Hepatitis C Virus Modulates Solute carrier family 3 member 2 for Viral Propagation

Hepatitis C virus (HCV) exploits an extensive network of host proteins to maintain chronic infection. Using RNA-Seq technology, we identified 30 host genes that were differentially expressed in cell culture grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected solute carrier family 3 member 2 (SLC3A2) for further investigation. SLC3A2, also known as CD98hc, is a member of the solute carrier family and encodes a subunit of heterodimeric amino acid transporter. SLC3A2 and LAT1 constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. In this study, we showed that HCV upregulated both mRNA and protein expression levels of SLC3A2 and this upregulation occurred through NS3/4A-mediated oxidative stress. HCV also elevated SLC3A2/LAT1 complex level and thus mammalian target of rapamycin complex 1 (mTORC1) signaling was activated. We further showed that L-leucine transport level was significantly increased in Jc1-infected cells as compared with mock-infected cells. Using RNA interference technology, we demonstrated that SLC3A2 was specifically required for the entry step but not for other stages of the HCV life cycle. These data suggest that SLC3A2 plays an important role in regulating HCV entry. Collectively, HCV exploits SLC3A2 for viral propagation and upregulation of SLC3A2 may contribute to HCV-mediated pathogenesis.

In an attempt to develop host-targeted agents (HTAs), we recently performed transcriptome sequencing (RNA-Seq) analysis and identified 30 host genes which were upregulated in HCV-infected cells 12 . In the present study, we selected SLC3A2 and showed that HCV upregulated SLC3A2 expression via oxidative stress. Furthermore, SLC3A2/LAT1 complex level was increased in the context of HCV replication, suggesting that HCV might regulate leucine transporter to promote cell proliferation via mammalian target of rapamycin complex 1 (mTORC1) signaling. We further showed that SLC3A2 was involved in the entry step of the HCV life cycle. These data suggest that HCV modulates SLC3A2 to provide a favorable milieu for viral propagation.

Results
HCV upregulates SLC3A2 expression level. Using RNA-Seq technology, we recently identified 30 host genes that were highly differentially expressed in the context of HCV infection 12 . Among these genes, we selected SLC3A2 to investigate the possible involvement of amino acid transporter function of SLC3A2 in HCV propagation. To validate the increase of SLC3A2 expression in HCVcc-infected cells, Huh7.5 cells infected with either mock or Jc1 13 were harvested at 2-day intervals. We showed that mRNA levels of SLC3A2 were noticeably increased at day 6, and its levels were doubled at day 8 in Jc1-infected cells compared with the level in mock-infected cells (Fig. 1A). Consistently, protein level of SLC3A2 was also gradually increased during the course of HCV infection (Fig. 1B). Moreover, both mRNA (Fig. 1C) and protein (Fig. 1D) levels of SLC3A2 were considerably upregulated in Huh7 cells harboring HCV subgenomic replicon as compared to those in interferon-cured and parental Huh7 cells. These data clearly indicate that HCV upregulates SLC3A2 expression. HCV upregulates SLC3A2 expression via redox signaling. It has been previously reported that HCV regulates ROS signaling cascade, leading to oxidative stress [14][15][16][17][18] . It is also known that SLC3A2 protects cells from oxidative stress by activating amino acid transporter system 6 . To investigate if the reactive oxygen species (ROS) is involved in upregulation of SLC3A2 in HCV infected cells, Huh7.5 cells were treated with increasing amounts of H 2 O 2 for 24 h and then both protein and mRNA levels of SLC3A2 were analyzed by either immunoblot analysis and qRT-PCR, respectively. Figure 2A shows that protein levels of SLC3A2 in Huh7.5 cells were gradually increased by H 2 O 2 . Consistently, mRNA levels of SLC3A2 were significantly increased by H 2 O 2 . To corroborate these results, Huh7.5 cells were either mock infected or infected with Jc1. The cells were then treated with either NAC, an antioxidant agent, or BAPTA-AM, an intracellular Ca 2+ chelator. As expected, both protein and mRNA expression levels of SLC3A2 were increased by HCV infection (Fig. 2B, lane 2). However, HCV-induced SLC3A2 expression levels were gradually decreased by NAC in a dose-dependent manner (Fig. 2B, lanes 3 and 4). Meanwhile, BAPTA-AM displayed no effect on protein and mRNA expression levels of SLC3A2 in HCV-infected cells (Fig. 2C). Taken together, these data suggest that oxidative stress is involved in upregulation of SLC3A2 in the context of HCV infection. SLC3A2 expression level is upregulated by NS3/4A. Since SLC3A2 expression levels are increased in both HCV infected cells and HCV replicon cells, nonstructural proteins may be responsible for this upregulation. To explore this hypothesis, Huh7.5 cells were transfected with either vector or increasing amounts of individual HCV protein expression plasmid and then SLC3A2 protein expression was analyzed by immunoblot analysis. As shown in Fig. 3A, NS3/4A markedly increased SLC3A2 protein expression level. Since SLC3A2 protein expression level was also minimally increased by E1E2, we further explored the possible involvement of E1E2 on SLC3A2 expression by infecting Huh7.5 cells with either VSVpp or HCVpp derived from either H77 or JFH1. Figure 3B shows that SLC3A2 protein expression was not upregulated by E1E2, indicating that HCV NS3/4A specifically upregulated SLC3A2 protein level. Consistently, mRNA level of SLC3A2 was also significantly increased by NS3/4A but not by other HCV proteins (Fig. 3C). It has been previously reported that NS3/4A showed an approximately 11-fold increase in ROS level in Huh7.5.1 cells 18 . To investigate whether SLC3A2 upregulation was modulated by NS3/4A-induced ROS, Huh7.5 cells transfected with NS3/4A plasmid were treated with NAC. As shown in Fig. 3D, protein level of SLC3A2 was markedly increased by NS3/4A. However, NS3/4A-induced SLC3A2 protein level was decreased to basal level by NAC in a dose-dependent manner (left panel). Consistently, mRNA level of SLC3A2 was significantly reduced by NAC (Fig. 3D, right panel), indicating that NS3/4 A played a crucial role in upregulation of SLC3A2. SLC3A2/LAT1 complex level was increased in the context of HCV infection. SLC3A2 (CD98) and LAT1 (SLC7A5) constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport 19 . We therefore wondered whether LAT1 expression level was also upregulated by HCV infection. For this purpose, Huh7.5 cells were either mock-infected or infected with Jc1 and then both mRNA and protein levels of LAT1 were determined. Figure 4A shows that mRNA levels of LAT1 were gradually increased during Jc1 infection (upper panel). Consistently, protein levels of LAT1 were markedly increased in the context of HCV infection (Fig. 4A, lower panel). We next investigated whether SLC3A2/LAT1 heterodimeric complex level was also modulated by HCV infection. We first examined whether SLC3A2 formed a heterodimeric complex with LAT1 in hepatic cells. For this purpose, Huh7.5 cells treated with either reducing or non-reducing buffer were subjected to native gel electrophoresis and analyzed by immunoblot analysis. As shown in Fig. 4B, the same size of heterodimeric complex was detected by either anti-SLC3A2 or anti-LAT1 antibodies in cells treated with non-reducing buffer (lane 2). However, this complex was not detectable under reducing condition (Fig. 4B, lane 1), suggesting that both SLC3A2 and LAT1 formed a heterodimeric complex via disulfide bonds. To determine how HCV modulated SLC3A2/LAT1 heterodimeric complex level, Huh7.5 cells were either mock-infected or infected with Jc1 and then total cell lysates harvested at the indicated time points were analyzed under non-reducing condition. Figure 4C shows that protein mass corresponding to the SLC3A2/LAT1 heterodimeric complex was detected in both mock-and Jc1-infected cells. Of note, SLC3A2/LAT1 complex level was markedly increased in the context of HCV infection.
HCV modulates SLC3A2/LAT1 complex to promote cell proliferation. It has been previously reported that HCV activates the mTORC1 signaling 20 , and SLC3A2/LAT1 heterodimeric amino acid transporter regulates mTORC1 signaling through transporting leucine into the cells 7,8 . To investigate the involvement of SLC3A2/LAT1 on amino acid transport, Huh7.5 cells treated with the indicated siRNAs were either left untreated or treated with L-leucine and then glutamate dehydrogenase (GDH) activity was determined. Because L-leucine stimulates GDH, L-leucine transport is measured by GDH activity. Figure 5A showed that GDH activity was significantly increased in the presence of L-leucine. Of note, L-leucine-induced GDH activity was significantly decreased in both SLC3A2-silenced cells and SLC3A2/LAT1-knockdown cells. To further investigate whether amino acid level is upregulated by HCV infection, Huh7.5 cells infected with either mock or Jc1 were treated with L-leucine and GDH activity was determined. We showed that GDH activity was increased with L-leucine treatment in mock-infected cells (Fig. 5B). Importantly, GDH activity was significantly increased in Jc1-infected cells and Jc1-induced GDH activity was further enhanced in the presence of L-leucine. These data indicate that HCV increases SLC3A2/LAT1 complex level to facilitate amino acid transport in HCV-infected cells. In addition, the mTORC1 pathway known to regulate cell proliferation is mainly mediated through eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) 21,22 . We showed that phosphorylation level of 4EBP1 was markedly increased in HCV-infected cells as compared with mock-infected cells (Fig. 5C). These data indicated that 4EBP1,  downstream target of mTORC1, was activated in HCV infected cells, suggesting that mTORC1 signaling was upregulated by SLC3A2/LAT1 heterodimeric amino acid transporter. To further corroborate this result, Huh7.5 cells infected with either mock or Jc1 were treated with BCH, an inhibitor of L-Leucine transporter, and then total cell lysates were immunoblotted with the indicated antibodies. Figure 5D shows that HCV-induced 4EBP1 phosphorylation level was gradually decreased with increasing amounts of BCH. We further showed that treatment of the same concentration of BCH displayed no effect on cell viability, demonstrating that the effect of BCH was specific to L-Leucine transporter (Fig. 5E). We further evaluated the role of SLC3A2/LAT1 complex in cell proliferation. Huh7.5 cells infected with either mock or Jc1 were treated with either vehicle (NH 4 OH) or BCH and then cell proliferation was analyzed by WST assay. As shown in Fig. 5F, BCH significantly decreased cell growth in HCV-infected cells as compared with mock-infected cells, suggesting that leucine uptake by SLC3A2/LAT1 transporter was required for cell proliferation. To further confirm this result, Huh7.5 cells infected with either mock or Jc1 were transfected with either negative siRNA or siRNAs targeting both SLC3A2 and LAT1 (double). Figure 5G verified that cell growth in HCV-infected cells was significantly decreased in SLC3A2 and LAT1 knockdown cells as compared with mock-infected cells. Collectively, these data suggest that HCV modulates SLC3A2/ LAT1 complex through mTORC1 signaling pathway to promote cell proliferation.

SLC3A2 is required for HCV propagation.
To explore the possible involvement of SLC3A2 in HCV propagation, Huh7.5 cells transfected with either negative control siRNA or SLC3A2-specific siRNA were infected with Jc1, and then both mRNA and protein levels were determined. Silencing of SLC3A2 significantly suppressed RNA expression level of HCV (Fig. 6A, upper panel). Consistently, protein expression levels of HCV were impaired in SLC3A2 knockdown cells (Fig. 6A, lower panel). To further investigate the role of SLC3A2 in HCV propagation, naïve Huh7.5 cells were infected with Jc1 in supernatant harvested from the primary infection, and then both RNA and protein levels of HCV were analyzed. As shown in Fig. 6B, viral infectivity determined by both viral RNA and protein levels in the second infection was markedly decreased by knockdown of SLC3A2. Moreover, treatment of the same concentration of siRNAs displayed no cytotoxicity in the cells, indicating that the silencing effect was specific to SLC3A2 (Fig. 6C). To rule out the off-target effect of a SLC3A2 siRNA, we generated an siRNA-resistant SLC3A2 mutant. Figure 6D showed that exogenous expression of the siRNA-resistant SLC3A2 mutant, but not of the wild-type SLC3A2, restored the HCV protein expression levels (lane 3 versus lane 4). Collectively, these data suggest that SLC3A2 is specifically required for HCV propagation.
SLC3A2 is not involved in the replication, translation, and virion production steps of the HCV life cycle. To investigate which step of the HCV life cycle was required for SLC3A2, we analyzed the SLC3A2 expression level in Huh7 cells harboring HCV subgenomic replicon by silencing of SLC3A2. As shown in Fig. 7A, knockdown of SLC3A2 displayed no effect on intracellular HCV RNA (left panel) and protein (right panel) levels in replicon cells derived from genotype 1b. We next investigated whether SLC3A2 was involved in HCV internal ribosome entry site (IRES)-mediated translation. For this purpose, Huh7.5 cells were transiently transfected with the indicated siRNAs together with pRL-HL and a β-galactosidase plasmid, and then luciferase activity was determined as we reported previously 12 . Figure 7B demonstrated that silencing of SLC3A2 exerted no effects on HCV IRES-mediated translation. To examine if SLC3A2 was required for a later stage of the HCV life cycle, Huh7.5 cells were first infected with Jc1 for 48 h and then further transfected with either a negative siRNA or a siRNA targeting SLC3A2 or an apolipoprotein E (ApoE) siRNA as a control. At 48 h after transfection, both RNA and protein levels were determined. Figure 7C shows that silencing of SLC3A2 displayed no effect on both RNA and protein levels of HCV (upper panel). To verify this result, naïve Huh7.5 cells were infected with HCV derived from culture supernatant harvested from the first infection. As shown in Fig. 7C (lower panels), silencing of SLC3A2 had no discernible effect on viral infectivity. All these data indicated that SLC3A2 was not involved in the replication, translation, and virion production steps of the HCV life cycle. SLC3A2 is required in the entry step of the HCV life cycle. Since SLC3A2 was not involved in replication and production steps, one remaining possibility was that SLC3A2 might play a role in the entry step of the HCV life cycle. To explore this possibility, we performed viral entry assays using HCVpp derived from either H77 or JFH1. The vesicular stomatitis glycoprotein G (VSVpp) was used as a control. As shown in Fig. 8A, HCVpp entry was impaired in SLC3A2 knockdown cells in both genotypes. However, VSVpp entry was not altered by knockdown of SLC3A2. To further verify the involvement of SLC3A2 in the HCV entry, Huh7.5 cells transfected with either negative or SLC3A2-specific siRNA were further transfected with either Flag-tagged wild-type SLC3A2 or siRNA-resistant SLC3A2 mutant plasmid. At 24 h after transfection, cells were infected with HCVpp derived from either H77 or JFH1. As shown in Fig. 8B, HCV entry was recovered by siRNA-resistant SLC3A2 mutant, but not by wild-type SLC3A2, indicating that SLC3A2 was specifically required for HCV entry. To further confirm this result, we performed viral entry assays using HCV like particle (HCV-LP) as we described previously 23 . In this system, HCV-LP can undergo single cycle infection without producing virions. We produced HCV-LP as depicted in Fig. 8C (left panel). Huh7.5 cells were transfected with either negative siRNA or siRNA targeting SLC3A2 and then infected with HCV-LP for 48 h. As shown in Fig. 8C (right panel), HCV-LP luciferase activity was significantly reduced in SLC3A2 knockdown cells as compared to the negative control. To further investigate the precise involvement of SLC3A2 in HCV entry, we divided HCV infection steps into attachment/binding cells were either mock infected (left panel) or infected with Jc1 (right panel). At 4 days postinfection, cells were transfected with either negative siRNA or siRNA mix targeting both SLC3A2 and LAT1 (double). At the indicated time points, cell proliferation was assessed by WST assay. and entry/fusion steps. To analyze if SLC3A2 was required during attachment/binding step, Huh7.5 cells were transfected with either negative control siRNA or SLC3A2-specific siRNA for 48 h and then incubated with Jc1 inoculum for 2 h at 4 °C. After washing with PBS, bound virions were determined by HCV RNA levels. As shown in Fig. 8D, silencing of SLC3A2 displayed no effect on HCV binding to the host cells (left panel). To determine if SLC3A2 was required during entry/fusion step, Huh7.5 cells transfected with either negative control siRNA or SLC3A2-specific siRNA for 48 h were incubated with Jc1 inoculum for 2 h at 4 °C and washed with PBS to remove unbound virions. Temperature was then shifted to 37 °C for 4 h to allow virions to internalize the host cells. The cells were trypsinized and non-internalized virions were washed away with PBS. Virion entry was determined by analyzing intracellular HCV RNA levels. Figure 8D demonstrated that HCV RNA level was significantly decreased in SLC3A2 knockdown cells (right panel), indicating that SLC3A2 is a host factor required at a post-binding step in HCV entry. Since SLC3A2 regulated HCV entry and mTORC1 signaling in complex with LAT1, we postulated that HCV envelope protein might be associated with SLC3A2. For this purpose, HEK293T cells were cotransfected with Flag-tagged SLC3A2 and either vector or V5-tagged E2 expression plasmid. Total cell lysates harvested at 36 h after transfection were immunoprecipitated with an anti-Flag antibody and bound protein was detected by an anti-E2 antibody. We showed that SLC3A2 interacted with HCV E2 protein (Fig. 8E). To verify this result, the same cell lysates were immunoprecipitated with an anti-V5 antibody and bound protein was analyzed by immunoblot assay using an anti-Flag antibody. Reciprocal coimmunoprecipitation experiment confirmed that E2 interacted with SLC3A2 (Fig. 8F). However, LAT1 did not interact with HCV E2 (Fig. 8G), further confirming that SLC3A2 specifically interacted with E2 protein. Collectively, these data suggest that HCV may coopt host SLC3A2 protein via E2 to facilitate viral entry.

Discussion
SLC3A2 (also known as CD98 heavy chain, CD98hc) and large neutral amino acid transporter 1 (LAT1) form a heterodimer and interact with integrins. SLC3A2 regulates the expression and distribution of the light chains to modulate amino acid transport, and overexpression of SLC3A2 promotes tumorigenesis 6 . Indeed, SLC3A2 was overexpressed in many cancer cell lines, including lung, colon, and breast 24 , and it is also known as a novel marker for renal cancer 25 . Our recent proteomic analysis data show that SLC3A2 is highly expressed in HCVcc-infected cells 12 . In the present study, we for the first time showed that both mRNA and protein expression levels of SLC3A2 were upregulated in the context of HCV replication. We also demonstrated that ROS significantly increased  SLC3A2 expression levels. It has been previously reported that HCV induces ROS production in hepatoma cells 16 . Using NAC, we verified that the upregulation of SLC3A2 expression in HCV-infected cells was mediated by oxidative stress. Since core, NS3/4A, and NS5A proteins are involved in ROS production [14][15][16] , why NS3/4A but not core and NS5A upregulate SLC3A2 expression needs further studies. We tempted to speculate that protease enzymatic activity of NS3/4A together with ROS may be involved in SLC3A2 upregulation by unknown mechanism.
SLC3A2 forms a complex with LAT1 for amino acid transport activity 4,5,7 . We demonstrated that LAT1 expression level was gradually increased during HCV infection and SLC3A2/LAT1 complex level was also elevated in Jc1-infected cells. SLC3A2/LAT1 complex plays a critical role in the uptake of extracellular leucine, which subsequently leads to mTORC1 activation 24,26 . By siRNA-mediated silencing of SLC3A2/LAT1, we verified that SLC3A2/LAT1 complex was involved in L-leucine transport. Importantly, L-leucine transport level was significantly increased in Jc1-infected cells as compared with mock-infected cells. All these data indicate that the increase in amino acid transport in HCV-infected cells may be due to enhanced SLC3A2/LAT1 expression level. We then investigated the phosphorylation level of 4EBP1, a downstream target protein of mTORC1, in HCV infected cells. As expected, phospho-4EBP1 level was remarkably increased by HCV infection and HCV-induced phospho-4EBP1 level was pronouncedly decreased by BCH, indicating that SLC3A2/LAT1 complex was upregulated by HCV. Furthermore, cell proliferation was significantly decreased in both BCH-treated and SLC3A2/ LAT1 knockdown cells, suggesting that HCV promoted cell proliferation by increasing leucine transport via mTORC1 signaling pathway for persistent infection.
Oxidative stress is an event of enhanced formation of ROS in the cells 27 . ROS plays an important role in determining the progression of HCV-induced liver pathogenesis 28 . Oxidative stress in HCV-infected cells occurs by a combination of chronic inflammation and proteins encoded by HCV. HCV core, NS3/4A, and NS5A proteins primarily cause an increase in ROS production [14][15][16] . In addition to ROS production, the mTORC1 can be also activated by HCV infection 17,18 . It has been previously reported that HCV NS5A activates mTOR signaling pathway to promote persistent infection 29 . mTORC1 consists of mTOR, raptor, mLST8, PRAS40, and DEPTOR. mTOR, core component of mTORC1/2, is a serine/threonine kinase that is involved in the regulation of multiple cellular processes, including cell growth, proliferation, protein synthesis, and survival. mTORC1 is activated by amino acids such as leucine and hormones, including leptin, insulin, and IGF-1 20 . However, the mechanism by which HCV hijacks mTORC1 signaling pathway has not been fully understood.
We then assessed the functional role of SLC3A2 in HCV propagation. Knockdown of SLC3A2 impaired both RNA and protein expressions of HCV, implying that SLC3A2 positively regulated HCV propagation. Using an HCVpp entry assay, we showed that HCV entry was impaired in SLC3A2 knockdown cells. By analyzing the precise involvement of SLC3A2 in HCV infection, we further showed that SLC3A2 was specifically required for post-binding step in the HCV entry. This result suggests that SLC3A2 may be a novel host cofactor involved in HCV entry. HCV entry is a complex process that requires multiple cell surface factors, including CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), occludin (OCLN), and Niemann-Pick C1-like 1 cholesterol absorption receptor (NPC1L1) [30][31][32][33][34] . Furthermore, HCV internalizes the host cells via clathrin-mediated endocytosis 35 . We also demonstrated that SLC3A2 interacted with HCV E2 protein. It has been previously reported that HCV E2 interacts with SR-BI at post-binding step to enhance viral infectivity 33 . Moreover, CD81 interacts with HCV E2 via a series of discontinuous amino acid residues in the large extracellular loop 36 and CD81 acts as a post-binding entry molecule 37 . Likewise, SLC3A2 may interact with E2 to facilitate HCV entry into the host cells. Interestingly, SLC3A2 has been also implicated in vaccinia virus entry 38 . Although SLC3A2 is identified as an additional cofactor involved in HCV entry, how SLC3A2 functions in conjunction with those already known cofactors in HCV entry needs further studies. In summary, HCV upregulated SLC3A2 expression levels via NS3/4A-mediated oxidative stress. Furthermore, HCV increased the SLC3A2/LAT1 heterodimeric amino acid transporter level to promote viral propagation. Our study provides some compelling evidence showing that SLC3A2 is an essential cellular cofactor required for HCV entry and thus SLC3A2 could be a potential therapeutic target to interrupt HCV propagation.
(***p < 0.001) from the activity for the negative control. (D) (Left panel) Huh7.5 cells were transfected with the indicated siRNAs for 48 h. The cells were incubated with Jc1 at 4 °C for 2 h for binding. The cells were washed with PBS and then bound virions were determined by analyzing HCV RNA levels by qRT-PCR. (Right panel) Huh7.5 cells were transfected with the indicated siRNAs. At 48 h after transfection, cells were incubated with Jc1 at 4 °C for 2 h. The cells were washed with PBS and then temperature was shifted to 37 °C for 4 h. The cells were trypsinized and washed twice with PBS to remove free virions. Internalized HCV virions were indirectly determined by analyzing HCV RNA levels by qRT-PCR. (E) HEK293T cells were cotransfected with Flagtagged SLC3A2 and either vector or V5-tagged E2 expression plasmid. At 36 h after transfection, total cell lysates were immunoprecipitated with an anti-Flag antibody and bound proteins were analyzed by immunoblot analysis using an anti-E2 antibody and secondary antibodies. To verify E2 protein, we used another kind of secondary antibody, E2(s), which did not recognize heavy chain. (F) Total cell lysates prepared as described in (E) were immunoprecipitated with anti-V5 antibody and bound proteins were detected by immunoblot analysis using an anti-Flag antibody. (G) HEK293T cells were cotransfected with Flag-tagged LAT1 and either vector or V5-tagged E2 plasmid. At 30 h after transfection, total cell lysates were immunoprecipitated with an anti-V5 antibody and bound proteins were analyzed by immunoblot analysis using an anti-Flag antibody.
WST assay. Cells seeded on 48-well plates were either transfected with the indicated siRNAs or treated with 2-aminobicyclo-(2,2,1)-heptanecarboxylic acid (BCH), a SLC3A2/LAT1 transporter inhibitor, for 96 h. Cell viability was measured by incubating cells with water-soluble tetrazolium salt (WST) (Dail Lab) for 1 h at 37 °C. The plate was shaken for 1 min and then aqueous layer in each well was transferred into 96-well plate. The absorbance was measured at 450/650 nm as reported previously 12 .
Luciferase reporter assay. Huh7.5 cells were cotransfected with a pRL-HL plasmid containing the Renilla luciferase gene under the cytomegalovirus (CMV) promoter and the firefly luciferase gene under the control of the HCV IRES and the plasmid indicated in the figures together with the pCH110 reference plasmid. After 48 h after transfection, cells were harvested and then dual-luciferase assays were performed as we described previously 23 .
HCV pseudoparticle entry assay. HCV pseudoparticles (HCVpp) with E1 and E2 glycoproteins derived from genotype 1a (H77) or genotype 2a (JFH1) and vesicular stomatitis virus pseudoparticles (VSVpp) were generated as we described previously 23 . Briefly, approximately 2.5 × 10 6 HEK293T cells were transfected with 2.5 μg of HCV E1E2 or VSV G envelope expressing plasmid, 7.75 μg of Gag-Pol (polymerase) packaging plasmid, and 7.75 μg of transfer vector encoding the firefly luciferase reporter protein by using polyethyleneimine (Sigma-Aldrich). At 48 h after transfection, supernatants containing HCVpp or VSVpp were collected. For the infection assay, Huh7.5 cells were transfected with siRNAs for 48 h and then infected with either HCVpp or VSVpp for 6 h. Cells were then replaced with fresh culture media. At 48 h postinfection, cells were harvested and luciferase activity was determined. Binding and entry assays. Approximately 0.6 × 10 5 Huh7.5 cells seeded on 12-well plate were transfected with either negative control siRNA or SLC3A2 siRNA for 48 h. For HCV binding assay, cells were incubated with Jc1 at 4 °C for 2 h to allow virions for binding but not to internalize the target cells. After washing cells with PBS, bound virions were measured by qRT-PCR. For HCV entry assay, cells were also incubated with Jc1 at 4 °C for 2 h, washed with PBS and then temperature was shifted to 37 °C for 4 h to allow virions to internalize the cells. Cells were then trypsinized and washed twice with PBS to remove non-internalized virions. HCV entry was indirectly determined by analyzing the intracellular HCV RNA levels by qRT-PCR 12,23 . Quantification of RNA. Quantitative real-time PCR (qRT-PCR) experiments were performed as we reported previously 12,23 . Single-cycle HCV infection. Single-cycle infectious HCV (HCVsc) was generated from a replicon trans-packaging system as previously described 23 . Briefly, Huh7.5 cells were cotransfected with pHH/SGR-Luc plasmid which carries a bicistronic HCV subgenomic replicon (NS3-NS5) firefly luciferase reporter with a Pol I promoter/terminator, and a HCV core-NS2 expression plasmid by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Culture medium containing HCV-like infectious particle (HCV-LP) was collected at 5 days after transfection. For single-cycle HCV infection assay, Huh7.5 cells were pretreated with either negative control siRNA or siRNA targeting SLC3A2 for 48 h and then infected with HCVsc. At 48 h postinfection, cells were harvested and luciferase activity was determined.
Immunoprecipitation. HEK293T cells were cotransfected with V5-tagged E2 and either Flag-tagged SLC3A2 or Flag-tagged LAT1. Total amounts of DNA were adjusted by adding an empty vector. At 30 h after transfection, cells were harvested and immunoprecipitation assay was performed as we described previously 12,23 . Cells were lysed in buffer containing 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.2% NP-40, and protease inhibitor cocktail, and then incubated for 10 min on ice. Cell lysates were centrifuged at 13,000 rpm for 10 min. The supernatant was then incubated overnight at 4 °C with the indicated antibody. The samples were further precipitated with 40 μl of protein A beads (Sigma-Aldrich) for 1 h at 4 °C. The beads were subsequently washed five times in washing buffer, and then bound proteins were detected by immunoblot analysis. SLC3A2/LAT1 transporter inhibitor assay. SLC3A2/LAT1 transporter inhibitor, 2-aminobicyclo-(2,2, 1)-heptane-carboxylic acid (BCH), was dissolved in 1N NH 4 OH and stored at room temperature. Final concentration of NH 4 OH in the medium was 200 μM. Huh7.5 cells were pre-incubated with various amounts of BCH in 1 h and then infected with Jc1 in the presence of BCH. At 48 h postinfection, cells were harvested and both RNA and protein levels were determined.
Cell proliferation assay. Cell proliferation was determined by WST assay. Huh7.5 cells were either mock-infected or infected with Jc1 for 4 days and then treated with either NH 4 OH (vehicle) or BCH. Cells were re-plated onto 96-well plates at a density of ~2 × 10 3 cells per well. At the indicated time intervals after chemical treatment, 10 μL of WST solution was added to each well to determine cell growth.
Analysis of GDH enzymatic activity. Leucine stimulates GDH activity in hepatocytes 40 . In order to measure L-leucine uptake in Huh7.5 cells, the enzymatic activity of GDH was analyzed by using a GDH Activity Colorimetric Assay kit (BioVision, Catalog #K729-100) according to the manufacturer's instructions. GDH activity was measured OD at 450 nm in a VersaMax Microplate Reader (Molecular Devices USA).