Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

David R Blais,Rodney K Lyn,Michael A Joyce,Yanouchka Rouleau,Rineke Steenbergen,Nicola Barsby,Lin-Fu Zhu,Adrian F Pegoraro,Albert Stolow,David L Tyrrell,John Paul Pezacki

doi:10.1074/jbc.m110.135483

Abstract

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

Highlights

Limited clinical treatments that are only successful in a small subset of patients [1]
Because the LDs are surrounded by a phospholipid monolayer [13, 14] and HCV replication complexes are likely enclosed by lipid bilayer membranes [15], the replication complexes are not likely to be directly associated with the membranes of LDs [11]
CES1 Is Differentially Active in Huh7 Cells Highly Replicating HCV—We examined the serine hydrolase Activity-based protein profiling (ABPP) in hepatoma cells replicating a genotype 1b HCV subgenomic replicon RNA [31] (Fig. 1A), using a FP-rhodamine ABPP probe (Fig. 1B) that has been shown to react with serine hydrolases in an activitydependent manner [19]

Summary

Introduction

Limited clinical treatments that are only successful in a small subset of patients [1]. We observe that HCV RNA and protein levels recover to nearly normal levassay revealed that HCV replication levels were 3 and 2 times els after transient CES1 knockdown (Fig. 3, A and B, and higher in Huh7CES1 cells when compared with Huh7 and supplemental S3) and that this recovery appears to happen

Results

Conclusion