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

ABSTRACT: Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3C^pro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. Thus, it is one of the nonstructural viral proteins essential for the replication. We found that the 3C^pros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3C^pro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3C^pros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3C^pro with RNA-dependent RNA polymerase and mature 3C^pro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3C^pro is the first 3C^pro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3C^pros of Picornavirales. Furthermore, it would be possible to use the specific inhibitors of DWV 3C^pro to control DWV infection in honey bees in future. IMPORTANCE The number of managed honey bee (Apis mellifera) colonies has considerably declined in many developed countries in the recent years. Deformed wing virus (DWV) vectored by the mites is the major threat to honey bee colonies and health. To give insight into the mechanism of DWV replication in the host cells, we studied the structure-function relationship of 3C protease (3C^pro), which is necessary to cleave a viral polyprotein at the specific sites to produce the mature proteins. We found that the overall structure, some inhibitors, and processing of 3C^pro are shared between Picornavirales; however, there is diversity in the catalytic triad. DWV 3C^pro is the first viral protease characterized among insect RNA viruses and reveals the evolutionary history of 3C^pro among Picornavirales. Furthermore, DWV 3C^pro inhibitors identified in our study could also be applied to control DWV in honey bees in future.

SUBMITTER: Yuan X

PROVIDER: S-EPMC9241925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.

Project description:Senecavirus A (SVA) is an emerging picornavirus infecting porcine of all age groups and causing foot and mouth disease (FMD)-like symptoms. One of its key enzymes is the 3C protease (3Cpro), which is similar to other picornaviruses and essential for virus maturation by controlling polyprotein cleavage and RNA replication. In this study, we reported the crystal structure of SVA 3Cpro at a resolution of 1.9 Å and a thorough structural comparison against all published picornavirus 3Cpro structures. Using statistical and graphical visualization techniques, we also investigated the sequence specificity of the 3Cpro. The structure revealed that SVA 3Cpro adopted a typical chymotrypsin-like fold with the S1 subsite as the most conservative site among picornavirus 3Cpro. The surface loop, A1-B1 hairpin, adopted a novel conformation in SVA 3Cpro and formed a positively charged protrusion around S' subsites. Correspondingly, SVA scissile bonds preferred Asp rather than neutral amino acids at P3' and P4'. Moreover, SVA 3Cpro showed a wide range tolerance to P4 residue volume (acceptable range: 67 Å3 to 141 Å3), such as aromatic side chain, in contrast to other picornaviruses. In summary, our results provided valuable information for understanding the cleavage pattern of 3Cpro. IMPORTANCE Picornaviridae is a group of RNA viruses that harm both humans and livestock. 3Cpro is an essential enzyme for picornavirus maturation, which makes it a promising target for antiviral drug development and a critical component for virus-like particle (VLP) production. However, the current challenge in the development of antiviral drugs and VLP vaccines includes the limited knowledge of how subsite structure determines the 3Cpro cleavage pattern. Thus, an extensive comparative study of various picornaviral 3Cpro was required. Here, we showed the 1.9 Å crystal structure of SVA 3Cpro. The structure revealed similarities and differences in the substrate-binding groove among picornaviruses, providing new insights into the development of inhibitors and VLP.

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

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