Abstract
Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH(2)-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser(633)Ogamma forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala(716), of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ( approximately 19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.
Highlights
Birnaviruses are non-enveloped viruses of ϳ60 –70 nm in diameter, which replicate in the cytoplasm of their host cells [1] and are characterized by their two double-stranded RNA genomic segments (A and B) [2]
The nucleophile Ser692 and general base Lys729 of blotched snakehead virus (BSNV) VP4 are rendered as van der Waals spheres
1), we designed a number of Infectious pancreatic necrosis virus (IPNV) VP4 constructs with a C terminus ending at Ala716 [23]
Summary
Birnaviruses are non-enveloped viruses of ϳ60 –70 nm in diameter, which replicate in the cytoplasm of their host cells [1] and are characterized by their two double-stranded RNA genomic segments (A and B) [2]. The polyprotein is processed through the proteolytic activity of VP4 to generate the proteins pVP2 and VP3 as well as VP4 [3]. PVP2 is further processed by VP4 to generate the capsid protein VP2 and several structural peptides. Infectious pancreatic necrosis virus (IPNV) is a well known pathogen in salmonid fish. It is responsible for infectious pancreatic necrosis, a disease characterized by severe damage to the internal organs and tissues [7]. Three cleavage sites within pVP2 are located after amino acid residues 442, 486, and 495. The main cleavage sites and those involved in the processing of pVP2 have been described by the consensus (Ser/Thr)-XAla2(Ser/Ala)-Gly motif (Fig. 1). The expression product from this plasmid VP4_514 – 716,K674A (VP4tri) is 204 residues long (including initial methionine) with a molecular mass of 21.7 kDa and a theoretical isoelectric point of
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