, thereby blocking antiviral RNAi. Turnip crinkle virus , a member of the Tombusviridae family, encodes a VSR, TCV P38, which also forms the viral capsid. Biochemical studies of the purified protein revealed that the TCV P38 VSR functions as a metastable dimer that binds double-stranded (ds) RNA with high affinity via an induced-fit mechanism of both binding partners. Consistent with its role as a VSR that interferes with antiviral RNAi at various stages, P38 distinguishes between siRNAs of different lengths. Consistent with its capsid-forming function, the protein binds longer dsRNAs cooperatively. Structural data obtained from an RNA-free capsid-like icosahedral crystal and modeling of Tombusviridae capsid proteins suggest that flexible interactions between the P (protruding)-domains of P38 are important determinants for forming both the VSR dimer and the capsid structure. Studies with protein mutants confirmed this and also revealed the central role of the S (shell)- and R (RNA-binding)-domains of TCV P38 in adaptive substrate binding. Our study provides comprehensive insights into the molecular and structural properties of a versatile viral “Janus head” protein.