Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. In this way, proteolysis enables temporal control over the viral life cycle. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. Despite the existence of X-ray crystal structures for the three proteins, the molecular mechanism for the different functionality of 3CD is not well understood due to the structure of the domains in 3CD being nearly identical to the isolated 3C and 3D proteins.<br/><br/>It has been proposed that the protein conformational dynamics of 3CD may be different than those of 3C and 3D. We tested this hypothesis by comparing the conformational dynamics of 3C, 3D, and 3CD using solution-state NMR spectroscopy. We used Carr-Purcell-Meiboom-Gill (CPMG) multiple quantum relaxation dispersion, methyl <sup>1</sup>H chemical exchange saturation transfer (CEST), and methyl spin-spin relaxation to probe conformational dynamics across multiple timescales. We also used methyl spin-spin relaxation to gain insight into the independence of the domains in solution. We have identified differences in conformational dynamics in functionally important regions that may be useful in understanding the differences in function between 3C, 3D, and 3CD. In particular, expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not seen in 3C and 3D alone despite having nearly identical lowest-energy structures.

Role of conformational dynamics of a viral polyprotein in expanding the functional viral proteome

ACS Spring Meeting 2021

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