Fig 2. ABHD5, but not the Chanarin-Dorfman syndrome mutants, supports both HCV assembly and release.

(a) Functional and structural organisation of ABHD5. Below are indicated the consensus sequences for the esterase/lipase motif (GXNXG, in green), and for the predicted NLS (in blue), phosphorylation sites (in red) and LPAAT motif (HX₄D, in purple). Note that the replacement of the nucleophilic serine in the typical GXSXG lipase motif by an asparagine prevents any hydrolase activity. The predicted hydrolase pseudo-catalytic triad [16] includes residues N¹⁵³, H³²⁷, and E²⁶⁰ or D³⁰¹. The lipid droplet-binding domain is defined according to Gruber et al. [40]. The mutations used in this study are shown on top of the schema with a color code corresponding to their targeted motif. Those mutations associated with the Chanarin-Dorfman syndrome (Q130P, E260K) are underlined. (b-e) Irrelevant (sh irr) or ABHD5-specific shRNAs (sh ABHD5) were transduced into cells together with shRNA-resistant wild-type or mutant ABHD5 (Q130P or E260K), or with an empty vector (pWPI-BLR). After 4 days, the cells were infected with JcR-2a for 72 h before transferring the supernatant onto target cells. (b) ABHD5 expression was detected by Western blot at the beginning (96h post-transduction) and at the end of HCV infection (72 h post-infection, corresponding to 7 days post-transduction). Detection of β-tubulin served as an internal control for protein load. A representative Western blot is presented in the bottom panel while the top panel depicts the quantification of ABHD5 expression, normalised for β-tubulin expression and for the mock-treated cells (sh irr and pWPI-BLR transduction) and averaged over 3 independent experiments. (c-e) In the same set of experiments as in panel b, HCV replication, progeny virion production and core secretion were analysed. Note that the mild effects of ABHD5 expression on HCV entry and replication in this assay are probably due to the late time point chosen (72h post-infection) where virus spread and re-infection starts affecting the readout. (f) Infectious titres were determined in the supernatants (extracellular infectivity) as well as in the cleared freeze-and-thaw lysates (intracellular infectivity) of infected cells with manipulated ABHD5 or ApoE expression.