**4. Discussion**

The development of an in vitro HCV infection system in 2005 [29,40,41] allowed the identification of viral proteins and cellular factors and pathways that regulate different aspects of the HCV infection (reviewed in [8,42,43]). Using this infection model, we and others have previously identified cellular factors required for initiation of RNA replication [17,32] and HCV assembly and secretion [15,38], and cellular pathways required for the induction [44,45] and evasion [36,46] of the innate immune system. In this study, we have analyzed the consequences of erlin-1 and erlin-2 protein down-regulation on the susceptibility of Huh-7 cells to HCV infection. Our data sugges<sup>t</sup> a role for erlin-1 protein early and late in the HCV life cycle.

Silencing of erlin-1 but not erlin-2 protein expression by siRNAs led to a strong reduction in the production of infectious progeny virus (Figure 1) suggesting that erlin-1 protein is required for efficient HCV infection. Single cycle infection experiments in *ERLIN1* down-regulated cells showed a modest 2–3 fold reduction in intracellular HCV RNA accumulation compared to a ~4–10 fold reduction in HCV protein accumulation and ~10-fold reduction in intracellular infectious virus accumulation (Figure 5 and Supplementary Figure S1). Further studies performed in HCV surrogate models that interrogate specific steps in the virus life cycle revealed that erlin-1 protein is required for the initiation of HCV RNA replication (Figure 3) without a ffecting earlier steps such as HCV cell entry (Figure 2) and HCV IRES dependent translation of the incoming RNA (Figure 3). Interestingly, *ERLIN1* silencing in subgenomic replicon cells (Figure 4) or in persistently infected cells (Figure 7) had no e ffect on the steady-state level of HCV RNA but revealed di fferences in the steady-state levels of HCV proteins. Collectively, these results suggested that erlin-1 protein plays a role in the initiation of RNA replication but it seems to be dispensable for the maintenance of RNA replication once it has been established. The formation of the first viral replication complexes and the initiation of RNA replication are thought to take place in the presence of very low levels of viral proteins. Therefore, it is conceivable that the initiation step is more likely to be dependent on host factors than later steps when the accumulation of viral factors required for RNA replication is higher and the virus has induced a more favorable environment for replication in the cell by inducing the membranous web [47]. Our results are consistent with this notion and add erlin-1 protein to the list of factors previously reported to show a di fferential requirement for the initiation and maintenance of the RNA replication processes [17,38,48]. Alternatively, these results might sugges<sup>t</sup> the existence of a di fferent threshold for the requirement of erlin-1 protein in the regulation of the initiation and maintenance of RNA replication i.e., higher threshold for initiation than for maintenance.

Analysis of virus accumulation in acutely (Figure 5 and Supplementary Figure S1) and persistently (Figure 7 and Supplementary Figure S2) infected *ERLIN1*-deficient cells showed a strong reduction in the production of both intracellular and extracellular infectious virus. Biophysical analysis of infectious virus particles produced in *ERLIN1*-deficient cells did not reveal any di fference in terms of their specific infectivity (FFU per RNA copies) or density profiles compared to the virus produced in control cells (data not shown) suggesting that *ERLIN1*-deficient cells produce qualitatively normal but quantitatively less infectious virus than *ERLIN1*-su fficient cells.

The disproportionate reduction in virus production compared to the decrease in HCV RNA levels suggested that erlin-1 protein regulates not only early steps leading to RNA and protein accumulation, but also later steps a ffecting virus production. In fact, direct targeting of RNA replication by an HCV polymerase inhibitor led to a proportional decrease of HCV RNA, protein and infectious virus, suggesting that the strong inhibition of virus production observed in *ERLIN1* down-regulated cells could not be solely explained by the modest reduction in the HCV RNA levels (Figure 6). As mentioned above, the magnitude of HCV NS3, NS5A and core protein reduction was disproportionate to the magnitude of HCV RNA reduction suggesting that *ERLIN1* down-regulation a ffects the steady-state levels of HCV proteins. The HCV-IRES translation results indicated that the translation of HCV RNA itself is not inhibited in *ERLIN1*-deficient cells (Figure 3) suggesting that the e ffect is likely to be at the level of protein stability or degradation and not at the translational level. This hypothesis is very appealing since erlin proteins have been implicated in the modulation of the ERAD pathway [23–26]. In contract to the reduced HCV protein accumulation observed in erlin-1 protein deficient HCV-infected cells (Figure 5), *ERLIN1* and *ERLIN2* deficiency produces an accumulation of cellular substrates that are typically degraded through the ERAD pathway [23–26]. These opposite e ffects on protein homeostasis (HCV vs. cellular proteins) may sugges<sup>t</sup> that cellular proteome changes specifically induced by *ERLIN1* down-regulation could indirectly be responsible, at least in part, of the e ffects observed in the HCV infection. Comparison of cellular proteomes of *ERLIN1*- and *ERLIN2*-deficient cells could identify cellular candidate proteins contributing to the e ffects observed in *ERLIN1*-deficient HCV-infected cells.

Recruitment of viral NS5A and core proteins to LDs is a prerequisite for virus assembly [10]. Indeed, interfering with viral protein tra fficking to LDs reduces virus production [13,49]. However, viral protein localization into LDs is not su fficient for virus particle assembly. For instance, a single mutation in the core protein (Y136A) of J6/JFH-1 virus has been associated with an increased accumulation of core in LD, decreased colocalization with E2 protein and reduced HCV particle assembly [50]. Confocal analysis of infected cells confirmed the reduced expression of HCV NS5A and core proteins and revealed a typical localization of these proteins surrounding LDs late in the infection, suggesting that *ERLIN1*-deficiency is affecting their abundance (Figures 5 and 7) but not their recruitment to the HCV assembly factories (Figure 9). Collectively these results sugges<sup>t</sup> that the reduction in HCV protein levels could be responsible for the strong inhibition of infectious virus production observed in erlin-1 down-regulated cells. Importantly, given the differential effect in HCV RNA levels and virus production, these results sugges<sup>t</sup> to us the existence of different HCV protein thresholds required to maintain RNA replication and assembly processes.

Another interesting aspect of these studies is that the HCV-related effects are specific to *ERLIN1* deficiency but not to *ERLIN2* deficiency despite their high similarity and functional cellular redundancy. This difference could be interpreted as a function of protein depletion efficiency achieved by the different siRNAs. *ERLIN1*-targeting siRNAs (1.3, 1.5 and 1&2) were in general more efficient (stronger reduction and longer-lasting effect) reducing erlin-1 protein expression than *ERLIN2*-targeting siRNAs (2.3 and 1&2) in silencing erlin-2 protein. The reason for this could be in part the longer half-life of erlin-2 compared to erlin-1 protein and the relative abundance of those proteins in Huh-7 cells, with erlin-2 being ~2–3 times more abundant than erlin-1 protein (data not shown). Alternatively, our results might indeed reflect a specific function of erlin-1 protein that is not shared by erlin-2 protein and that would be the first function exclusively assigned to erlin-1 protein. Importantly, our results identify erlin-1 protein as a new positive modulator of HCV infection and together with the results described by Inoue et al. in the SV40 infection system [28] point to the need of investigating the role of erlin proteins in other viral infections, especially in those in which their life cycle is tightly associated with the ER e.g., dengue virus or zika virus infection.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/12/1555/s1, Figure S1: Erlin-1 protein down-regulation interferes with HCV in single-cycle infection experiments, Figure S2: Erlin-1 protein down-regulation impairs infectious virus production in an ongoing HCV infection cell culture system.

**Author Contributions:** Conceptualization, L.G. and U.G.; methodology, C.W.-B., J.C., A.G.-M., P.G.-Z., M.D.H., and U.G.; investigation, C.W.-B., J.C., A.G.-M., P.G.-Z. and U.G.; formal analysis, C.W.-B., J.C. and U.G.; writing—original draft preparation, U.G.; writing—review and editing, A.G.-M., M.D.H., L.G. and U.G.; supervision, U.G.; funding acquisition, U.G.

**Funding:** This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, gran<sup>t</sup> numbers RYC-2014-15805 and SAF2016-75169-R (AEI/FEDER, UE) to U.G. and A.G.-M. is supported by an FPU fellowship (FPU17/03424) from the Spanish Ministry of Education, Culture and Sports.

**Acknowledgments:** We are thankful to Frank Chisari (The Scripps Research Institute, La Jolla, CA, USA) for inspiration, guidance and support and in whose laboratory these experiments were begun, to Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan) for providing JFH-1 cDNA, to François-Loïc Cosset (Ecole Normale Superieure, Lyon, France) for providing the HCVpp system, to Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany) for providing JFH-1 replicons and to Mansun Law (The Scripps Research Institute, La Jolla, USA) and Charles M. Rice (Rockefeller University, New York, USA) for providing antibodies against E2 and NS5A, respectively. We would like to acknowledge Sylvia Gutierrez Erlandsson and the Advanced Light Microscopy Facility personnel for exceptional technical assistance. We are also thankful to Frank Chisari and Pablo Gastaminza (Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain) for critically reading the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
