Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

Ragunath Singaravelu,David R Blais,Craig S Mckay,John Paul Pezacki

doi:10.1186/1477-5956-8-5

Abstract

BackgroundHepatitis 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≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.ResultsThe PS4≡ 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≡ 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.ConclusionsThese 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.

Highlights

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
We investigated the ability of Activitybased protein profiling (ABPP) approaches using the non-directed PS4≡ probe to profile enzyme activity in Huh-7 cells harboring the genotype 1b HCV subgenomic replicon RNA (Figure 1) [26,27]
The selection of the PS4≡ probe was based on its activity-based reactivity towards a large but manageable fraction of the proteome [21,28] and on its relatively small size, which should minimize non-covalent probeprotein interactions and allow cellular uptake and distribution for in situ labeling

Summary

Introduction

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. With no vaccines yet available and clinical treatments that have only limited success, many HCV-infected individuals develop chronic hepatitis which eventually progresses into liver steatosis, cirrhosis and hepatocellular carcinoma. As the HCV genome is a single-stranded RNA molecule of only ~9.6 kb (Figure 1A) that encodes for ten mature viral proteins, HCV relies heavily on host factors for its. High throughput techniques, such as gene expression profiling and proteomic approaches have led to the identification of several host-viral interactions [10,11]. We have previously applied some of these directed probes to study the differential activity of host cell proteases during HCV replication [19]

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Conclusion