Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.


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Detailed Experimental Procedures 28-34 References for Supporting Information 35 Figure S1. Mass spectrometry analysis of DHCR24 and DHCR24* isolated from Huh7.5 and Huh7.5-SGR cells. Samples were separated by SDS-PAGE. One half of the gel was silverstained (left) and peptides (marked by arrows) were excised for mass spectrometry. The other half of the gel (right) was transferred to PVDF membrane and probed with anti-Flag antibody as described in Experimental Procedures. The molecular weight markers were used to align the two images. Mass spectrometry analysis of the excised bands, marked "a" and "b," detected multiple peptides derived from DHCR24. Lower signal was detected for peptides prior to residue 113, but a definitive cleavage site for the "b" sample could not be identified. Peak intensities for the indicated N-terminal charged peptide fragment ions in the "a" and "b" samples are reported in tables on pages 3 and 4. Raw data sets have been uploaded to MassIVE (accession number MSV000085492. The MBP-DHCR24 protein was produced by heterologous expression in an E. coli-derived in vitro translation system. Incubation of the MBP-DHCR24 fusion protein with recombinant NS3-4A resulted in formation of a faster-migrating species (red arrow head). This reaction was blocked in the presence of telaprevir ("inh"). Figure S3. Characterization of Huh7.5 DHCR24 KO cells by PCR, immunoblot, and Sanger sequencing. A) Genomic DNA from Huh7.5 CRISPR-Cas9 knockout of DHCR24 (clone 3) shows no band corresponding to wild type (WT) DHCR24 in PCR assays using forward primer: 5 '-AGA  TGC CTA GTG TGT TGG GAA T-3' and reverse primer: 5'-AAA GGG TTC CCC TGA CCT ATT  A-3'. B) Immunoblot analysis of lysates from candidate Huh7.5 DHCR24 KO clones using an Nterminal DHCR24 antibody (residues 68-85). No band was observed for clone 3. C) Nucleotide region of WT DHCR24 between exon 1 and 2. D) Region between exon 1 and 2 excised by CRISPR-Cas9. E) Sanger sequencing results of clone 3 band from agarose gel (shown in A) depicting removal of the designated region, resulting in removal of ~280 nucleotide bases in DHCR24 in clone 3. This clonal cell line is referred to as Huh7.5-DHCR24 KO in the text. Figure S4. Co-occurrence of NS3-4A and DHCR24 associated with the ER of Huh-7.5[VEEV/NS3-5B] cells. Immunofluorescence detection of NS3-4A, DHCR24, and the ER marker protein disulfide isomerase (PDI) in Huh-7.5[VEEV/NS3-5B] cells. These cells have been transduced with a noncytopathic subgenomic replicon derived from Venezuelan equine encephalitis virus. They are an established system for expression of the NS3-5B polyprotein at physiological levels with appropriate polyprotein processing and allow for studies of protein expression, processing, and localization over several weeks when maintained under selective conditions (36). A) DHCR24 (magenta) and NS3-4A (green) were observed as a fraction cooccurring in the endoplasmic reticulum (ER). B) DHCR24 (magenta, gamma = 1.2) and the ER marker protein disulfide isomerase (PDI, green, gamma=1.4) co-occur in the ER. Insert scale bar = 10 µm. Original scanned immunoblot for Figure 1D with ladder. Note that MW marks were unfortunately not preserved for the actin blot. The location of the actin band relative to core and NS5A was consistent between this and other immunoblots in which the MW markers were recorded. We have therefore indicated the approximate location of the 46 kDa marker relative to actin on comparable immunoblots and indicated this with an "*" mark.
Original scanned immunoblots for Figure 2 with respective MW ladders. The areas highlighted by boxes in Figure 2C were discussed in the main study. While there is no marker indicated in the image presented in Figure 2E (excised during processing), the relative locations of the upper and lower bands are consistent with DHCR24 (higher) and DHCR24* (lower) observed in other experiments. Figure 4B with respective MW ladders.
A3. Generation of DHCR24 KO Huh7.5 cells. DHCR24 KO Huh7.5 cells were generated by CRISPR/Cas9-mediated genome editing as previously described (55). DHCR24 has two isoforms that largely differ in the N-terminal region encoded in Exon 1. For this reason, Exon 2 was chosen as the target sequence. The removal introduced a frame shift resulting in a non-functional gene and partial removal of the N-terminal DHCR24 antibody epitope (DHCR24 residues 68-84). Two guide RNAs targeting removal of the splicing junction between intron1/exon2 encoded in exon 2 of DHCR24 (gRNA1: 5'-GCTTGTGGTGTAGCAATATGT-3' and gRNA2: 5'-GCTCACTGTCTCACTACGTGTG-3') were employed.
The CRISPR-DHCR24 double gRNA construct was transfected into Huh7.5 cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer's protocolin a T-75 flask seeded at a density of 2.1 x10 6 cells/mL following the manufacturer's protocol. The cells were cultured overnight at 37°C with 5% CO 2 . Following transfection, the medium was replaced with medium containing a final concentration of 4 µg/mL puromycin (InvivoGen) to select for Huh7.5 cells containing transfected pCRISPR-DHCR24 2x-gRNA-DHCR24 KO . Cells viable after selection with puromycin over several days were subjected to monoclonal selection by dilution of the cells to 10 cells/10 mL resuspended in sterile-filtered 50% preconditioned media for monoclonal limiting dilution in a 96-well plate in the absence of puromycin. Medium in the wells was replaced every 7 days with non-conditioned media until the appearance of single colonies (~5 weeks) was observed. Upon observation of single colonies, wells containing single colonies were amplified and characterized by Sanger sequencing and immunoblot analysis for the elimination of the Nterminal DHCR24 epitope ( Figure S3). Genome editing was confirmed by PCR. Diagnostic primers: Forward primer: 5'-AGA TGC CTA GTG TGT TGG GAA T-3' and Reverse primer: 5'-AAA GGG TTC CCC TGA CCT ATT A-3'.

B2.
In vitro plasmids. pMALc5x_maltose binding protein (MBP)-DHCR24 and pMALc5x-DHCR24 (full-length)-super folded green fluorescent protein (sfGFP) containing an ampicillin resistant cassette were purchased from GenScript USA Inc. The super-folded (sf)GFP cassettes were based on constructs that have previously been expressed and described (56). The various surrogate DHCR24 peptide constructs spanning residues 56-110, 52-66, and 85-99, and the 85-99-C91P mutant were expressed as fusions between an N-terminal MBP and sfGFP with a Cterminal DYKDDDDK (Flag-Tag). Expressions constructs were generated by overlapping extension PCR using Q5 High-Fidelity DNA Polymerase (NEB) and were subcloned into a pET28b plasmid containing a kanamycin resistance cassette. These plasmids were transformed into Rosetta2 (DE3) pLysS host strain (Novagen) following the manufacturer's protocol.

C1. Preparation of samples for immunoprecipitation of DHCR7-Flag and DHCR24-Flag.
Transfection of Huh7.5 cells, or Huh7.5 cells stably harboring SGR or SGR-GFP under G418 selection (750 µg/mL), were transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific) within 30 minutes of seeding. For a T75 flask, Huh7.5 cells were seeded at 60% confluency and transfected with 20 µg of either DHCR7-Flag or DHCR24-Flag, 20 µL of Lipofectamine 2000 (Thermo Fisher Scientific), and one mL of Opti-MEM (Gibco) with incubation times followed according to manufacturer's protocol. Sixteen hours later, the cell culture medium was removed and replaced with fresh medium. Forty eight hours after the addition of transfection reagents, the cells were lysed with RIPA buffer (Boston BioProducts) and HALT protease inhibitor cocktail (Roche) and stored at -20ºC until analysis.
C2. Immuno precipitation and immunoblot analysis of immunoprecipitates. Samples for immunoprecipitation were thawed on ice and clarified by centrifugation (18,000 x g) for five minutes. Protein lysate was collected by vigorously pipeting cell pellets in RIPA buffer supplemented with Halt protease cocktail inhibitor (Roche), and protein content was quantified by Bradford assay (Pierce). 50 µg of protein (each of two biological replicate) was applied to the magnetic anti-DYKDDDDK resin (Clontech), which was incubated with supernatant overnight at 4ºC with a tube rotisserie. After incubation, the resin was separated magnetically and washed six times with RIPA buffer. Sample was boiled and eluted with 1X SDS denaturing loading dye to elute immunoprecipitated protein and subjected to separation by SDS-PAGE followed by transfer to a PVDF membrane (semidry transfer at 10 volts for 1.5 hours). The membrane was washed with TBST followed by blocking in 5% milk (TBST, w/v). Post-blocking, the membranes were incubated with their respective primary antibodies overnight at 4°C rocking in the dark. The membranes were then washed with TBST, followed by incubation with a species-specific secondary antibody. After washing with TBST, the membrane was incubated with a chemiluminescent reagent (Pierce) and exposed to an X-ray film for imaging.

D2.
In vitro protein synthesis. MBP-DHCR24 (full-length) in a pET28b vector (Novagen) was expressed using the PURExpress in vitro synthesis kit (NEB E6800S) as previously described (Shimazu 2001). Briefly, the pET28_MBP-DHCR24 plasmid was prepared using a maxi-prep kit (Qiagen), eluted in ultra-pure water (Invitrogen), and subsequently used in the in vitro translation reaction. To the in vitro translation reaction mixture, RNAsin (Promega) and 450 ng of purified plasmid were added and incubated for 4 hours (37 °C, 250 RPM). The reaction mixture was concentrated using a 0.5 mL Ultra Centrifugal Filter MWCO of 50 kDa to eliminate transcription and translation proteins from the kit. The protein mixture was flash frozen in liquid nitrogen and stored at -80°C. E2. Immunoblot analysis to of NS3-4A cleavage assays. 100 µg of the reaction mixture (25 µL) was analyzed by SDS-PAGE using a 8-16% TGX Mini-protean gel (BioRad). The gel was transferred onto nitrocellulose membrane (BioRad Trans-blot Turbo). The membrane was washed in TBST and blocked with 5% milk (TBST, w/v). After incubation overnight with primary antibodies at 4 °C, membranes were washed with TBST and incubated with their respective species-specific secondary antibodies. After washing with TBST, blots were developed with enhanced chemiluminescence reagents (Thermo Fisher Scientific), and the chemiluminescence signal was processed using the Amersham Imager 600 (GE Healthcare).

F. Lipid analyses
F1. Lipid isolation. Cells were washed once with 1X PBS and harvested by trypsinization, addition of medium to quench trypsin, and centrifugation (1000 x g, 5 minutes, 4 °C). Cell pellets were washed twice with cold 1X PBS, resuspended in 1 mL of PBS and processed with a Dounce homogenizer using 2:1:1 CHCl 3 :CH 3 OH:PBS buffer. The organic and aqueous layers were separated by centrifugation (2000 x g, 5 minutes, 4°C). The organic layer was removed, concentrated under nitrogen gas (N 2 ), and then dissolved in 90 µL CHCI 3 prior to analysis by LC-MS or GC-MS. 30 µL of this sample was used for LC-MS. Absolute quantitation of sterol levels was accomplished by isotope dilution mass spectrometry and adding deuterated ketocholesterol-d7 (5 µL of 10 µM stock) as an internal quantitation standard. For GC-MS samples, samples were dried down with the internal standard (ketocholesterol-d7).

F3. Gas Chromatography-Mass Spectrometry. GC/MS analysis was performed on a Thermo
Scientific TRACE 1310 Gas Chromatograph equipped with a Thermo Scientific Q Exactive Orbitrap mass spectrometry system. 50 μL of the (BSTFA+10% TMCS)/pyridine (1/1 v/v) derivatized product was added into each vial, vortexed well, and heated at 70˚C for 30 min. One µL of the sample was injected into a Thermo fused-silica capillary column of cross-linked TG-5SILMS (30 m x 0.25 mm x 0.25 µm). The GC conditions were as follows: inlet and transfer line temperatures, 290˚C; oven temperature program, 50˚C for 0 min, 24˚C/min to 325˚C for 5.7 min; inlet helium carrier gas flow rate, 1 mL/min; split ratio, 5. The electron impact (EI)-MS conditions were as follows: ion source temperature, 310˚C; full scan m/z range, 30 -750 Da; resolution, 60,000; AGC target, 1e6; maximum IT, 200ms. Data were acquired and analyzed with Thermo TraceFinder 4.1 software package.

G. Mass Spectrometry Analysis of Protein Bands
Excised gel bands were cut into approximately 1 mm 3 pieces and submitted to the Taplin Mass Spectrometry Facility for subsequent processing and analysis. The samples were reduced with 1 mM DTT for 30 minutes at 60ºC and then alkylated with 5 mM iodoacetamide for 15 minutes in the dark at room temperature. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure (57). Gel pieces were washed and dehydrated with acetonitrile for 10 min, followed by removal of acetonitrile. Pieces were then completely dried in a Speed-Vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/µl modified sequencinggrade trypsin (Promega, Madison, WI) at 4ºC. Samples were then placed in a 37 ºC room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vac (~1 hr). The samples were then stored at 4 ºC until analysis.
On the day of analysis the samples were reconstituted in 5 -10 µL of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6 µm C18 spherical silica beads into a fused silica capillary (100 µm inner diameter x ~30 cm length) with a flame-drawn tip (58). After equilibrating the column each sample was loaded via a Famos auto sampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As each peptide was eluted, they were subjected to electrospray ionization followed by entry into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Eluting peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein or translated nucleotide databases with the acquired fragmentation pattern by the software program, Sequest (ThermoFinnigan, San Jose, CA) (59). The modification of 79.9663 mass units to serine, threonine, and tyrosine was included in the database searches to determine phosphopeptides. Phosphorylation assignments were determined by the Ascore algorithm (60). All databases include a reversed version of all the sequences and the data was filtered to between a 1-2% peptide false discovery rate.