Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease.
Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2' positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2' positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.
Robel
Ivonne
I
The Calicilab, Institute of Virology, Dresden University of Technology, Dresden, Germany.
Gebhardt
Julia
J
Mesters
Jeroen R
JR
Gorbalenya
Alexander
A
Coutard
Bruno
B
Canard
Bruno
B
Hilgenfeld
Rolf
R
Rohayem
Jacques
J
D001665:Binding Sites; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D053818:Chymases / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D029341:Sapovirus / Q000737:chemistry* / Q000201:enzymology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
D010455:Peptides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D053818:Chymases