Activity, specificity, and probe design for the smallpox virus protease K7L.

Area of Science:

• Virology
• Biochemistry
• Molecular Biology

Background:

• The K7L gene product of the smallpox virus is a protease involved in viral protein maturation.
• K7L belongs to protease Clan CE, a diverse group of cysteine proteases found across different organisms.

Purpose of the Study:

• To characterize the recombinant K7L protease, including its expression, biochemical mechanism, substrate preference, and regulation.
• To investigate factors influencing K7L's catalytic activity and substrate specificity.
• To develop tools for studying K7L's role in the viral life cycle and for antiviral drug design.

Main Methods:

• Recombinant high-level expression of the K7L protease.
• Biochemical assays to determine substrate preference and catalytic mechanism.
• Analysis of factors enhancing K7L activity, including homodimerization, P25K protein, glycerol, RNA, and DNA.
• Library-based peptide preference analyses to design an activity-based probe.

Main Results:

• The AG-X motif is necessary but not sufficient for K7L cleavage; extended peptide sequences (P7 and P8 positions) are required for optimal activity.
• K7L's catalytic activity is enhanced by homodimerization, the substrate protein P25K, and glycerol.
• RNA and DNA enhance K7L cleavage of P25K protein, suggesting a role in protein-substrate interaction.
• An activity-based probe was developed to selectively label K7L in cellular lysates.

Conclusions:

• K7L protease activity is regulated by specific substrate sequences and protein-protein interactions.
• Nucleic acids may play a role in augmenting K7L's interaction with its protein substrates.
• The developed activity-based probe and understanding of K7L function provide a basis for designing novel antiviral strategies.