title: Functional dissection of the alphavirus capsid protease: sequence requirements for activity.
abstract: BACKGROUND
  The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus.
  
  
  RESULTS
  Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity.
  
  
  CONCLUSIONS
  We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.
authors.affiliation: Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, D-18057 Rostock, Germany. saijo.thomas@uni-rostock.de
authors.forename: Saijo
authors.initials: S
authors.lastname: Thomas
authors.forename: Jagdish
authors.initials: J
authors.lastname: Rai
authors.forename: Lijo
authors.initials: L
authors.lastname: John
authors.forename: Stephan
authors.initials: S
authors.lastname: Günther
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
authors.forename: Brigitte M
authors.initials: BM
authors.lastname: Pützer
authors.forename: Stephan
authors.initials: S
authors.lastname: Schaefer
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D002646:Chikungunya virus / Q000201:enzymology* / Q000235:genetics*; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D036022:Capsid Proteins; D010447:Peptide Hydrolases