Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons.
ABSTRACT Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.
Article: Activity-based proteome profiling of hepatoma cells during hepatitis C virus replication using protease substrate probes.[show abstract] [hide abstract]
ABSTRACT: Activity-based protein profiling (ABPP) offers direct insight into changes in catalytic activity of enzyme classes in complex proteomes, rather than protein or transcript abundance. Here, ABPP was performed in Huh7 hepatoma cell lines with a group of ABPP probes composed of an N-acetylated amino acid, that mimic the P(1) position in protease peptide substrates. Five different probes bearing distinct amino acids (Ser, Thr, Phe, Glu and His) labeled 54 differentially active proteins, including proteases, other hydrolases, oxidoreductases and isomerases. Four of the six protease families were targeted based on their P(1) substrate preferences. The broader specificity of the labeling observed could be explained by the substrate-based targeting nature and the electrophilic properties of the ABPP probes. When applied to Huh7 cells stably replicating hepatitis C virus (HCV) subgenomic replicon RNA, four proteins showed reduced activity, while three proteins had increased activity during HCV replication. These differentially active hits included carboxylesterase 1, cathepsin D, HSP105, protein disulfide isomerase 1 and A6, chaperonin containing TCP1 and isochorismatase domain containing 1, which demonstrated substrate preferences by being labeled by specific substrate probes. This illustrates the broader activity-based profiling capabilities of these substrate-based probes to reveal novel enzyme candidates and their potential roles during HCV replication.Journal of Proteome Research 12/2009; 9(2):912-23. · 5.11 Impact Factor
Article: Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe.[show abstract] [hide abstract]
ABSTRACT: 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. 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. 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.Proteome Science 02/2010; 8:5. · 2.33 Impact Factor