**3. Results**

### *3.1. Erlin-1 Protein Is a Host Factor Required for E*ffi*cient HCV Infection*

To investigate the potential role of erlin-1 and erlin-2 proteins in the HCV lifecycle we took advantage of siRNAs that have being previously validated and used to characterize the cellular functions of erlin proteins [24,27]. We transfected Huh-7 cells with siRNAs targeting specifically erlin-1 (i.e., siErlin 1.5) or erlin-2 (i.e., siErlin 2.3) individually, or siRNAs targeting both erlin proteins simultaneously (siErlin 1&2), siRNAs targeting HCV as a positive control (siHCV) and a non-targeting siRNA as a negative control (siCtrol). siRNA transfected cells were inoculated at low multiplicity of infection (moi = 0.2) with a cell culture-adapted HCV virus (D183v) [34] and their susceptibility to infection was assessed by measuring virus production three and five days later. Analysis of the supernatants of erlin-1 down-regulated HCV-infected cells showed ~ 7–10 fold reduction of progeny virus production compared to non-targeting control siRNA-transfected cells that were infected in parallel (Figure 1A).

Remarkably, erlin-2 protein down-regulation did not reduce HCV virus production. As expected the HCV-targeting siRNA prevented the production of infectious HCV virus. Consistent with the effect on virus production, the accumulation of intracellular HCV proteins (e.g., NS3 and NS5A) was also reduced in erlin-1, but not in erlin-2, down-regulated cells (Figure 1B). Despite a small defect in the cellular proliferation rate observed in siErlin 1.5-transfected cells (Figure 1C,D), the cellular respiration capacity of the different siRNA-transfected cells was comparable to that of control cells (Figure 1E). Collectively, these results sugges<sup>t</sup> that the effects observed in erlin-1 down-regulated cells (in siErlin 1.5 and siErlin 1&2) are not due to a reduction in cell viability and most probably reflect a specific requirement of erlin-1 protein for efficient HCV infection.

**Figure 1.** Erlin-1 protein down-regulation impairs efficient HCV infection. Huh-7 cells were transfected with individual siRNAs targeting erlin-1 (siErlin 1.5), erlin-2 (siErlin 2.3), both erlins simultaneously (siErlin 1&2), HCV (siHCV) or a non-targeting siRNA control (siCtrol) as indicated in Material and Methods section. Transfected cells were infected 36 h later with JFH-1 D183 virus at low multiplicity of infection (moi = 0.2). (**A**) The extracellular infectivity present in supernatants of infected cells was determined on days three and five post-inoculation by titration assay. Infectivity titers are represented as the FFUs per ml of supernatant in logarithmic scale and are displayed as the average and standard deviation (Mean; SD; n = 3). The horizontal black dotty line represents the limit of detection (LoD) of the assay. (**B**) Three days after virus inoculation cellular erlin-1, erlin-2 and EEA1 (as loading control) and viral NS3 and NS5A protein expression was determined in HCV-infected cell extracts by WB. Relative protein levels were determined by densitometry and are shown below each panel. The ratio of intensities of each protein and EEA1 in siCtrol-transfected cells was set as 100 and it was used to calculate the relative amount of each protein in each sample. Results in panels A and B are representative of two independent experiments, each one performed in triplicate. (**C**) Cells plated in 96-well plate format were transfected with siRNAs and the cell biomass was quantitated by crystal violet staining at different times post-transfection. Data are displayed as the average and standard deviation of two independent experiments with three replica wells per condition at each time point in

each experiment (Mean; SD; n = 6). ( **D**) Cells plated in 96-well plate format were transfected and infected as described above and were used to perform MTT assays three days after HCV inoculation. Data are displayed as the average and standard deviation (Mean; SD; n = 7) relative to the levels in siCtrol condition, set as 100. Results are representative of two independent experiments with seven replica wells per condition in each experiment. (**E**) The cellular respiration capacity was calculated as described in Material and Methods section. Data are displayed as the average and standard deviation (Mean; SD; n = 3) relative to that of siCtrol, set as 1. Statistical significance was determined using: Two-way ANOVA followed by a Bonferroni posttest for panels A and C (\*\* *p* < 0.01; \*\*\* *p* < 0.001), and One-way ANOVA followed by the Dunnett's Multiple Comparison Test for panels D and E (\* *p* < 0.05).

### *3.2. Erlin-1 Protein Is Not Required for HCV Cell-Entry*

The multiple cycle infection experiments described above suggested that erlin-1 protein plays an important role in the HCV lifecycle. Next, we set out to determine the step in the virus life cycle in which erlin-1 protein was required. Many aspects of HCV cell entry have been studied using HCV-pseudotyped retroviral vectors. Therefore, we used retroviral particles pseudotyped with the JFH-1 envelope glycoproteins E1 and E2 (HCVpp) or the vesicular stomatitis virus protein G (VSVpp), as control, to determine their ability to infect erlin-1 down-regulated cells. siRNA-transfected Huh-7 cells were infected with the corresponding retroviral particles and the intracellular luciferase activity was measured forty-height hours later. Down-regulation e fficiency was verified in parallel cultures by WB (Figure 2A). As shown in Figure 2B, transfection of *ERLIN1*-targeting siRNAs had no e ffect on viral entry e fficiency despite strong reduction in erlin-1 protein expression achieved in those cells. As expected *ERLIN2*- and HCV-specific siRNAs had no e ffect on the HCVpp entry process. These results sugges<sup>t</sup> that reduced erlin-1 protein expression does not a ffect HCV cell entry.

**Figure 2.** Erlin-1 protein down-regulation does not inhibit HCV cell-entry. Huh-7 cells were transfected with individual siRNAs as described in Material and Methods. ( **A**) 36 h later one set of wells was harvested for erlin protein analysis by WB. Relative protein expression levels were determined by densitometry as described in the legend of Figure 1 and they are shown below each panel. (**B**) Another set of wells was inoculated with HCVpp (grey bars) or particles harboring the VSV-G glycoprotein (VSVpp; white bars) as control. 48 h after inoculation the intracellular luciferase activity was measured. Results are displayed as percentage of the luciferase activity present in siCtrol-transfected cell extracts for each virus particle. Data are displayed as the average and standard deviation (Mean; SD; n = 3). The luciferase values (units per well) in siCtrol-transfected cell extracts were 9 × 10<sup>3</sup> ± 5.1 × 10<sup>2</sup> and 1.2 × 10<sup>4</sup> ± 2 × 10<sup>2</sup> for HCVpp and VSVpp, respectively. These results are representative of two independent experiments, each one performed in triplicate. The average erlin-1 protein expression level of the two experiments is shown in black bars. One-way ANOVA followed by Dunnett's Multiple Comparison Test analysis of the HCVpp data did not show any statistically significant e ffect on the luciferase levels among di fferent siRNAs. Instead, the entry of VSVpp was significantly higher in siHCV- and siErlin 2.3-transfected cells (\* *p* < 0.05; \*\* *p* < 0.01).

### *3.3. Erlin-1 Protein Down-Regulation Impairs the Establishment of HCV RNA Replication but Does Not A*ff*ect Primary Translation or Maintenance Of Replication*

To test whether erlin-1 protein regulates early steps downstream HCV entry such as the primary translation of the incoming RNA or the establishment of RNA replication, we used a highly sensitive approach based on the transfection of an in vitro transcribed JFH-1 subgenomic RNA bearing a luciferase reporter gene (JFH-1 Rluc/SGR RNA). In this system the luciferase activity measured at early time points after transfection (i.e., at six hours post-transfection) derives exclusively from the translation of transfected RNA, while the accumulation of luciferase at later time points (e.g., at 48, 72 or 96 h post-transfection) is the result of both translation and RNA replication processes. Consistent with this, luciferase activity derived from JFH-1 Rluc/SGR wt RNA was equivalent in the presence (white bars) or absence (black bars) of the HCV polymerase inhibitor 2-C-methyladenosine (2 mAd) six hours after transfection (Figure 3A). However, it was clearly reduced in 2 mAd-treated cells at 96 h post-transfection.

**Figure 3.** Erlin-1 protein down-regulation impairs the initiation of HCV RNA replication without affecting HCV IRES-dependent translation. Huh-7 cells were transfected with individual siRNAs as described in Material and Methods. 48 h later cells were transfected with in vitro transcribed bicistronic renilla luciferase (Rluc)-containing wt or replication-deficient (GND mutant) sub-genomic (SGR) HCV RNAs. As control for the inhibition of HCV RNA replication process, transfected cells were treated with the HCV polymerase inhibitor 2-C-methyladenosine (2 mAd). (**A**) Quantitation of renilla luciferase activity derived from Rluc/SGR wt RNA in the presence (white bars) or absence (black bars) of 2 mAd at early (6 h) and late (96 h) time points. Data are displayed as the average and standard deviation (Mean; SD; n = 3) of luciferase values relative to that of control cells at 96 h post-transfection (hpt), that was set as 100. (**B**) Time course of renilla luciferase activity derived from RLuc/SGR wt RNA in siRNA-transfected cells. For each independent experiment, Rluc activity was normalized to cell density and was displayed as a percentage of that determined in siCtrol-transfected cell extracts at 96 hpt. Data shown are averages and standard deviation of two independent experiments, each one performed in

triplicate (Mean; SD; n = 6). The luciferase activity in siCtrol-transfected cell extracts at 96 h post-transfection was 1.3 × 10<sup>7</sup> ± 9.1 × 10<sup>5</sup> light units per well. To assess the specificity of Rluc activity, cells were treated with 2 mAd (red line) or were transfected with siRNAs targeting directly HCV RNA (orange line). Statistical analysis performed using Two-way ANOVA followed by Bonferroni posttest showed statistically significant di fferences in: 2 mAd-treated, siHCV-, siErlin 1.5- and siErlin 1&2-transfected cells at 48-, 72- and 96-hpt, and siErlin 2.3-transfected cells at 72- and 96-hpt compared to siCtrol-transfected cells (\*\* *p* < 0.01; \*\*\* *p* < 0.001). ( **C**) Erlin protein down-regulation was determined at the time of Rluc/SGR RNA transfection by WB and densitometry (shown below each panel) as described in the legend of Figure 1. ( **D**) Luciferase activity of replication-deficient (GND mutant) Rluc/SGR RNA was determined at six hours post-transfection. For each independent experiment Rluc activity was normalized to cell density. Data shown are averages and standard deviation of two independent experiments, each one performed in triplicate (Mean; SD; n = 6). Statistical analysis performed using One-way ANOVA followed by Dunnett's Multiple Comparison Test showed statistically significant differences in siErlin 2.3- and siHCV-transfected cells compared to siCtrol-transfected cells (\*\*\* *p* < 0.001). No di fferences were observed in erlin-1 downregulated cells compared to siCtrol-transfected cells.

Luciferase activity measured at early time points (6 and 24 h post-transfection) was similar in all siRNA-transfected cell lines, suggesting that primary translation is not a ffected by the levels of erlin-1 or erlin-2 protein expression (Figure 3B). Interestingly, the luciferase activity in erlin-1 down-regulated cells (siErlin 1.5- and 1&2-transfected cells) was around 50% lower than that of control cells at 48 and 72 h post-transfection. Moreover, the luciferase activity measured at 96 h time point showed greater di fferences in siErlin 1&2-transfected cells (30% of siCtrol) than in siErlin 1.5-transfected cells (60% of siCtrol), correlating with erlin-1 down-regulation e fficiencies achieved by the corresponding siRNAs (Figure 3C). These results sugges<sup>t</sup> that erlin-1 protein regulates the establishment of HCV RNA replication without a ffecting the primary translation. Supporting this notion, luciferase activity measured six hours after the transfection of a replication deficient JFH-1 RLuc/SGR GND mutant RNA was comparable in erlin-1 down-regulated cells and control cells (Figure 3D). Altogether, these data sugges<sup>t</sup> that erlin-1 protein regulates the initiation of RNA replication without a ffecting the translation of incoming HCV RNA.

Next, we analyzed the e ffect of erlin-1 protein down-regulation on the maintenance of HCV RNA replication in the stably replicating JFH-1 subgenomic replicon cells (SGR cells). SGR cells were transfected with the indicated siRNAs and three days later the intracellular erlin protein levels and HCV protein and RNA levels were analyzed by WB and RT-qPCR, respectively. As expected, transfection of the HCV-targeting siRNA produced a significant reduction of HCV proteins (Figure 4A) and RNA (Figure 4B). However, no e ffect was observed when siRNAs targeting erlin-1 protein were transfected despite the 50–70% reduction in erlin-1 protein expression achieved. Similar results were obtained when JFH-1 full-length replicon cells were used (data not shown). These results sugges<sup>t</sup> that erlin-1 protein does not regulate the ongoing HCV RNA replication process.

**Figure 4.** Erlin-1 protein down-regulation does not interfere with ongoing HCV RNA replication. JFH-1 sub-genomic replicon (SGR) bearing Huh-7 cells were transfected with siRNAs as described in Material and Methods. Three days after transfection cells were harvested for analysis. (**A**) Cellular erlin-1, erlin-2 and EEA1, and HCV NS3 and NS5A protein accumulation were determined by WB and densitometry (shown below each panel) as described in the legend of Figure 1. (**B**) The intracellular HCV RNA content was quantitated in infected cell extracts three days after siRNA transfection by RT-qPCR. Data were normalized relative to *GAPDH* mRNA levels in the same samples and are displayed as percentage of the HCV RNA present in siCtrol-transfected cells. The HCV RNA content in siCtrol-transfected cell extracts was 1.4 × 10<sup>6</sup> ± 1.2 × 10<sup>5</sup> copies per μg of total RNA. Data shown are averages of three independent experiments, each one performed in triplicate (Mean; SD; n = 9). Only the HCV RNA level in siHCV-transfected cells was significantly lower than that in siCtrol-transfected cells as determined by One-way ANOVA followed by Dunnett's Multiple Comparison Test (\*\*\* *p* < 0.001).

### *3.4. Erlin-1 Protein Down-Regulation Interferes with HCV Protein and Intracellular Infectious Virus Accumulation*

Collectively the results from the experiments described above suggested that reduction of erlin-1 protein expression impairs HCV infection by inhibiting the establishment of RNA replication (Figure 3) without affecting HCV entry (Figure 2), HCV IRES dependent primary translation (Figure 3) or ongoing HCV RNA replication (Figure 4). Consistent with those results, quantitation of the intracellular HCV RNA levels 48 h after a high moi (moi = 3) infection showed a modest but statistically significant two to three-fold decrease in erlin-1 down-regulated cells compared to control cells (Figure 5A). Interestingly, the accumulation of intracellular (Figure 5B) and extracellular (Figure 5C) infectious virus was strongly reduced (around ten-fold) in those same cells. These results were confirmed with a different *ERLIN1*-targeting siRNA (siErlin 1.3 in Supplementary Figure S1). Similarly, the accumulation of HCV core, NS3 and NS5A proteins was reduced by four to ten-fold in erlin-1 down-regulated cells (Figure 5D). The disproportionate effect on the infectivity levels compared to the effect on the intracellular HCV RNA level suggested that erlin-1 protein is required not only for the establishment of RNA replication as shown in Figure 3, but also for a post-replication step in the virus lifecycle. In fact, the assembly rate of infectious virus particles (Figure 5E) but not the secretion rate (Figure 5F) was reduced in erlin-1 down-regulated cells compared the control cells. Supporting the latest, siRNAs that target directly the HCV genome (i.e., siHCV) inhibited HCV RNA accumulation and downstream HCV protein accumulation as well as intra- and extra-cellular infectious virus accumulation to the same extent (around 15–20 fold).

**Figure 5.** Erlin-1 protein down-regulation interferes with HCV in single-cycle infection experiments. Huh-7 cells were transfected with siRNAs as described in Material and Methods. 36 h later transfected cells were inoculated with JFH-1 D183 virus at high multiplicity of infection (moi = 3). 48 h after virus inoculation cellular extracts were prepared for intracellular RNA (**A**), infectivity (**B**) and protein (**D**) analysis and supernatants were collected for extracellular infectivity (**C**) analysis. Assembly (**E**) and secretion (**F**) rates were calculated using data from panels A, B and C. The RNA and infectivity results are displayed as percentage of the levels in siCtrol-transfected cells. The HCV RNA and the intracellular and extracellular infectivity levels in siCtrol-transfected cells were: 1.2 × 10<sup>7</sup> ± 1.3 × 10<sup>6</sup> HCV RNA copies per μg of total RNA, 3.5 × 10<sup>4</sup> ± 6.1 × 10<sup>3</sup> ffus per well, and 1.3 × 10<sup>5</sup> ± 2 × 10<sup>4</sup> ffus per ml of supernatant, respectively. Data shown are averages of three independent experiments, each one performed in triplicate (Mean; SD; n = 9). One-way ANOVA followed by Dunnett's Multiple Comparison Test was used to determine the statistical significance (\*\*\* *p* < 0.001).

Moreover, dose-dependent and parallel reductions in HCV parameters (i.e., intracellular HCV RNA, protein and infectious virus, and extracellular infectious virus) were observed when cells were infected in the presence of different doses of the HCV polymerase inhibitor 2 mAd (Figure 6). These results imply that the disproportionate effect in virus infectivity levels observed in erlin-1 down-regulated cells could not be explained solely by the modest reduction in intracellular HCV RNA accumulation suggesting an independent requirement for erlin-1 protein at a post RNA replication step.

**Figure 6.** Dose-dependent reduction on HCV parameters upon treatment with a replication inhibitor. Huh-7 cells were inoculated with JFH-1 D183 virus at high multiplicity of infection (moi = 3) in the absence or presence of increasing amounts (0, 0.1, 0.33 and 1 μM) of the HCV polymerase inhibitor 2 mAd. 48 h later supernatants were collected, cell extracts were harvested and samples were subjected to the same analysis described in Figure 5. Note the proportional and dose response reduction of all viral parameters (RNA (**A**), infectivities (**B**,**C**) and protein (**D**)) in 2 mAd-treated cells and the absence of any significant effect in the assembly (**E**) and secretion (**F**) rates. The HCV RNA and the intracellular and extracellular infectivity levels in untreated cells were: 2 × 10<sup>7</sup> ± 2 × 10<sup>6</sup> HCV RNA copies per μg of total RNA, 2 × 10<sup>4</sup> ± 2.5 × 10<sup>3</sup> ffus per well, and 1.6 × 10<sup>5</sup> ± 2.5 × 10<sup>4</sup> ffus per ml of supernatant, respectively. Data shown are averages of two independent experiments, each one performed in duplicate (Mean; SD; n = 4). One-way ANOVA followed by Dunnett´s Multiple Comparison Test analysis was used for statistical significance analysis (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001).

To prove unequivocally that erlin-1 protein is a rate limiting factor for a later step in the HCV virus life cycle we took advantage of the possibility of manipulating erlin-1 protein expression after the HCV infection is fully established. This approach allowed us to avoid the effect of erlin-1 protein down-regulation in the establishment of RNA replication described above. siRNAs were transfected into persistently HCV-infected cells and four days later the intracellular HCV RNA (Figure 7A) and the intracellular (Figure 7B) and extracellular (Figure 7C) infectious virus accumulation were quantitated by RT-qPCR and titration assays, respectively. While siRNAs targeting HCV genome significantly suppressed HCV RNA and infectious virus accumulation, *ERLIN1*-targeting siRNAs reduced infectious virus production by 70–80% with a modest 10–20% reduction on HCV RNA levels. This was reflected in a lower assembly rate (Figure 7E), but not in the secretion rate (Figure 7F) in erlin-1 down-regulated cells. These results were confirmed with a different *ERLIN1*-targeting siRNA (siErlin 1.3 in Supplementary Figure S2). HCV protein analysis showed stronger defect in NS3 than in NS5A and core protein accumulation in erlin-1 down-regulated cells (Figure 7D). These results strongly sugges<sup>t</sup> that erlin-1 protein regulates later event(s) that lead to infectious virus production.

**Figure 7.** Erlin-1 protein down-regulation impairs infectious virus production in an ongoing HCV infection cell culture system. Persistently infected Huh-7 cells were transfected with siRNAs as described in Material and Methods. Four days after siRNA transfection cellular extracts were prepared for intracellular RNA (**A**), infectivity (**B**) and protein (**D**) analysis and supernatants were collected for extracellular infectivity (**C**) analysis. Assembly (**E**) and secretion (**F**) rates were calculated using data from panels A, B and C. The RNA and infectivity results are displayed as percentage of the levels in siCtrol-transfected cells. The HCV RNA and the intracellular and extracellular infectivity levels in siCtrol-transfected cells were: 5.8 × 10<sup>5</sup> ± 7 × 10<sup>4</sup> HCV RNA copies per μg of total RNA, 2.3 × 10<sup>3</sup> ± 2.8 × 10<sup>2</sup> ffus per well, and 1 × 10<sup>4</sup> ± 1.1 × 10<sup>3</sup> ffus per mL of supernatant, respectively. Data shown are averages of three independent experiments, each one performed in triplicate (Mean; SD; n = 9). One-way ANOVA followed by Dunnett's Multiple Comparison Test was used to determine the statistical significance (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001).

### *3.5. Erlin-1 Protein Down-Regulation Increases LD Accumulation in Huh-7 Cells*

We have previously described that down-regulation of erlin proteins lead to an intracellular fatty acids and cholesterol increase in Hela cells [27]. Furthermore, it is well known that HCV requires LDs for assembly of infectious virus and that HCV induces LD accumulation in infected cells [10]. Therefore, we analyzed the effect of erlin protein down-regulation on LD accumulation in HCV-infected Huh-7 cells. To do so, erlin down-regulated and control cells were infected with JFH-1 D183 virus at high moi (moi = 3) and fixed forty-eight hours later for analysis. Fixed cells were incubated with LipidTox, a reagen<sup>t</sup> that stains specifically neutral lipids, and they were analyzed by confocal microscopy. As shown in Figure 8A, reduction in erlin-1 and erlin-2 protein expression produced an increase in LD accumulation compared to control cells. Quantitation of fluorescence intensity in individual cells confirmed those results and revealed a statistically significant increase in LD accumulation in cells where *ERLIN1* and *ERLIN2* were simultaneously down-regulated (i.e., siErlin 1&2) compared to that of control cells (Figure 8B). These results sugges<sup>t</sup> that erlin protein down-regulation increases the intracellular accumulation of LDs not only in Hela cells, as previously described [27], but also in Huh-7 cells.

**Figure 8.** Erlin-1 protein down-regulation increases LD content in Huh-7 cells. siRNA-transfected Huh-7 cells were inoculated with JFH-1 D183 virus at high multiplicity of infection (moi=3) and the intracellular LD content was analyzed 48 h after infection as described in Material and Methods. (**A**) Representative confocal image sections of intracellular LD accumulation (in green) in siRNA-transfected HCV-infected and uninfected cells. Nuclei (in blue) were counterstained with Hoechst dye. (**B**) Quantitation of the total LD fluorescence intensity signal per cell from confocal image sections. ImageJ software was used to quantitate the total LD fluorescence intensity signal in twenty individual cells of each condition. Each dot in the graph represents the LD fluorescence intensity of an individual cell and the horizontal lines show the average LD fluorescence intensity in each group of data. One-way ANOVA followed by Dunnett's Multiple Comparison Test was used to determine the statistical significance (\*\*\* *p* < 0.001).

### *3.6. Erlin-1 Protein Deficiency Does Not Impair HCV Core and NS5A Protein Re-Localization to LDs*

Late in the infection both core and NS5A proteins are known to be associated to LDs to facilitate virus assembly [10]. Thus, we analyzed the distribution of core and NS5A proteins in *ERLIN* downregulated cells by confocal microscopy. As shown in Figure 9A, core and NS5A proteins localized to LDs similarly in both control and *ERLIN* down-regulated cells. A quantitative analysis of the localization of core and NS5A proteins surrounding LDs (Figure 9B) confirmed that the reduced infectious virus accumulation observed in *ERLIN1*-deficient cells was not due to a mislocalization of core or NS5A proteins during infection.

**Figure 9.** Erlin-1 protein down-regulation does not impair the localization of HCV core and NS5A proteins surrounding LDs. siRNA-transfected Huh-7 cells were inoculated with JFH-1 D183 virus at high multiplicity of infection (moi = 3) and the intracellular HCV protein and LD content was analyzed as described in Material and Methods. (**A**) Representative confocal images of the intracellular localization of HCV core (in red) and NS5A (in orange) proteins surrounding LDs (in green) in *ERLIN* down-regulated (siErlin 1&2) and control (siCtrol) HCV-infected cells. Nuclei (in blue) were counterstained with Hoechst dye. The panels on the right side show merged images of the four channels of each image. White boxes in the upper right side of each panel show zoom-in images of single cells for a more detailed observation. (**B**) The fluorescence intensity signal of core and NS5A proteins associated to LDs was quantitated in twenty randomly selected HCV-infected cells of each condition. Each dot in the graph represents the percentage of the fluorescence intensity signal of core or NS5A protein associated to LDs in a given cell, while the horizontal lines show the average of all data points in each group. One-way ANOVA followed by Dunnett's Multiple Comparison Test was used to determine the statistical significance (\* *p* < 0.05; \*\*\* *p* < 0.001).
