Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe.
BACKGROUND
Hepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4 identical with) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.


RESULTS
The PS4 identical with probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled in situ compared to the in vitro labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4 identical with targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation.


CONCLUSIONS
These findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of in situ ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.
Singaravelu
Ragunath
R
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario, K1A 0R6, Canada.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada.
Blais
David R
DR
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario, K1A 0R6, Canada.
McKay
Craig S
CS
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario, K1A 0R6, Canada.|Department of Chemistry, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada.
Pezacki
John Paul
JP
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario, K1A 0R6, Canada.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada.|Department of Chemistry, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada.