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. 2020 Jul 8;295(35):12426–12436. doi: 10.1074/jbc.RA120.013455

Figure 4.

Figure 4.

Expression of active NS3-4A protease reduces conversion of desmosterol-d6 to cholesterol-d6 in Huh7.5-DHCR24KO cells. Conversion of desmosterol-d6 to cholesterol-d6 and formation of DHCR24* were monitored in Huh7.5-DHCR24KO cells. The extent of conversion in Huh7.5-DHCR24KO cells expressing DHCR24 alone was set as 100%. A, the conversion of desmosterol-d6 to cholesterol-d6 was monitored by GC-MS analysis of extracted lipidomes. Each bar of the graph represents an average of 6 biological replicates, with error bars representing the standard deviation. Although no conversion of desmosterol-d6 to cholesterol-d6 is observed in the Huh7.5-DHCR24KO cells expressing a GFP control protein, ectopic expression of DHCR24-FLAG is sufficient to restore intracellular conversion of desmosterol-d6 to cholesterol-d6. Co-expression of active NS3-4A with DHCR24-FLAG reduces this reaction significantly, and this is correlated with formation of DHCR24*. NS3-4A′s effect on the intracellular reaction is abrogated in the presence of telaprevir (p = 0.0061) or when NS3 bears the H57A mutation in the protease active site (p = 0.0186). There was no significant (ns) difference in conversion of desmosterol-d6 to cholesterol-d6 between the Huh7.5-DHCR24KO cells expressing DHCR24 alone, telaprevir-treated cells co-expressing DHCR24 with NS3-4A (p = 0.2064) or cells co-expressing DHCR24 with the inactive NS3-4A H57A mutant (p = 0.1189). B, DHCR24-FLAG species were immunoprecipitated and analyzed by immunoblots with anti-FLAG and anti-NS3 antibodies. Analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cell lysates performed as an internal control is shown. Co-expression of active NS3-4A with DHCR24-FLAG results in formation of DHCR24*, which is correlated with reduced conversion of desmosterol-d6 to cholesterol-d6. DHCR24 residues 92-516, which correspond to the soluble catalytic domain, has mobility comparable to DHCR24*-FLAG, but ectopic expression of this construct does not support intracellular conversion of desmosterol-d6 to cholesterol-d6. Note that the α-FLAG blot was cut between the GFP and DHCR24(92-516) lanes to remove the intervening lane with the molecular weight ladder.