Expression, purification, and in vitro activity of an arterivirus main proteinase

Abstract

To allow the biochemical and structural characterization of the chymotrypsin-like "main proteinase" (non-structural protein 4; nsp4) of the arterivirus prototype Equine Arteritis Virus (EAV), we developed protocols for the large-scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase was expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag. Following purification, the nsp4 moiety of MBP-nsp4 was successfully used for structural studies [Barrette-Ng, I.H., Ng, K.K.S., Mark, B.L., van Aken, D., Cherney, M.M., Garen, C, Kolodenko, Y., Gorbalenya, A.E., Snijder, E.J., James, M.N.G, 2002. Structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole. J. Biol. Chem. 277, 39960-39966]. Furthermore, both forms of the EAV proteinase were shown to be proteolytically active in two different trans-cleavage assays. Recombinant nsp4 cleaved the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates. In a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the P9-P7' residues of six nsp4 cleavage sites was investigated. The peptide representing the EAV nsp7/8 junction was used to optimize the reaction conditions (pH 7.5, 25mM NaCl, 30% glycerol at 30 degrees C), which resulted in a maximum turnover of 15% of this substrate in 4h, using a substrate to enzyme molar ratio of 24:1. The assays described in this study can be used for a more extensive biochemical characterization of the EAV main proteinase, including studies aiming to identify inhibitors of proteolytic activity.

Structure and Cleavage Specificity of the Chymotrypsin-Like Serine Protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

“…Just like other reports on peptide-based cleavage assays, 21,22 our enzyme also shows a remarkable difference in efficiency towards different peptides. Of the nine potential substrates investigated, only three can be cleaved to the extent of being detectable on an HPLC system (see Table 2).…”

“…The unusual properties of both the S1 subsite and the oxyanion hole observed in our structure, as we discussed above, might well explain the low in vitro catalytic activity of PRRSV nsp4 compared to that of other 3C-like proteases in similar experiments. 21,22 Another interesting point is that in the neighborhood of the substrate-binding site, Cys111 is located in close proximity to Cys115 but has its side chain directed away from that of the latter, so that a disulfide bond cannot be formed. Disulfide bonds are rare in proteins that function in the cytosol, but for instance in coronavirus nonstructural proteins, several cases have been observed.…”

Characterization of Equine Humoral Antibody Response to the Nonstructural Proteins of Equine Arteritis Virus

“…Incomplete disassociation of these nsps from cellular proteins during SDS-PAGE could have caused some of these extra bands with higher molecular masses. Consistent with previous reports, the recombinant nsp4 (predicted as 21 kDa and 25 kDa with modifications in mammalian and bacterial expression systems, respectively) migrated as a protein of approximately 30 kDa, a phenomenon described as aberrant migration of this protein during SDS-PAGE (49,53). Additionally, the two bands of nsp4 visible by Western immunoblotting analysis using nsp4-specific rabbit antisera have been reported previously (53).…”

Identification and characterization of Iflavirus 3C-like protease processing activities

“…Like the 3CL proteases of norovirus and EAV, the EoV 3CL protease is relatively sensitive to varying Na þ ionic concentrations (Someya et al, 2005;van Aken et al, 2006), because high concentrations of Na þ strongly inhibited the proteolytic activity of EoV 3CL. In contrast, the 3CL proteases of mouse hepatitis virus and porcine reproductive and respiratory syndrome virus (PRRSV) are insensitive to 100-500 mM of Na þ (Seybert et al, 1997;Xu et al, 2010).…”