Background & aims: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production.
Methods: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches.
Results: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle.
Conclusions: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.
Keywords: AADAC; Activity-based protein profiling; ApoB; Apolipoprotein; Arylacetamide deacetylase; BSA; Carboxylesterase; DMEM; Dulbecco’s Modified Eagle Medium; E600; ELISA; ER; FBS; FP; HCV; Hepatic lipid metabolism; Hepatitis C virus; JFH; Japanese fulminant hepatitis; LD; Lipase; Lipid droplet; MOI; MTP; OA; ORO; Oil Red O; PBS; PDI; RNA; Serine esterase; TG; TGH; Triacylglycerol; Triacylglycerol hydrolase; VLDL; Very low density lipoprotein; apolipoprotein B-100; arylacetamide deacetylase; bovine serum albumin; diethyl-p-nitrophenyl phosphate; endoplasmic reticulum; enzyme-linked immunosorbent assay; fetal bovine serum; fluorophosphonate; hepatitis C virus; lipid droplet; microsomal triglyceride transfer protein; multiplicity of infection; oleic acid; phosphate buffered saline; protein disulfide isomerase; qRT-PCR; quantitative reverse transcriptase-polymerase chain reaction; ribonucleic acid; triacylglycerol; triacylglycerol hydrolase; very low density lipoprotein.
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