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Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

ABSTRACT: Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales) is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1) includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2) is predicted to contain the domains for a movement protein (MP) and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1) and in trans (P2). Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q)/(G or S).

SUBMITTER: Mann KS

PROVIDER: S-EPMC5407059 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

Mann Krin S KS Walker Melanie M Sanfaçon Hélène H

Frontiers in microbiology 20170427

Strawberry mottle virus (SMoV, family <i>Secoviridae</i>, order <i>Picornavirales</i>) is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1) includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminu ...[more]

PMID: 28496438

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Project description:Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a bipartite genome with each RNA encoding one polyprotein. All characterized secovirids encode a single protease related to the picornavirus 3C protease. The SMoV 3C-like protease was previously shown to cut the RNA2 polyprotein (P2) at a single site between the predicted movement protein and coat protein (CP) domains. However, the SMoV P2 polyprotein includes an extended C-terminal region with a coding capacity of up to 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we identified a novel cleavage event at a P↓AFP sequence immediately downstream of the CP domain. Following deletion of the PAFP sequence, the polyprotein was processed at or near a related PKFP sequence 40 kDa further downstream, defining two protein domains in the C-terminal region of the P2 polyprotein. Both processing events were dependent on a novel protease domain located between the two cleavage sites. Mutagenesis of amino acids that are conserved among isolates of SMoV and of the related Black raspberry necrosis virus did not identify essential cysteine, serine, or histidine residues, suggesting that the RNA2-encoded SMoV protease is not related to serine or cysteine proteases of other picorna-like viruses. Rather, two highly conserved glutamic acid residues spaced by 82 residues were found to be strictly required for protease activity. We conclude that the processing of SMoV polyproteins requires two viral proteases, the RNA1-encoded 3C-like protease and a novel glutamic protease encoded by RNA2.IMPORTANCE Many viruses encode proteases to release mature proteins and intermediate polyproteins from viral polyproteins. Polyprotein processing allows regulation of the accumulation and activity of viral proteins. Many viral proteases also cleave host factors to facilitate virus infection. Thus, viral proteases are key virulence factors. To date, viruses with a positive-strand RNA genome are only known to encode cysteine or serine proteases, most of which are related to the cellular papain, trypsin, or chymotrypsin proteases. Here, we characterize the first glutamic protease encoded by a plant virus or by a positive-strand RNA virus. The novel glutamic protease is unique to a few members of the family Secoviridae, suggesting that it is a recent acquisition in the evolution of this family. The protease does not resemble known cellular proteases. Rather, it is predicted to share structural similarities with a family of fungal and bacterial glutamic proteases that adopt a lectin fold.

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.

Dataset Information

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

Publications

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

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