**1. Introduction**

Hepatitis C virus (HCV) is an enveloped virus belonging to the Flaviviridae family [1]. It is estimated that around 71 million people are chronically infected, many of whom will develop cirrhosis, end-stage liver disease and hepatocellular carcinoma [2].

The HCV genome is around 9.6 kb positive-sensed single-stranded RNA containing a unique long open reading frame (ORF) that encodes a single polyprotein of approximately 3000 amino acids [3]. The 5 and 3 nontranslated regions (NTR), flanking the ORF, contain essential sequences for RNA stability, translation and replication [4–6]. A highly structured internal ribosomal entry site located in the 5 NTR drives the translation of the polyprotein that is co- and post-translationally processed by both host and viral proteases leading to the expression of three structural (core, E1 and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) [7].

HCV entry into hepatocytes starts with the interaction of viral and cellular factors, present in the viral membrane, with several receptors present in the plasma membrane of the hepatocytes (reviewed

in [8]). These interactions trigger the internalization of HCV via a receptor-mediated endocytosis process that is followed by the fusion of viral and cellular membranes and the release of the viral genome into the cytosol [9]. The incoming HCV RNA is then transported to intracellular membranes derived from the endoplasmic reticulum (ER) where primary translation and polyprotein processing occur. The viral replicase complex together with cellular factors is responsible for the RNA replication process, which starts by the generation of negative-sense RNA intermediates that are used as templates for the production of new positive-sense RNA molecules. Newly synthesized HCV genomes are then used as templates for translation, more rounds of RNA replication or packaging into progeny virus.

Lipid droplets (LD) have emerged as an important platform for HCV virus assembly [10], which is a highly coordinated process with several intermediate steps including: recruitment of NS5A and core proteins into LDs, encapsidation of HCV RNA into core particles and membrane envelopment of HCV RNA-containing core particles [11–15]. After budding into the ER lumen HCV exits the cell through the cellular secretory pathway. Given the tight association to the ER throughout di fferent steps in its life cycle, it is not surprising that several ER and ER-related proteins have been identified as host factors regulating the HCV infection e.g., signal peptide peptidase [16], Sigma-1 receptor [17].

The erlin proteins, erlin-1 and erlin-2, are endoplasmic reticulum membrane lipid raft-associated proteins that belong to a larger family of proteins containing a conserved stomatin, prohibitin, flotillin, HflK/C (SPFH) domain which is proposed to organize membrane microdomains [18–20]. Both erlins are closely related as they share ~80% identity at the amino acid level [20,21] and they are evolutionarily conserved with homologous proteins found in *Caenorhabditis elegans* and *Arabidopsis thaliana* [21]. Erlin proteins are located in detergent resistant membranes (DRM) where they form high molecular weight complexes containing erlin homo- and hetero-oligomers as well as other cellular proteins [22]. Early reports described the function of erlin-1 and erlin-2 proteins in the endoplasmic reticulum associated degradation (ERAD) of inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) [23,24]. Afterwards other reports suggested that erlin-2 protein is required for the sterol-induced degradation of cholesterol biosynthetic enzyme HMG-CoA reductase [25] and for the processing of amyloid β-peptide (Aβ) precursor (APP) into Aβ by γ-secretase in the brain [26]. Besides their function in the ERAD pathway erlin proteins have been shown to regulate cholesterol homeostasis. They are cholesterol-binding proteins that interact with the sterol regulatory element binding protein (SREBP)-Scap-Insig complex restricting SREBP activation and leading to an intracellular accumulation of lipids and cholesterol [27]. More recently, Inoue and Tsai reported the first link between erlin proteins and viral infections [28]. They showed that erlin 1 and erlin 2 proteins are both required for polyomavirus SV40 infection by facilitating B12 transmembrane J-protein mobilization to specific foci in the ER, a prerequisite for the ER to cytosol transport of SV40, thus enabling the establishment of infection [28].

In view of the cellular functions and ER localization of erlin proteins, and considering the dependence of HCV on lipid metabolism and the ER for its life cycle, we decided to investigate the potential role of erlin proteins in HCV infection. In this study we describe the discovery that erlin-1 protein regulates the initiation of HCV RNA replication, the accumulation of viral proteins and therefore, the production of infectious virus, adding erlin-1 to the list of host factors required for efficient HCV infection.

### **2. Materials and Methods**

### *2.1. Cells, Plasmids, Antibodies and Reagents*

The origin of Huh-7 [29], Huh-7.5.1 [29], Huh-7.5.1 subclone 2 (Huh-7.5.1c2) [15] and HEK-293T [30] cells have been described previously. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Cellgro; Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS) (Cellgro), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) in 5% CO2 at 37 ◦C. The sub-genomic and full-length JFH-1 stable replicon Huh-7 cell lines were cultured in medium supplemented with 400 or 200 μg/mL of G418, respectively, as described previously [15]. The JFH-1 genome-containing plasmid has been previously described [31]. The JFH-1 Rluc/SGR wt or JFH-1 Rluc/SGR GND plasmids carry bicistronic constructs containing the luciferase reporter gene in the first cistron and wild-type (wt) or replication-deficient (encoding a GDD-to-GND mutation in NS5B) JFH-1 sub-genomic replicon in the second cistron, respectively [32]. The rabbit polyclonal antibody for the detection of cellular erlin-2 protein was in-house generated and a ffinity purified against the full-length immunogen [27]. The rabbit polyclonal antibody HPA011252 against erlin-1 protein was obtained from Sigma-Aldrich (St. Louis, MO, USA). The recombinant human IgG anti-E2, the mouse monoclonal 9E10 anti-NS5A and the rabbit polyclonal MS5 anti-NS5A antibodies were kindly provided by M. Law (Scripps Research, La Jolla, CA, USA), C. M. Rice (Rockefeller University, New York, NY, USA) and M. Houghton (University of Alberta, Edmonton, AB, Canada), respectively. The monoclonal mouse antibodies against EEA1, HCV core (clone C7-50) and NS3 (clone 2E3) proteins were obtained from BD Transduction Laboratories (Franklin Lakes, NJ), Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BioFront Technologies (Tallahassee, FL, USA), respectively. The HCV RNA replication inhibitor 2-C-methyladenosine (2 mAd) was used at 10 μM final concentration and was a gift from W. Zhong (Gilead Sciences, Foster City, CA, USA). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich. Protease and phosphatase inhibitors were purchased from Roche (Indianapolis, IN, USA).

### *2.2. Silencing of Erlin Proteins by siRNA Transfection*

siRNAs targeting human *ERLIN1* (siErlin 1.5: CCACAAATAGGAGCAGCAT [27]) or *ERLIN2* (siErlin 2.3: GCCTCTCCGGTACTAACAT [27]) individually, or *ERLIN1* and *ERLIN2* simultaneously (siErlin 1&2: AGAAGCAATGGCCTGGTAC [27]), and the non-targeting control siRNA (siCtrol: ACTGTCACAAGTACCTACA [24]), as well as the siRNA targeting HCV genome (siHCV: ACCTCAAAGAAAAACCAAA [17]) were all previously described. All the siRNAs were purchased from Integrated DNA Technologies (IDT, San Diego, CA, USA). Typically, Huh-7 cells were plated at a density of 1 × 10<sup>5</sup> cells per well on a 6-well plate. 24 h later cells were transfected with 20 pmol of the corresponding siRNA per well using Dharmafect 4 transfection reagen<sup>t</sup> following manufacturer's instructions (GE Healthcare Dharmacon Inc, Pittsburgh, PA, USA). In brief, 20 pmol of siRNA was added into 200 μL of OptiMEM containing tube and 1 μL of Dharmafect 4 reagen<sup>t</sup> was added into a second tube containing another 200 μL of OptiMEM. These mixtures were incubated for 5 min at room temperature before they were mixed together and incubated for another 20 min at room temperature. During the incubation time the culture medium of the Huh-7 cells was discarded and 1.6 mL of complete DMEM medium was added to each well. After 20 min of incubation the siRNA-Dharmafect complexes-containing medium (400 μL) was added to the corresponding wells. Fresh medium was applied after overnight incubation and the cells were assayed at the indicated times after transfection as indicated in each experiment. Cell viability was determined by evaluating cell biomass at di fferent times after siRNA transfection by crystal violet staining and colorimetry at 570 nm, as previously described [33]. As a complementary approach, the cytotoxic e ffect of siRNAs was evaluated by quantifying the mitochondrial activity of siRNA-transfected cells (in MTT assays) and by determining the cellular respiration capacity. The cellular respiration capacity was calculated as the mitochondrial activity relative to the total protein content of the cells, measured by BCA in wells that were transfected in parallel.

### *2.3. Preparation of Viral Stocks and Infections*

The original JFH-1 virus was generated by transfection of an in vitro transcribed full-length JFH-1 HCV RNA into Huh-7 cells and viral stocks were produced by inoculation of fresh Huh-7 cells at a multiplicity of infection (moi) of 0.01 as described [29]. Production of high titer cell culture adapted JFH-1 day 183 virus (D183) [34] was achieved by inoculation of highly susceptible Huh-7.5.1c2 at low moi. These D183 virus stocks were used in all low and high moi infection experiments (moi = 0.2 or 3) as described previously [15]. Intracellular infectious HCV particles were obtained by 5 freeze-thaw cycles of infected cells as described [35]. The infectivity titers present in culture supernatants and cell extracts were determined by end-point dilution and fluorescence focus forming unit (FFU) assay in Huh-7.5.1 cells, as described previously [36].

### *2.4. Analysis of HCV Cell Entry Using HCVpp*

Retroviral particles pseudotyped with the JFH-1 E1 and E2 envelope proteins or the VSV G glycoprotein, as control, were produced in HEK293T cells [37]. For HCV entry experiments, Huh-7 cells (2 × 10<sup>4</sup> cells) were plated on 48-well plates and twenty hours later they were transfected with the indicated siRNAs as described above. Thirty-six hours later, cells were inoculated with pseudotyped retroviral particles (HCVpp or VSVpp) diluted to produce similar luciferase activity levels. Forty-eight hours later, cells were lysed and luciferase activity was detected and quantitated in a luminometer using a commercial kit (Luciferase Assay Kit; Promega, Madison, WI, USA). Luciferase results were normalized to cell density and relative infection values were calculated as a percentage of cells transfected with control siRNA. In parallel, cultures transfected with siRNAs were used: (i) to analyze knockdown e fficiency by Western-blotting (WB) and (ii) to confirm the reduced susceptibility of erlin-1-deficient cells to HCV infection.

### *2.5. In Vitro Transcription and HCV RNA Transfection*

The subgenomic JFH-1 replicon plasmid bearing a luciferase reporter gene (JFH-1 Rluc/SGR wt) and the corresponding replication-deficient construct (JFH-1 Rluc/SGR GND) have been described previously [32]. After digestion with XbaI restriction enzyme the linearized plasmids were in vitro transcribed using the T7 MEGAscript (Ambion, Austin, TX, USA) kit following the manufacturer's instructions. siRNAs targeting the indicated genes were transfected into Huh-7 cells as described above. Twenty hours later the cells were trypsinized and cells were seeded in wells of 12-well plate at a density of 1 × 10<sup>5</sup> cells per well. The following day *in vitro* transcribed subgenomic HCV RNAs were introduced into those cells by transfection using TransIT mRNA Transfection Kit (Mirus Bio LLC, Madison, WI, USA) as recommended by the manufacturer. At the indicated time points the cells were lysed. An aliquot of each lysate was used to quantitate the luciferase activity in a luminometer using a commercial kit (Promega). Another aliquot from each lysate obtained at six hours post-transfection was used to quantitate the HCV RNA levels by reverse transcription real-time quantitative PCR (RT-qPCR) as described below. The HCV RNA values were used to control for di fferences in the transfection efficiency between samples and were used to normalize the luciferase values. Luciferase results were normalized to cell density at each time point and the relative values were calculated as a percentage of cells transfected with control siRNA. In parallel, cultures transfected with siRNAs were used: (i) to analyze knockdown e fficiency by WB and (ii) to confirm the reduced susceptibility of erlin-1-deficient cells to HCV infection.

### *2.6. siRNA Transfection Experiments in Acutely HCV Infected Cells*

Huh-7 cells were transfected with the indicated siRNAs as described above. Thirty-six hours later, transfected cells were infected with JFH-1 D183 virus at low or high moi (0.2 or 3, respectively) as described before [15]. Five hours after HCV inoculation, cells were washed twice with PBS and medium was replaced. At the indicated times after infection supernatants were collected and extracellular infectivity was determined by end-point dilution in Huh-7.5.1 cell line. Cell extracts were harvested as indicated above and the intracellular infectivity, HCV RNA, and viral proteins were analyzed by titration, RT-qPCR and WB, respectively, as described [38]. Erlin protein down-regulation e fficiency was confirmed by WB and densitometry analysis at the time post-infection indicated in each figure legend.

### *2.7. siRNA Transfection Experiments in Persistently Infected Cells*

Huh-7 cells were infected with JFH-1 virus at a low moi = 0.01 as described [29]. Infected cells were passaged for 3 weeks before siRNAs were transfected. Three days after siRNA transfection supernatants were discarded, cells were washed twice with PBS and medium was replenished. Twenty-four hours later supernatants were collected and progeny virus released during those last twenty-four hours was determined by end-point dilution in Huh-7.5.1 cells as described above. Cell extracts were harvested and the intracellular infectivity, HCV RNA and proteins and erlin protein down-regulation were analyzed by titration, RT-qPCR and WB, respectively.

### *2.8. Protein Analysis by WB*

Cell extracts were prepared in RIPA bu ffer supplemented with protease and phosphatase inhibitors and, after protein quantitation by bicinchorinic acid (BCA) assay (Thermo Scientific, Waltham, MA, USA), equivalent amounts of total protein (typically 20–25 μg) were separated in polyacrylamide-SDS gels by electrophoresis and they were transferred to Immobilon membranes (Millipore, Billerica, MA, USA) for blotting. The membranes were blocked for 1 h at room temperature with PBS-5% milk and incubated overnight at 4 ◦C with the primary antibodies diluted in 1% milk-0.1% Tween 20 in PBS. Primary antibodies were used at the following dilutions: EEA1 at 1:2000; erlin-1, erlin-2 and NS3 at 1:1000; NS5A at 1:500; and Core at 1:200 dilution. After washing at least 4 times for 15 min with 0.1% Tween 20 in PBS, membranes were incubated with a 1:10,000 dilution of goat-anti-rabbit or goat-anti-mouse IgG conjugated to horseradish peroxidase prepared in 1% milk-0.1% Tween 20 in PBS. After washing at least 4 more times with 0.1% Tween 20 in PBS, membranes were developed using the SuperSignal-West Pico or Femto chemiluminescence reagents (Thermo Scientific) and autoradiography. Densitometry of non-saturated films was performed using Fiji/Image J analysis software [39] and the results, shown beneath each panel, are expressed relative to control cells using EEA1 protein expression for normalization and as loading control. Two-fold serial dilutions of siCtrol samples were used to produce standards for relative quantitation.
