The Picornavirus Precursor 3CD Has Different Conformational Dynamics Compared to 3Cpro and 3Dpol in Functionally Relevant Regions

Abstract

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. 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 the 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. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures.

DWV 3C Protease Uncovers the Diverse Catalytic Triad in Insect RNA Viruses

“…It is likely that DWV polyprotein is cleaved at Q2118 to release the 97 kDa precursor of 3C pro and RdRP. However, we need to assume that the 97 kDa precursor is active as a protease similar to picornavirus 3CD precursor ( 51 ) and that cleavage should occur in cis , at least during the early stage of polyprotein synthesis until a sufficient 97 kDa precursor and 42 kDa mature 3C pro s accumulate in DWV-infected cells. Because the 97 kDa precursor remains abundant in the virus-infected honey bee cells ( Fig.…”

DWV 3C protease uncovers the diverse catalytic triad in insect RNA viruses

“…It is likely that DWV polyprotein is cleaved at Q2118 to release the 97 kDa precursor of 3C pro and RdRP. However, we need to assume that the 97 kDa precursor is active as a protease similar to picornavirus 3CD precursor (Winston and Boehr, 2021) and that cleavage should occur in cis , at least during the early stage of polyprotein synthesis until a sufficient 97 kDa precursor and 42 kDa mature 3C pro s accumulate in DWV-infected cells. Because the 97 kDa precursor remains abundant in the virus-infected honey bee cells (Fig.…”

Running With Scissors: Evolutionary Conflicts Between Viral Proteases and the Host Immune System

“…Here and in subsequent sections, we will predominantly discuss activities of the 3C, 3CL, and NS3 proteases of Picornaviridae , Coronaviridae , and Flaviviridae , respectively, due to their known roles in cleaving mammalian host factors. It is important to point out that because the polyprotein is sequentially processed, and not always to completion, protease activity may also be carried out when 3C, 3CL, or NS3 remains fused or associated with additional viral proteins ( 27 , 28 ). This is especially true in the Flaviviridae , where the NS3 protease usually functions in association with NS2B (in the case of flaviviruses such as dengue and Zika viruses) or NS4A [in the case of hepatitis C virus (HCV)] ( 26 , 29 ).…”