Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process

SUMMARY

Hepatitis C virus (HCV) enters hepatocytes via various entry factors, including scavenger receptor BI (SR-B1), cluster of differentiation 81 (CD81), epidermal growth factor receptor (EGFR), claudin-1 (CLDN1), and occludin (OCLN). As CLDN1 and OCLN are not readily accessible due to their tight junctional localization, HCV likely accesses them by either disrupting cellular polarity or migrating to the tight junction. In this study, we image HCV entry into a three-dimensional polarized hepatoma system and reveal that the virus sequentially engages these entry factors through actin-dependent mechanisms. HCV initially localizes with the early entry factors SR-B1, CD81, and EGFR at the basolateral membrane and then accumulates at the tight junction in an actin-dependent manner. HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR. Thus, HCV uses a dynamic and multi-step process to engage and enter host cells.

In Brief

HCV entry is complex and involves multiple host factors. By imaging the infection of polarized hepatoma organoids with fluorescent HCV virions, Baktash et al. define the sequence of events during entry. They determine that HCV virions traffic with early receptors to tight junctions and then internalize in an EGFR-dependent manner.

Graphical Abstract

INTRODUCTION

Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped, positive-sense RNA virus of approximately 9,600 nt. The HCV virion is frequently referred to as a lipoviroparticle, containing cellular apolipoproteins (Apos) and a lipid composition similar to very-low-density lipids (VLDLs) (Bartenschlager et al., 2011). The virus has a highly restricted tropism, preferentially infecting hepatocytes, the uniquely polarized epithelial cells of the liver. Hepatocytes have disproportionately large basolateral surfaces that access the bloodstream via contact with sinusoidal capillaries; lateral association with adjacent hepatocytes form extended sheets (Perrault and Pé cheur, 2009). The apical domains of adjacent hepatocytes, defined and maintained by tight junctions, form a network of bile canaliculi (Easter et al., 1983).

HCV entry into hepatocytes is a highly complex, multistep process involving numerous cellular factors. Initial attachment is proposed to occur via interactions of virion-associated ApoE with cell-associated glycosaminoglycans (Jiang et al., 2012), LDL receptor (Owen et al., 2009), and syndecan-1 (Shi et al., 2013). HCV E2 then interacts with two entry factors that are generally accepted to function as receptors: SR-B1 and the tetraspanin CD81. Disrupting the interaction of E2 with SR-B1 or CD81 via siRNAs or blocking antibodies inhibits HCV entry (Bartosch et al., 2003; Pileri et al., 1998; Scarselli et al., 2002; Zhang et al., 2004).

Additionally, HCV entry into hepatocytes requires the tight junction proteins CLDN1 and OCLN (Evans et al., 2007; Liu et al., 2009; Ploss et al., 2009; Zheng et al., 2007). HCV virions and envelope proteins have not been shown to interact directly with CLDN1 or OCLN, suggesting either that the entry factors play an indirect role in HCV entry or, alternatively, that conformational changes in the envelope proteins, likely resulting from initial receptor engagement, are required for HCV binding to CLDN1 and OCLN. In support of the latter interpretation, antibodies directed to extracellular domains of CLDN1 and OCLN block HCV entry (Evans et al., 2007; Fofana et al., 2013; Sourisseau et al., 2013). There is also genetic evidence for an interaction between E1 and CLDN1 (Hopcraft and Evans, 2015). If, as these studies suggest, HCV associates with CLDN1 and OCLN, the virion cannot readily access these required host factors from the bloodstream. One model proposes that HCV migrates to the tight junction following engagement of the early receptors SR-BI and CD81 (Evans et al., 2007). Another alternative involves HCV association with extrajunctional forms of CLDN1 and OCLN, potentially due to disruption of tight junction barrier function (Harris et al., 2008; Mee et al., 2009).

Additional proposed entry cofactors include epidermal growth factor receptor (EGFR) (Lupberger et al., 2011), very-low-density lipoprotein receptor (Ujino et al., 2016), Niemann-Pick C1-like 1 (Sainz et al., 2012), transferrin receptor (Martin and Uprichard, 2013), serum response factor-binding protein 1 (Gerold et al., 2015), E-cadherin (Li et al., 2016), CD36 (Cheng et al., 2016), cell-death-inducing DFFA-like effector B (Wu et al., 2014), and ankyrin repeat domain 1 (Than et al., 2016). EGFR interacts with CD81 (Diao et al., 2012; Zona et al., 2013), and EGFR inhibitors limited CD81 diffusion and CD81-CLDN1 interaction in unpolarized hepatocytes in the absence of infection (Zona et al., 2013). Zona et al. (2013) consequently hypothesized that EGFR signaling may be required for HCV migration to the tight junction.

We previously developed single-particle tracking of fluorescent HCV virions, which was used in conjunction with RNA interference analysis to define HCV entry in unpolarized Huh-7.5 cells (Coller et al., 2009). This analysis identified numerous host proteins as cofactors of HCV entry: components of clathrin-mediated endocytosis, the actin cytoskeleton, ubiquitylated receptor sorting, and endosomal acidification pathways. HCV particles were fluorescently labeled with the lipophilic dye DiD, and live cell imaging of HCV cotrafficking with CD81, CLDN1, and the identified entry cofactors was performed. Perhaps surprisingly, HCV internalization occurred without preference for cell-cell junctions (Coller et al., 2009). Thus, either HCV entry does not occur at tight junctions or unpolarized Huh-7.5 cells, lacking discrete tight junctions, are an insufficient model to study HCV entry.

In this study, we performed HCV single-particle imaging in three-dimensional (3D), extracellular matrix (ECM)-embedded Huh-7.5 cells (Molina-Jimenez et al., 2012). The hepatoma cells formed spherical organoids with polarization that closely resembles hepatocyte architecture in vivo. We find that DiD-HCV particles colocalize with early entry factors CD81, SR-B1, and EGFR atthebasolateralmembraneandthat,overtime,thesefluorescent HCV particles accumulate at the tight junction, localizing with CLDN1 and OCLN in an actin-dependent manner. EGFR signaling was not required for DID-HCV accumulation at the tight junction, as has been previously suggested, but EGFR was instead essential for particle internalization via clathrin-mediated endocytosis.

RESULTS

Hepatoma Organoids Display Polarity and Are Susceptible to HCV Infection

Polarized hepatocytes display a distinct morphology wherein the basolateral surface is exposed to extracellular virus traveling via the bloodstream, while the apical membrane, which forms the bile canaliculus, is flanked by tight junctions (Figure 1A). Huh-7.5 hepatoma cells are poorly polarized when grown in standard two-dimensional (2D) cultures, as indicated by localization of the tight junction protein zona occludens-1 (ZO-1) throughout the plasma membrane (Figure 1B). In contrast, Huh-7.5 cells grown in ECM form 3D polarized cultures that resemble hepatocytes in vivo (Molina-Jimenez et al., 2012). The Matrigel-based ECM abuts the basolateral membrane; similarly, the sinusoidal face of hepatocytes is in contact with stellate-cell-secreted ECM in vivo. We seeded Huh-7.5 cells on coverslips at single-cell density in ECM and incubated for 7 days, resulting in sphere-like organoids of 10–20 cells. We tested the polarity of Huh-7.5 organoids by examining the localization of hallmark apical, basolateral, and tight junctional markers (Figure 1B). In contrast to the unpolarized 2D Huh-7.5 cells, Huh-7.5 organoids displayed a restricted ZO-1 subcellular localization at internal membrane interfaces that is consistent with tight junctions. The localization of MRP2, an apical protein that defines the bile canaliculus, was similarly restricted in Huh-7.5 organoids. Na⁺K⁺-ATPase, a basolateral marker, displayed an expected localization to lateral faces (between cells) as well as the external membranes.

Figure 1. ECM-Embedded Huh-7.5 Cells Display Hallmarks of Polarization and Are Susceptible to HCV Infection.

(A) Diagrammatic of Huh-7.5 organoids and polarized membrane compartments.(B) Top left: Huh-7.5 cells were grown under 2D conditions before being fixedand stained for ZO-1. Nuclei are stained with DAPI (blue). Huh-7.5 organoids were fixed and probed with indicated antibodies. Bottom right: Huh-7.5 organoids were infected with HCV for 48 hr and probed for ZO-1 (green) and NS5A (red). (C and D) HCV replication (C) and virus production (D) were quantified in 2D or 3D Huh-7.5 cells at the indicated times. Error bars, SD. See also Figure S1.

To determine whether the hepatoma organoids display functional characteristics of polarization, we tested whether they retained the bile analog 5-chloromethylfluorescein diacetate (CMFDA) at the apical membrane. CMFDA freely diffuses into cells, where it is converted to a cell-impermeant form after cleavage by esterases. Hepatoma organoids showed enrichment of CMFDA at the apical bile canaliculi, suggesting that the polarized cells are capable of export into the bile canalicular space. Thus, hepatoma organoids display the appropriate localization of cellular markers and functional characteristics of polarized hepatocytes.

We next tested whether Huh-7.5 organoids were susceptible to HCV infection. Cells were infected with HCV for 48 hr and then fixed and probed with an NS5A-specific antibody. NS5A expression was readily detected in all cells within the organoid (Figure 1B, bottom right). Additionally, we compared kinetics of HCV replication and infectious virus production in 2D versus 3D Huh-7.5 cells. Viral RNA copies (Figure 1C) and infectious virus titers (Figure 1D) increased over the indicated time course with no significant difference in HCV replication between 2D and 3D cultures, validating the use of the 3D cultures to characterize HCV infection.

HCV entry factor localization in Huh-7.5 organoids was then examined (Figure S1). EGFR primarily localized to the basolateral membrane, while SR-B1 and CD-81 localized to both basolateral and apical domains. CLDN1 and OCLN were restricted to the internal membrane interfaces as would be expected for tight junctional proteins. To verify tight junctional localization of the HCV receptors, we immunoprobed the Huh-7.5 organoids for OCLN and ZO-1. OCLN and ZO-1 are fully colocalized, confirming that in this polarized system, the HCV tight junctional entry factors reside exclusively in and define the tight junction.

Single-Particle Imaging of HCV Entry into Polarized Hepatoma Organoid Cultures

We previously published single-particle tracking of HCV infection of 2D Huh-7.5 cells (Coller et al., 2009). DiD-labeled HCV was purified by density gradient ultracentrifugation to enrich for highly infectious virions. High levels of DiD colocalization with HCV core indicated specific labeling of virions. Successful immunodepletion of DiD-HCV with E2 antibodies demonstrated that they were free virions not contained within microvesicles such as exosomes. Entry and uncoating of DiD-HCV in Huh-7.5 cells required CD81 and endosomal acidification (Bartosch et al., 2003; Blanchard et al., 2006; Coller et al., 2009; Koutsoudakis et al., 2006; Meertens et al., 2006). In the current study, the purified DiD-HCV stocks had a specific infectivity of 4.9 ± 4 (Figure 2A), with 96% of DiD-HCV colocalized with core (Figure 2B), similar to our previous study. We also co-labeled DiD-HCV with various components of the viral envelope to demonstrate specific labeling in our preps (Figure S2A). Particles showed almost complete colocalization of DiD with either core and E2 or core and ApoE. In addition, to ensure DiD-HCV samples were pure and not contained within exosomes, we performed electron microscopy (Figure S2B). Particles of the expected size and shape were observed without the presence of exosomes. Immunogold E2 labeling confirmed that these particles contained the envelope glycoprotein E2.

Figure 2. DiD-HCV Accumulates at the Tight Junction during Infection of Huh-7.5 Organoids.

(A) Comparison of specific infectivity (SI): HCV supernatant, concentrated HCV supernatant, DiD-HCV, and purified DiD-HCV.(B) Purified DiD-HCV (red) was spotted onto poly-L-lysine treated coverslips, fixed, and probed with core antibody (green).(C) Huh-7.5 organoids were pretreated with or without 10 mg/mL CD81 blocking antibody JS-81 for 1 hr, infected with DiD-HCV for 1 hr at 4C, shifted to 37°C (t = 0) for the indicated amount of time (in minutes), fixed, and probed for ZO-1 (green). Nuclei are stained with DAPI (blue). Insets display the tight junctional region: ZO-1 (left) and DiD-HCV (right).(D) Quantitation of (C). n = total DiD puncta imaged at each time point. Error bars, SD. (E) Huh-7.5 organoids were preincubated for 2 hr with 10 mg/mL JS-81 blocking antibody or control IgG with HCV for 6 hr, washed twice, and replaced with fresh media. RNA was isolated at 6 and 36 hr and quantified via RT-PCR, normalized first to their respective cellular RNA levels and then to control IgG at 6 hpi. Experiments were performed in triplicate; error bars, SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S2 and S3, and Movies S1, S2, S3, and S4.

To determine whether HCV migrates to the tight junction in polarized cell systems, we visualized DiD-HCV colocalization with ZO-1 over a time course of infection. Organoids were infected with HCV and immunoprobed for ZO-1 over the indicated time course (Figure 2C, top panel). At the temperature shift (t = 0), DiD-HCV localized to the external (basolateral) membrane, to the tight junction, and at intermediate points between. As the time course of infection progressed, DiD-HCV showed increasing accumulation at the tight junction, with 93% tight junctional localization at 90 min post temperature shift (Figure 2D). This suggests that DiD-HCV traffics from the basal membrane to the tight junction in hepatoma organoids.

HCV infection can be inhibited through blocking antibodies to CD81 with earlier kinetics than with blocking antibodies specific to CLDN1 or OCLN, indicating that HCV-CD81 interaction occurs prior to a requirement for CLDN1 or OCLN (Evans et al., 2007; Sourisseau et al., 2013). If HCV-CD81 engagement were required for migration of HCV to the tight junction, then CD81 blocking antibodies should prevent the tight junctional accumulation of DiD-HCV. We tested this prediction by incubating the hepatoma organoids with the CD81 blocking antibody prior to infection with DID-HCV in a parallel entry time course. The CD81 blocking antibody prevented accumulation of DiD-HCV at the tight junction, with most particles localized basolaterally (Figure 2C, bottom panel). At all time points, there was a significant decrease in DiD-HCV/ZO-1 colocalization (Figure 2D), indicating that CD81 is critical for tight junctional accumulation of HCV. HCV RNA levels in cells incubated with CD-81 blocking antibody showed a significant decrease as compared to cells incubated with control IgG (Figure 2E). Altogether, the results demonstrate that, unlike previous work in unpolarized systems, DiD-HCV relocalizes to the tight junction in polarized hepatoma organoids. The tight junctional trafficking of DiD-HCV and subsequent infection is dependent on the HCV-CD81 interaction.

In order to track HCV entry in live cells, we used Huh-7.5CD81-GFP cells, enabling visualization of basolateral and apical domains. Huh-7.5-CD81-GFP organoids were infected with DiD-HCV and imaged every 15–30 s to capture DiD-HCV interactions with the organoid. DiD-HCV particles demonstrate heterogeneous behavior. On the basal face, particles displayed a range of movement, including static, random, and processive (Figures S3A and S3B; Movie S1). We observed virions moving basal to lateral, along lateral faces, and lateral to tight junction transitions (Figures S3C–S3E; Movies S2 and S3). Particles accumulated at the tight junction over time (Figure S3F; Movie S4) as with the fixed samples (Figure 2C). These observations support the conclusion that HCV traffics along the basolateral membrane to the tight junction.

Localization of DiD-HCV with Its Entry Factors

We next determined the membrane localization of DiD-HCV with its entry factors. We hypothesized that early entry factors, such as CD81 and SR-B1, should initially colocalize with HCV at the basolateral membrane, as it is the first (and only) accessible cellular surface to extracellular virus. Additionally, CD81 should maintain DiD-HCV colocalization throughout the entry process, as we previously observed CD81 co-trafficking and internalization with DiD-HCV in 2D Huh-7.5 cells (Coller et al., 2009). We repeated the DiD-HCV time course of infection, probing with indicated entry factor antibodies. Samples were imaged and quantified for DiD-HCV entry factor colocalization at the basolateral and internal (tight junctional) domains. We observed high colocalization of DiD-HCV with CD81, SR-B1, and EGFR at both the external basolateral membrane and interior domains (Figure 3A). 73%–98% of DiD-HCV was localized with the individual entry factors at the basolateral and internal membranes (Figure 3C). This suggests that an HCV receptor complex containing SR-B1, CD81, and EGFR forms at the basolateral membrane and then migrates to the tight junction.

Figure 3. Colocalization of DiD-HCV with Entry Factors.

(A and B) Huh-7.5 organoids were infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for 0 to 90 min, fixed, and probed for CD-81, SR-B1, or EGFR (A) or CLDN1 or OCLN (B). Nuclei were stained with DAPI (blue). Arrows indicate the portion of the image enlarged in insets. Dotted arrow or arrowhead in SR-B1 panel indicates colocalization. Left: HCV entry factor (green); right: DiD-HCV (red). In images shown, all DiD particles colocalize with selected entry factor with the exception of OCLN, in which 3 of 4 particles colocalize. (C) Percent DiD-HCV colocalization with each entry factor at the basolateral or internal/tight junctional portion of the cluster as determined by z stack analysis. n values (total DiD signal) range from 81 to 276 for each sample; error bars, SD. ND, not detected.

We previously observed colocalization of DiD-HCV with CLDN1 outside of intracellular membranes in 2D Huh-7.5 cells (Coller et al., 2009), raising the possibility either that HCVCLDN1 interactions occur outside of the tight junction or, alternatively, that the 2D Huh-7.5 cells were not forming tight junctions (as we observed in Figure 1B). In probing the colocalization of DiD-HCV with CLDN1 and OCLN during infection of the hepatoma organoids, we did not detect any colocalization of DiD-HCV with CLDN1 or OCLN at the basolateral membrane (Figure 3C). However, we observed extensive colocalization of DiD-HCV with CLDN1 and OCLN in the internal, tight junction membrane (Figures 3B and 3C). These data demonstrate that the HCV association with CLDN1 and OCLN is restricted to the tight junction of polarized hepatocytes. It also suggests that CLDN1 and OCLN play direct roles in HCV entry.

DiD-HCV Accumulation at the Tight Junction Is Dependent on Actin

We previously defined a series of roles for actin in HCV entry into 2D Huh-7.5 cells (Coller et al., 2009). DiD-HCV traveled from filopodial projections to the plasma membrane via retrograde actin flow followed by actin nucleation and virion internalization in association with actin stress fibers. However, most DiD-HCV particles at the plasma membrane of 2D Huh-7.5 only colocalized transiently with actin and displayed primarily random movements that are characteristic of diffusion. This suggested that events driving active lateral movement of HCV at the plasma membrane failed to occur in unpolarized Huh-7.5 cells. In order to investigate the role of actin in polarized cell entry, we repeated the time course of HCV infection in hepatoma organoids and stained for F-actin. DiD-HCV localized with actin basolaterally, often in association with actin-rich clusters (Figure 4A, left panel, indicated by arrow). DiD-HCV also colocalized with actin filaments parallel to the basolateral membrane, which likely represent virions in transit to the tight junction (Figure 4A, middle panel). DiD-HCV displayed full colocalization with actin at the tight junction, a site of actin enrichment (Figure 4A, right panel, and 4B). We observed no global reorganization of the actin cytoskeleton during infection.

Figure 4. DiD-HCV Traffics in Association with Actin.

(A) Huh-7.5 organoids were infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for 0 to 90 min, fixed, and probed with phalloidin. Nuclei were stained with DAPI (blue). Representative images of actin colocalization with DiD-HCV localized to basolateral (left), actin filament (middle), and internal (right) portions of the cluster; insets displaying actin colocalization labeled with dashed arrows. Left: actin (green); right: DiD-HCV (red). (B) Percentage of DiD-HCV particles colocalized with phalloidin-labeled actinat each domain. (C) Huh-7.5 organoids were incubated for 1 hr with either 10 mM cytochalasin D or DMSO vehicle control. Cells were then incubated with DiD-HCV for 1 hr at 4C, shifted to 37°C for 90 min, fixed, and stained with phalloidin. DiD-HCV particles were then quantified for their localization within the cluster. n = total DiD signal; mean ± SD. ***p < 0.001. See also Figure S4.

To determine whether actin was required for DiD-HCV relocalization in hepatoma organoids, we pretreated cells with 10 mM cytochalasin D (CytoD), an inhibitor of actin polymerization, or DMSO control, infected with HCV, and stained for F-actin (Figure S4A). 88% of DiD-HCV particles accumulated at the internal membranes of DMSO-treated organoids, consistent with tight junction localization. In CytoD-treated organoids, there was a significant reduction of internally localized DiD-HCV (36% of total), with the majority of DiD-HCV remaining at the basolateral membrane (Figure 4C). Additionally, co-staining of DiD-HCV with CMFDA during the entry time course showed that CMFDA was retained at the bile canaliculus during HCV infection, indicating that tight junctional integrity is maintained during HCV entry (Figure S4B). The results indicate that DiDHCV localizes extensively with actin at all membranes and that the trafficking of DiD-HCV to the tight junction requires an intact actin cytoskeleton.

EGFR Is Required for DiD-HCV Internalization, but Not Migration to the Tight Junction

The role of EGFR in HCV entry is somewhat unclear. In unpolarized 2D Huh-7.5 cells, it was proposed that EGFR was required either for membrane diffusion of CD81 and subsequent CLDN1 association (Zona et al., 2013) or for HCV internalization (Diao et al., 2012). We first confirmed that EGFR signaling is intact in Huh-7.5 organoids. Treatment of the organoids with EGF resulted in phosphorylation of EGFR residues that was blocked by the EGFR inhibitor erlotinib (Figure S5C). We then examined the effect of erlotinib on DiD-HCV colocalization with CLDN1. In DMSO-treated cells, DiD-HCV colocalization with CLDN1 peaked 90 min after temperature shift and then decreased at 360 min, likely due to internalization and uncoating of DiD-HCV (Figures 5A and 5B). Erlotinib-treated cells showed similar DiDHCV colocalization with CLDN1 at 90 min post shift, indicating that EGFR signaling is not required for DiD-HCV trafficking to the tight junction and association with CLDN1 in hepatoma organoids. However, we did observe an obvious difference at 360 min post shift: DID-HCV remained colocalized with CLDN1 in the presence of erlotinib (Figures 5A and 5B). This indicates that erlotinib blocks the internalization and uncoating of DiDHCV and that DID-HCV remains associated with CLDN1 at the tight junction.

Figure 5. Inhibition of EGFR Signaling with Erlotinib Prevents Internalization, but Not Tight Junctional Localization, of DiD-HCV.

(A–D) Huh-7.5 organoids were incubated with 15 mM Erlotinib (Erl) or DMSO for 1 hr, infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for the indicated times, fixed, and probed for claudin-1 (A) or core (C). (A) Tight junction region is shown in the inset. Left: claudin (green), Right: DiD-HCV (red). (B) Quantitation of (A). (C) Arrows indicate DiD-HCV particles enlarged in insets; dashed arrows and arrowhead represent colocalization with core. Left: core (green); right: DiD-HCV (red). Lack of DiD-HCV colocalization with core antibody, seen in DMSO treated cluster at 360 min post shift, indicates an uncoating event. (D) Quantitation of (C). (E and F) Huh-7.5, shEGFR, or shEGFR+EGFR organoids were infected with DiD-HCV as above, fixed at indicated times, and stained for ZO-1. (E) Tight junction region is shown in the inset. Left: ZO-1 (green); right: DiD-HCV (red). (F) Quantitation of (E). n = total DiD signal, mean ± SD. **p < 0.01, ***p < 0.001. See also Figure S5.

We tested this possibility by analyzing the effects of erlotinib in our previously published DiD-HCV uncoating assay (Coller et al., 2009). Intact virions are visualized as a colocalization of DiD with core, while DiD in the absence of core signifies an uncoated particle, in which the lipophilic dye has intercalated the endosomal membrane, where fusion has occurred. We examined the effects of erlotinib on HCV uncoating over a time course of infection. At early time points (0 and 90 min post temperature shift), virtually all DiD-HCV colocalized with core, indicating that they are intact virions (Figures 5C and 5D). At 360 min post shift, only 16% of DiD-HCV and core colocalized in DMSO-treated cells, indicating that the majority of DiD-HCV had undergone uncoating. In contrast, we observed high levels of DiD-HCV and core colocalization in erlotinib-treated cells throughout the time course, indicating that virions did not uncoat. In parallel, we confirmed that erlotinib treatment inhibited HCV replication in hepatoma organoids without impacting cell viability (Figures S5A and S5B). Thus, erlotinib prevents DiD-HCV internalization at the tight junction.

As erlotinib is not a highly selective EGFR inhibitor, we confirmed a role for EGFR in HCV internalization using a genetic approach. We created a Huh-7.5 cell line expressing EGFR shRNA (shEGFR) and a cell line that was virally transduced to complement this defect via an shRNA-resistant EGFR (shRNA+EGFR) (Figure S5D). The shEGFR cell line was defective in HCV entry in that HCV RNA and infectious virus production was decreased following HCV infection; however, when entry was bypassed via electroporation of HCV RNA, infectious virus production was unaffected. The restoration of EGFR expression via shRNA-resistant EGFR rescued the entry defect (Figures S5E–S5G). We next infected Huh-7.5 or shEGFR organoids with DiD-HCV and analyzed the colocalization of DiD-HCV with the tight junction marker ZO-1 over a time course of infection. Similar to erlotinib treatment of Huh-7.5 cells, we observed that DiD localized to the tight junction of shEGFR cells but failed to internalize (Figures 5E and 5F).

EGFR Is Associated with and Required for Internalization of the Viral Particle via ClathrinMediated Endocytosis

As EGFR signaling is required for HCV entry, we investigated whether activated, phosphorylated EGFR is associated with DiD-HCV. After extended serum starvation, organoids were infected with DiD-HCV over a time course, fixed, and probed for either EGFR or phospho-EGFR (at amino acid 1045). DiD-HCV colocalized with EGFR throughout the time course, while it preferentially colocalized with phospho-EGFR at later time points of infection (90 and 120 min) (Figures 6A and 6C). While DiD-HCV colocalized with EGFR at both the external basolateral and the internal membrane domains, phospho-EGFR colocalization with DiD-HCV particles was generally restricted to internal membrane domains near the tight junction (Figures 6D and 6E). DiDHCV/phospho-EGFR colocalization increased over time, with phospho-EGFR forming cups underneath DiD-HCV particles (Figure 6B). Internalized particles can also be seen to localize within phospho-EGFR-containing vesicles (Figure 6A). We then validated the EGFR phosphorylation kinetics by immunoblot. EGFR phosphorylation of tyrosines 1045, 1148, and 1173 peaks at 90–150 min after infection, while organoids incubated with mock-treated media showed no increase in EGFR activation (Figure 6F). Thus, although DiD-HCV colocalizes with EGFR at the basolateral membrane, EGFR is selectively activated following HCV receptor accumulation at the tight junction. The kinetics of EGFR phosphorylation mirror the kinetics of HCV internalization (and not earlier time points associated with DiDHCV trafficking to the tight junction).

Figure 6. Activated EGFR Is Associated with DID-HCV at the Tight Junction prior to Internalization.

(A–E) Huh-7.5 organoids were infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for the indicated times, fixed, and probed for either total EGFR or phosphoEGFR 1045. (A, B, and D) Arrows indicate area enlarged in insets; dashed arrows indicate DiD colocalization while solid arrows denote DiD-HCV particles lacking colocalization. (A) DiD-HCV colocalization over a time course of infection. (B) DiD-HCV colocalization with phospho-EGFR at 120 min post shift. Lower inset is merged and enlarged in upper inset. (D) DiD-HCV colocalization at 0 min post shift, analyzed for localization of DiD-HCV particles. (C and E) Quantification of (A) and (C), respectively. (F) Huh-7.5 organoids were serum starved, infected with concentrated HCV for 1 hr at 4C, shifted to 37°C, processed with Matrigel cell recovery solution, and lysed at the indicated times. Lysate samples were immunoblotted for the specified proteins. (G) Wild-type, shEGFR, and shEGFR cells expressing wild-type or mutant EGFR were seeded into 6-well plates, lysed, and analyzed via immunoblot with indicated antibodies. (H) Wild-type, shEGFR, or complemented cells were seeded onto 96-well plates, infected with HCV for 48 hr, and then analyzed for relative HCV RNA levels. n = total DiD signal, mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

We next mutagenized the tyrosines to alanine to determine whether they were required for HCV infection. shRNA-resistant wild-type or mutant EGFR was used to complement the shEGFR organoids via lentiviral transduction. The cells were then infected with HCV and assayed for HCV replication. The shEGFR cells had decreased HCV replication, which was rescued by the wild-type EGFR and EGFR mutated at tyrosine 1045 (Figure 6H). EGFR with mutated tyrosine 1148 or 1173 failed to complement HCV replication, indicating that they are required for HCV infection (Figure 6H). The difference in HCV RNA levels was not due to differential EGFR expression (Figure 6G).

We next investigated the function of EGFR in HCV internalization. DiD-HCV enters 2D Huh-7.5 cell via clathrin-mediated endocytosis to early endosomes (Coller et al., 2009); however, it remained a formal possibility that polarization might alter this process. Huh-7.5 organoids were infected with DiD-HCV particles over a time course and probed for clathrin light chain (LC), a component of clathrin triskelions. At the temperature shift (t = 0), some DiD-HCV-clathrin-LC colocalization was evident (Figures S6A and S6B), mostly occurring basolaterally. At 90 min post shift, when most DiD-HCV particles are localized to the tight junction, clathrin LC association with DID-HCV particles increased, suggesting the initiation of clathrin-mediated endocytosis. Colocalization of DiD-HCV and clathrin LC decreased at 360 min, a time at which most DiD-HCV particles have uncoated (Figures S6A and S6B).

EGFR utilizes clathrin-mediated endocytosis during its internalization (McMahon and Boucrot, 2011); we therefore asked whether EGFR activation is required for recruitment of the clathrin machinery to DiD-HCV puncta. We performed a time course of infection in the presence of erlotinib or DMSO and then probed for clathrin LC. DiD-HCV had increased colocalization with clathrin LC, which was abrogated by erlotinib treatment (Figures 7A and 7B). Similarly, shEGFR cells were defective in clathrin LC colocalization with DiD-HCV as compared to the parental Huh-7.5 cells. (Figures 7C and 7D). This suggests that EGFR activation is required for the recruitment and assembly of clathrin components during HCV clathrin-mediated endocytosis.

Figure 7. HCV Requires EGFR for Colocalization with Clathrin and Rab5a-Positive Endosomes.

Huh-7.5 organoids were incubated with either erlotinib or vehicle control (VC) for 1 hr, if indicated, infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for stated times, fixed, and probed for clathrin light chain (clathrin LC) (A and C) or Rab5a (E), both in green. Arrows mark areas enlarged in insets; dashed arrows and arrowheads denote colocalization with labeled host factor. (C) Organoids seeded with Huh-7.5 or Huh-7.5-shEGFR cells were infected with DiD-HCV as above, fixed, and stained for clathrin LC. (B, D, and F) Quantitation of (A), (C), and (E), respectively. n = total DiD signal; error bars, SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6.

To determine whether DiD-HCV internalizes to early endosomes in an EGFR-dependent manner, we infected the Huh7.5 organoids with DiD-HCV over a time course in the presence of DMSO or erlotinib and probed for Rab5a colocalization. We observed minimal colocalization of DiD-HCV and Rab5a at t = 0, which is expected since infection at 4°C prevents particle internalization. Rab5a-DiD-HCV colocalization peaked at 150 min and then decreased somewhat over time. (Figures 7E and 7F). Erlotinib prevented DiD-HCV/Rab5a colocalization at all time points, indicating that EGFR is required for internalization of DiD-HCV into early endosomes.

Ammonium chloride (NH₄Cl), which inhibits endosomal acidification, has been shown to block HCV entry (Tscherne et al., 2006) and prevents DiD-HCV uncoating (Coller et al., 2009). To investigate whether HCV uncoating requires endosomal acidification in polarized cells, we repeated the DiD-HCV uncoating assay in the presence and absence of NH₄Cl. In the absence of NH₄Cl, DiD-HCV colocalized with core at 0 and 150 min and then displayed decreased colocalization at 360 min, indicating that most particles have uncoated (Figures S6C and S6D). However, in NH₄Cl-treated cells, DiD remained colocalized with core throughout the time course, indicating that uncoating of DiDHCV requires endosomal acidification (Figures S6C and S6D). We validated that NH₄Cl inhibits HCV replication in hepatoma organoids without impacting cell viability (Figures S6E and S6F). Thus, HCV uncoating and replication in polarized 3D Huh-7.5 cells require endosomal acidification.

DISCUSSION

HCV entry is an unusually complex process with many distinct host cofactors that either directly or indirectly modulate it. The differential subcellular localizations of these cofactors highlight the importance of cell polarity in studying HCV entry. Primary hepatocytes, while potentially providing the closest proxy for the in vivo viral life cycle, are highly heterogeneous and irregular, do not support robust levels of viral replication, and often have a short cell culture lifespan (Bartenschlager and Lohmann, 2001; Wilson and Stamataki, 2012). In contrast, Huh-7.5 hepatoma cells that are widely used in HCV research support robust levels of HCV replication. Although when embedded in Matrigel, they produce ideal in vivo-like polarity and architecture, it must be acknowledged that some gene expression/signaling pathways may be dysregulated in these cells (Durantel and Zoulim, 2007). However, as we show, EGFR signaling is functional in Huh-7.5 organoids (Figure S5C).

Polarized hepatoma organoids have many advantages over 2D Huh-7.5 cells in imaging HCV entry. (1) The entry cofactors have an appropriately restricted localization at their respective membrane compartments (Figure S1). HCV first encounters the ECM-associated basolateral membrane; virions in vivo most likely access hepatocytes’ basolateral surfaces in the context of stellate-cell-secreted ECM. (2) The organoids establish bona fide tight junctions as defined by the secretion and retention of bile analogs at the apical bile canaliculus (Figure 1F). This restricts the virions’ access to tight junctional entry cofactors CLDN1 and OCLN in the absence of virally induced signaling, replicating conditions in vivo. (3) The highly distinct localization patterns of the early basolateral receptors versus the tight junction entry factors enable a simplified interpretation of virus-receptor colocalization as compared to unpolarized cells, in which the receptors diffuse randomly and likely form spatiotemporally inappropriate complexes. (4) Our previous single-particle tracking studies of DiD-HCV movements at the plasma membrane of 2D unpolarized Huh-7.5 cells found that the majority of the virions randomly diffuse at the plasma membrane, indicating the absence of signaling processes to direct virion migration (Coller et al., 2009). In the polarized organoids, virions actively travel 25 mm in the organoids from the basolateral membrane to the tight junction in association with actin, indicating an active signaling process to drive HCV receptor lateral migration. (5) Finally, we anticipate that properly polarized hepatoma cell models will reveal important differences at other stages of the viral life cycle. For instance, the Huh-7.5 organoids also have a polarized secretory pathway that likely impacts viral egress (data not shown).

Single-particle imaging of HCV infection of the polarized hepatoma organoids presented an opportunity to directly test models of HCV entry. Evans et al. (2007) identified CLDN1 as a late HCV entry factor and proposed that HCV may migrate to the tight junction following interactions with SR-B1 and CD81. This model is conceptually similar to the entry pathway of Coxsackie virus B3 (Coyne and Bergelson, 2006). Alternatively, HCV could disrupt the tight junction to gain access to CLDN1 or OCLN. Since a physical interaction of HCV virions or structural proteins with CLDN1 and OCLN has not been demonstrated, it was also possible that HCV has an indirect requirement for CLDN1 and OCLN in HCV entry. We find that, indeed, HCV virions traffic to the tight junction in a manner that requires CD81 (Figures 2 and S3). Initially, HCV colocalizes with its early receptors SR-B1 and CD81 in addition to EGFR (Figure 3). Physical interactions between CD81 and EGFR have been reported (Zona et al., 2013). We demonstrated that EGFR is also localized with the HCV receptor complex. The HCV receptor complex is detected at the basolateral membrane and at internal localizations consistent with the tight junction, indicating lateral movement of HCV receptors to the tight junction (Figure 3). This migration is associated with actin and requires actin polymerization (Figure 4).

Surprisingly, given prior reports, EGFR was not required for tight junction accumulation of DiD-HCV or CLDN1 colocalization, although it did inhibit HCV entry (Figure 5). This is in contrast to studies performed in unpolarized 2D Huh-7.5 cells that found that erlotinib inhibited CD81-CLDN1 interactions, as assayed via fluorescent resonance energy transfer (FRET) (Zona et al., 2013). It is difficult to interpret FRET experiments in unpolarized cells using overexpressed receptor-fluorescent protein fusions, however, since the receptors were not properly localized or studied in the context of HCV infection. In this study, EGFR is not phosphorylated with either the kinetics or the localization to influence HCV receptor migration to the tight junction (Figure 6).

It is clear that EGFR is required for HCV virion internalization and uncoating (Figures 5, 6, 7,and S6). DiD-HCV remained localized at the tight junction with ZO-1 for R6 hr in the absence of EGFR (Figure 5). This suggests that HCV, SR-B1, and CD81 bring EGFR to the tight junction to stimulate HCV receptor internalization at the tight junction. EGFR is known to be co-opted by other viruses to induce internalization. Larger viruses, such as vaccinia and respiratory syncytial virus, utilize EGFR-based macropinocytosis for entry (Krzyzaniak et al., 2013; Mercer et al., 2010); influenza A virus uses an as-yet-unidentified mechanism for EGFR-based internalization (Eierhoff et al., 2010). Furthermore, we found that phosphorylation of EGFR residues 1143 and 1173 are critical for HCV infection. This is consistent with data implicating components of the MAPK pathway in HCV entry (Lupberger et al., 2011).

Based on these and other data, we propose a revised model for HCV entry into polarized hepatocytes (Figure S7). HCV first associates with hepatocytes via interactions of the virion-associated ApoE with attachment factors, including LDLR, heparan sulfate, and syndecan-1 (Barth et al., 2003; Jiang et al., 2012; Owen et al., 2009). Once the virion is associated with its attachment factors, HCV E2 binds SR-BI and then CD81 (Pileri et al., 1998; Scarselli et al., 2002); the HCV receptor complex also associates with EGFR at the basolateral membrane, likely via the CD81-EGFR interaction. This complex then migrates to the tight junction in an actin-dependent, but EGFR-independent, fashion. Once at the tight junction, HCV encounters the late receptor CLDN1. The interaction of CD81 and CLDN1 localizes the HCV receptor complex in proximity to OCLN, which is likely associated with internalization in concert with EGFR. We speculate that HCV interaction with its early receptors produces a conformation that enables a direct E1/E2-OCLN interaction. Alternatively, HCV may not need to directly interact with EGFR or CLDN1, since CD81 is capable of binding them in the absence of HCV infection (Diao et al., 2012). EGFR is selectively activated at or near the tight junction. EGFR helps recruit clathrin-coated vesicles, which internalizes HCV and then matures into early endosomes. Following endosomal acidification, the virion uncoats, releasing its genome into the cytoplasm.

At least one important question remains in regard to this model: which signaling pathways, if not EGFR, stimulate HCV receptor migration to the tight junction?

STAR+METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Prof. Glenn Randall, PhD (grandall@bsd.uchicago.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell Culture

Huh-7.5 cells (Blight et al., 2002) were maintained in Dulbecco’s modified high glucose media (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 0.1 mM nonessential amino acids (GIBCO), and 1% penicillin-streptomycin (GIBCO). Cells were grown at 37C with 5% CO₂. To induce polarization, Huh-7.5 cells were embedded in Matrigel (Growth Factor Reduced, Phenol Red-free; Sigma). Matrigel was first thawed on ice. Huh-7.5 cells were then trypsinized and diluted in DMEM + 10% FBS to a final concentration of 1 ×10⁵ cells/mL. Equal volumes of Matrigel and diluted cells were mixed and seeded into plates. The Matrigel solution was allowed to polymerize for 30 min at 37C before adding DMEM + 10% FBS to cover. Cells were cultured for 6–8 days, changing media every other day.

Pseudoparticle Transduction

A construct containing shEGFR, pLKO.1-puro shEGFR, was obtained from Sigma (SCHLDN MISSION shRNA DNA clone Oligo TRCN0000010329). pBABE-EGFR (Addgene plasmid # 11011) was a gift from Matthew Meyerson (Greulich et al., 2005). EGFR and its mutants were moved into the pLVX construct for increased transduction efficiency.

pBABE-EGFR was digested with BglII and SalI. pBABE-EGFR was used as a template for two separate, overlapping PCR reactions: 1) BglII forward primer (5′- AAT CCC TGCC AGC GAG ATC TC) and mutation reverse primer; (2) mutation forward primer and SalI reverse primer (5′- ACA CAT TCC ACA GGG TCG ACC). Primers to create the mutations on the EGFR background are as follows: Y1045F reverse (5′- TCT GAG CTG AAT CGC TGC AAG AAG), Y1045F forward (5′- CAG CGA TTC AGC TCA GAC CCC AC), Y1148F reverse (5′- TCC TGC TGG AAG TCA GGG TTG TCC), Y1148F forward (5′- CCC TGA CTT CCA GCA GGA CTT CTT TC), Y1173F reverse (5′- CCC TTA GGA ATT CTG CAT TTT CAG CTG TG), Y1173F forward (5′- GCA GAA TTC CTA AGG GTC GCG C). The two amplified segments were inserted into the digested pBABE vector via the In-Fusion Cloning kit. For insertion into pLVX, the vector was digested with EcoRI and XbaI. Wild-type and mutant EGFR constructs were amplified with pLVX overhangs, forward primer (5′- GGA TCT ATT TCC GGT GAA TTC ATG CGA CCC TCC GGG ACG G) and reverse primer (5′- ATC CGC GGC CGC TCT AGA TCA TGC TCC AAT AAA TTC ACT GCT TTG TGG C), then amplified fragments were inserted into the digested pLVX vector with the In-Fusion cloning kit.

To produce retroviral stocks, 293T cells were transfected with MMLV gag-pol, vesicular stomatitis virus glycoprotein G, and either the shEGFR or EGFR plasmids. Plasmids were transfected using Lipofectamine 2000 (Thermo Fisher), as per manufacturer’s guidelines. Supernatants were harvested 48 hr post-transfection, filtered through a 0.22 mM filter, then used to transduce cells. Huh-7.5 cells were incubated with pseudoparticles and 8 mg/mL polybrene for 5 hr. Cells transduced with shEGFR were puromycin selected, then expanded from single cell clones. For the complemented cell line, shEGFR stable cells were transduced with EGFR pseudoparticles and polybrene. Cells were probed for EGFR expression via western blot to check for successful complementation. For live cell imaging, cells were transduced with CD81-GFP lentivirus (Coller et al., 2009).

METHOD DETAILS

Highly Infectious Virus Preparation

Stocks of HCV genotype 2a RNA (infectious clone pJFHxJ6-CNS2C3) were generated as previously described (Coller et al., 2009, 2012; Mateu et al., 2008). Briefly, viral supernatants were collected for up to 5 passages after electroporation, then filtered through a 0.22 micron nitrocellulose filter and stored at 4°C protected from light. Viral titer was determined via limiting dilution and subsequent immunohistochemical staining with a monoclonal NS5A antibody (9E10, generous gift of Charles Rice, Rockefeller University) as described (Randall et al., 2006).

Viral stocks were concentrated via PEG (polyethylene glycol 8000; Fisher) precipitation (Blight et al., 2002; Coller et al., 2009). Viral supernatant was mixed with PEG (final concentration 8%) and incubated overnight at 4C. Following centrifugation (20 min, 8000xg), pellet was resuspended in 15 mLs of the original supernatant; resuspended sample was spun down again (15 min, 8000xg) and pellet was resuspended in supernatant for a final concentration of 1/100 of the starting volume. 5 mL of DiD (Invitrogen), was added to 1 mL concentrated virus and incubated for 90 min with shaking, protected from light. Labeled virus was layered onto a 10%–60% weight/volume iodixanol gradient (OptiPrep, Sigma) in sterile water and centrifuged for 16.5 hr (34,000 RPMs at 4C). The gradient was separated into 1 mL fractions; each fraction was subsequently analyzed for HCV RNA levels (following Trizol-LS extraction; Invitrogen) and infectious virus (as described in materials and methods). Fractions with the best specific infectivity were added to Amicon Ultra 100k filters (Millipore) and spun for 20 min at 14000xg. Filters were then inverted in a new tube and spun for 2 min at 2000xg. The resulting supernatants were pooled for use in imaging studies.

Cell Recovery from Matrigel

Matrigel-polarized cells were harvested with Matrigel cell recovery solution (Corning). Briefly, cells were shaken for 1 hr with 500 mL cell recovery media, then centrifuged at 300 g for 5 min. Supernatant was removed, and the pellet was washed twice in PBS.

HCV RNA Quantitation

Following cell recovery, RNA was extracted using RNeasy 96 kit (QIAGEN); RNA copy number was determined via quantitative realtime RT-PCR as previously described (Randall et al., 2007) HCV and 18S RNA copy numbers were determined via comparisons to concentration standards. Absolute HCV RNA was normalized to the sample’s 18S RNA, then to the (normalized) 6-hr vehicle control for relative HCV RNA levels.

Inhibitors

CD-81 blocking antibody (JS-81, BD PharMingen) and its mouse IgG1 isotype control (Novus Biologicals) were used at 10 mg/mL. Inhibitor concentrations were as follows: 10 mM Cytochalasin D (Sigma), 15 mM Erlotinib (Santa Cruz) and 20 mM Ammonium Chloride (Fisher). Chemical inhibitors used in HCV replication analysis were incubated with Matrigel-embedded cells 5 days post-plating for 2 hr prior to addition of HCV (MOI = 3).

Electron Microscopy

Concentrated virus was labeled, gradient purified, and concentrated with Amicon filters as described above. Resulting supernatant was mixed with equal parts 4% paraformaldehyde. Sample was pipetted onto 400 mesh carbon-coated glow-discharged gold grids. Grids were soaked in PBS for 20 min, blocked with 1% BSA in PBS for 20 min, then incubated with anti-E2 CBH5 (diluted 1:100 in block solution) in humidified chamber for 3.5 hr. Grids were then washed six times (10 min each) in PBS, then blocked in 0.5% BSA in PBS for 20 min. Grids were probed with 12 nm colloidal gold-conjugated anti-mouse IgG secondary antibody (Jackson Immuno Research; diluted 1:10 in 0.5% BSA in PBS) in humidified chamber for 1 hr. Grids were washed three times with PBS (10 min each), fixed in 1% glutaraldehyde in PBS for 10 min, then washed three times with water (5 min each). Grids were stained with 1% uranyl acetate and air-dried. Samples were examined under 300kV with an FEI Tecnai F30 transmission electron microscope.

Infectious Time Course and Fixed Cell Immunofluorescence Microscopy

Matrigel-cell mixtures were prepared as described above, and 75 mL of the solution was placed onto coverslips in 24-well plates. Matrigel-embedded samples were preincubated on ice for 20 min. If infected, DiD-labeled HCV, mixed 1:1 with supplemented DMEM + 10% FBS, was added to cells and incubated, covered, on ice for an additional hour. Cells were then transferred to 37C (time of temperature shift: t = 0) and fixed at various points after the shift in 3.6% paraformaldehyde (PFA) for 20 min at room temperature. Cells were permeabilized with room temperature 0.5% Triton x-100 in PBS for 10 min, and then rinsed with 0.1 M Glycine in PBS 3 times for 10 min each. Cells were incubated for 2 hr in blocking solution (0.1% BSA, 0.2% Triton x-100, 0.005% Tween-20, and 20% goat serum in PBS). Coverslips were incubated overnight at 4°C with primary antibodies diluted in blocking solution. (1:500 anti-ZO-1, Invitrogen; 1:1000 anti-Na⁺K⁺-ATPase, Abcam; 1:400 anti-MRP2, Abcam; 1:1000 anti-NS5A 9E10; 1:300 anti-CD81 sc-7637, Santa Cruz; 1:300 anti-SR-B1 NB400–101, Novus; 1:400 anti-EGFR sc-120, Santa Cruz; 1:400 anti-OCLN, Invitrogen; 1:100 anti-CLDN1 sc-81796, Santa Cruz; 1:350 anti-Core, Virostat; 1:200 anti-Clathrin LC, Santa Cruz; 1:900 anti-Rab5a, Abcam; 1:75 anti-phospho-EGFR 1045, Cell Signaling) Following overnight incubation, the Matrigel was allowed to reform for 10 min at room temperature without shaking. Coverslips were washed 3 times, 20 min each, with wash buffer (0.1% BSA, 0.2% Triton x-100, and 0.005% Tween-20 in PBS). Alexa Fluor conjugated secondary antibody (488 or 594) was diluted 1:1000 in blocking solution and incubated with the Matrigel-embedded cells for 1 hr at room temperature, then rinsed 3 times with wash buffer (as above). Actin staining utilized the same protocol (PFA fixation, permeabilization, glycine wash, and block), followed by incubation for 1 hr at room temperature with Alexa Fluor 488 Phalloidin (1:40, Invitrogen), then washed 3 times in wash buffer. CMFDA labeling (Invitrogen) occurred prior to fixation: CMFDA was incubated with the cells (1:1000) for 1 hr, then washed and incubated for an additional hour with DMEM +10% FBS. Cells were then fixed and stained as above. For all samples, coverslips were mounted with ProLong Gold AntiFade with DAPI nuclear stain (Invitrogen) following the final rinse.

For immunostaining of DiD-HCV particles, purified particles were added to poly-lysine treated coverslips and incubated at 37°C for 2 hr, then fixed in 3.6% paraformaldehyde for 30 min. Samples were washed with PBS, permeabilized in PBS with 0.2% Triton x-100 for 15 min, washed in PBS containing 0.1% Tween 20 (PBS/Tween), then blocked with 10% goat serum in PBS/Tween for 1 hr. Coverslips were incubated for 1 hr with primary antibody in blocking solution (1:100 anti-core, Virostat; 1:100 anti-E2 CBH5; 1:250 anti-Apo-E, Abcam). Following incubation with primary antibodies, samples were washed twice with PBS and incubated with fluorescently conjugated secondary antibodies at 1:1000 in blocking solution (488 or 350, AlexaFluor) for 1 hr. Coverslips were washed three times with PBS, then mounted with ProLong Gold AntiFade (Invitrogen).

Live Cell Imaging

Huh-7 cells expressing CD81-GFP were mixed with Matrigel; 400 mL of the solution was seeded into a 48-well plate and maintained as described. After 7 days, organoids were extracted from Matrigel and resuspended in 1 mL imaging media containing DMEM-F12 without phenol red (GIBCO) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 0.1 mM nonessential amino acids (GIBCO), 1% penicillin-streptomycin (GIBCO), and 50 mM HEPES (GIBCO). Organoids were then replated onto collagen-treated 35 mm imaging dishes with interlocking lids (Ibidi), 200 mL of the cell solution per plate. Imaging media was added to the dishes, and organoids were allowed to adhere for 6–8 hr. Just prior to imaging, DiD-labeled HCV was added to imaging dishes. Dishes were incubated on ice for 1 hr, then placed on an enclosed stage heated to 37°C on a Leica SP5 Tandem Scanner Spectral 2-Photon confocal microscope.

Cells were visualized using a 63x NA 1.4 oil-immersion objective. CD81-GFP was imaged with an Argon laser and a HyD detector set for the 495–535 nm wavelength range; DiD was imaged with a HeNe laser and a HyD detector using the 600–670 nm wavelength range. Videos were acquired through sequential exposures every 30 s up to 90 min post-temperatures shift. Images were processed using ImageJ.

Cell Viability Assay

Cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Results were normalized to vehicle control in chemical inhibitor studies.

Western Blot Analysis

Unpolarized cells were serum starved for 4 hr, then treated (if indicated) with 200 nM EGF (Life Technologies) for 30 min before lysis. For immunoblot analysis in polarized cells, Huh-7.5 cells were seeded into Matrigel (500 mL/well) and cultured for 8 days as described above. Cells were serum starved 8 hr prior to experiment. Cells were infected with PEG-concentrated HCV, then incubated on ice for 1 hr before transferring to 37°C incubator. 1 hr prior to lysis, cells were extracted from Matrigel (as described above). Following the last wash, supernatant was removed, and the pellet was lysed

All cells were lysed in 100 mL 5% NP40 buffer (150 mM NaCl, 20 mM Tris-HCL [pH 7.5], 10% glycerol, 2 mM EDTA) supplemented with 1 mM protease inhibitors (cOmplete Mini, Roche) and 1 mM sodium orthovanadate (Fisher). Proteins were separated on a 4 to 20% SDS-PAGE gel (BioRad) and transferred to PVDF. Membrane was incubated in blocking solution (10% BSA and 0.1% Tween-20 in 1xPBS), followed by overnight incubation at 4°C with primary antibodies (1:1000 anti-actin, Sigma; 1:500 anti-EGFR, Cell Signaling; 1:800 anti-phospho-EGFR 1045, Cell Signaling; 1:1000 anti-phospho-EGFR 1148, Cell Signaling; anti-1:1000 phospho-EGFR 1173, Cell Signaling) diluted in 5% BSA (0.1%Tween-20, 1xPBS). Blots were then incubated for 1 hr with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and rabbit anti-mouse, Thermo Fisher), followed by detection using SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher) and exposure to film.

QUANTIFICATION AND STATISTICAL ANALYSIS

Confocal Microscopy Analysis

Fixed cell imaging was performed on an Olympus DSU Spinning Disc Confocal with a 100X NA 1.45 oil-immersion objective. Using Slidebook imaging software, images were captured with a Hamamatsu back thinned EM-CCD camera set to an intensification of 255. DiD-labeled HCV and Alexafluor 594 were visualized with the DsRed filter set; Alexafluor 488 was visualized with the EGFP filter set. Z stacks of the organoids were acquired using slices taken every 0.3 mm. Following acquisition, images were processed with ImageJ (NIH). Z stacks were normalized on Slidebook, then imported using BioFormats (LOCI). DiD puncta were assayed for their localization within the organoid and/or colocalization with selected antibodies; ‘n’ values reflect the total number of DiD puncta quantified per treatment. Images were quantified for colocalization using RGB profiler (Christophe Laummonerie) and colocalization highlighter. Briefly, images were separated by channel, background was removed, and slices of the z stack were analyzed for colocalization via colocalization highlighter. Thresholds were standardized for each image set. Some puncta were further clarified (positionally) with RGB profiler. Images presented in the figures were duplicated out of the Z stack, separated into individual channels, adjusted for contrast and smoothed, then reassembled.

Statistical Analysis

Data shown as mean ± standard deviation. Statistical significance was determined using two-tailed Student’s t test. Details of statistical significance and n values can be found in the figures or corresponding figure legends.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-NS5A antibody (clone 9E10)	Laboratory of Charles Rice	N/A
Mouse monoclonal anti-CD-81 (clone JS-81)	BD PharMingen	Cat#555675
Mouse IgG1 Kappa Unconjugated Isotype Control	Novus Biologicals	Cat#NBA1–21302; RRID: AB_1660646
Mouse monoclonal anti-E2 (clone CBH5)	Laboratory of Steven Foung	N/A
Goat polyclonal 12 nm colloidal gold-conjugated anti-mouse IgG	Jackson ImmunoResearch	Cat#115–205-166; RRID: AB_2338734
Rabbit polyclonal anti-ZO-1	Invitrogen	Cat#18–7430; RRID: AB_2533048
Mouse monoclonal anti-Na+K+-ATPase (clone 464.6)	Abcam	Cat#ab7671; RRID: AB_306023
Mouse monoclonal anti-MRP2 (clone M2 III-6)	Abcam	Cat#ab3373; RRID: AB_303751
Mouse monoclonal anti-CD81(clone 1.3.3.22)	Santa Cruz	Cat#sc-7637; RRID: AB_627190
Rabbit polyclonal anti-SR-B1	Novus Biologicals	Cat#NB400–101; RRID: AB_10107658
Mouse monoclonal anti-EGFR (clone 528)	Santa Cruz	Cat#sc-120; RRID: AB_627492
Mouse monoclonal anti-Occludin (clone OC-3F10)	Invitrogen	Cat#33–1500; RRID: AB_2533101
Mouse monoclonal anti-Claudin1 (clone XX7)	Santa Cruz	Cat#sc-81796; RRID: AB_2083306
Mouse monoclonal anti-core	Virostat	Cat#1851
Mouse monoclonal anti-Clathrin LC (clone CON.1)	Santa Cruz	Cat#sc-12735; RRID: AB_627264
Rabbit polyclonal anti-Rab5a	Abcam	Cat#ab18211; RRID: AB_470264
Rabbit polyclonal anti-phospho-EGFR 1045	Cell Signaling Technology	Cat#2237; RRID: AB_331710
Rabbit polyclonal anti-ApoE (clone EP1374Y)	Abcam	Cat#ab52607; RRID: AB_867704
Rabbit polyclonal anti-actin	Sigma	Cat#A2066; RRID: AB_476693
Rabbit polyclonal anti-phospho-EGFR 1148	Cell Signaling Technology	Cat#4404; RRID: AB_331127
Rabbit monoclonal anti-phospho-EGFR 1173 (clone 53A5)	Cell Signaling Technology	Cat#4407
Rabbit polyclonal anti-EGFR	Cell Signaling Technology	Cat#2232; RRID: AB_331707
Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, rabbit anti-mouse)	Thermo Fisher Scientific	Cat#31460, RRID: AB_228341; 31455, RRID: AB_228418
Alexa Fluor secondary antibodies (488, 594)	Thermo Fisher Scientific	Cat#R37114, RRID: AB_2556542; R37118, RRID: AB_2556546; R37115, RRID: AB_2556543; R37119, RRID: AB_2556547
Bacterial and Virus Strains
HCV genotype 2a (infectious clone pJFHxJ6- CNS2C3)	Laboratory of Arash Grakoui	N/A
Chemicals, Peptides, and Recombinant Proteins
Matrigel	Sigma	Cat#E6909
Matrigel Cell Recovery Solution	Corning	Cat#354253
Lipofectamine 2000	Thermo Fisher Scientific	Cat#11668027
Vybrant DiD Cell-Labeling solution	Thermo Fisher Scientific	Cat#V22887
Cytochalasin D	Sigma	Cat#C8273
Erlotinib Hydrochloride	Santa Cruz	Cat#sc-202154
Ammonium Chloride	Fisher	Cat#AC199975000
Alexa Fluor 488 Phalloidin	Invitrogen	Cat#A12379
Cell Tracker Green CMFDA Dye	Thermo Fisher Scientific	Cat#C7025
EGF	Life Technologies	Cat#PHG0313
Critical Commercial Assays
RNEasy 96 kit	QIAGEN	74181
CellTiter-Glo Luminescent Cell Viability Assay	Promega	G7570
SuperSignal West Femto Maximum Sensitivity Substrate	Thermo Fisher Scientific	34095
Experimental Models: Cell Lines
Human: Huh-7.5 cells	Laboratory of Charles Rice; cell origin is of male gender	Accession#CVCL_7929
Human: 293T cells	ATCC; cell origin is likely of female gender	CRL-3216
Oligonucleotides
pBABE external primer, BglII forward: AAT CCC TGCC AGC GAG ATC TC	This paper	N/A
pBABE external primer, SalI reverse: ACA CAT TCC ACA GGG TCG ACC	This paper	N/A
Y1045F reverse primer: TCT GAG CTG AAT CGC TGC AAG AAG	This paper	N/A
Y1045F forward primer: CAG CGA TTC AGC TCA GAC CCC AC	This paper	N/A
Y1148F reverse primer: TCC TGC TGG AAG TCA GGG TTG TCC	This paper	N/A
Y1148F forward primer: CCC TGA CTT CCA GCA GGA CTT CTT TC	This paper	N/A
Y1173F reverse primer: CCC TTA GGA ATT CTG CAT TTT CAG CTG TG	This paper	N/A
Y1173F forward primer: GCA GAA TTC CTA AGG GTC GCG C	This paper	N/A
Insertion into pLVX forward primer: GGA TCT ATT TCC GGT GAA TTC ATG CGA CCC TCC GGG ACG G	This paper	N/A
Insertion into pLVX reverse primer: ATC CGC GGC CGC TCT AGA TCA TGC TCC AAT AAA TTC ACT GCT TTG TGG C	This paper	N/A
Recombinant DNA
pBABE-EGFR	Greulich et al., 2005	Addgene Plasmid #11011
pLKO.1-puro shEGFR	Sigma	SCHLDN MISSION shRNA DNA clone Oligo TRCN0000010329
pLVX	Clontech	pLVX-puro
gag-pol	Clontech	pLenti HTX gag/pol
Vesicular stomatitis virus glycoprotein G	Clontech	pLenti HTX VSV-G
CD81-GFP	Coller et al., 2009	CD81-GFP
Software and Algorithms
RGB Profiler	Christophe Laummonerie; ImageJ	https://imagej.nih.gov/ij/plugins/rgbprofiler.html
Colocalization Highlighter	Pierre Bourdoncle; ImageJ	N/A

Highlights.

• HCV entry can be imaged in three-dimensional hepatoma organoids

• HCV initially colocalizes with early receptors at the basolateral membrane

• HCV virions accumulate at the tight junction in an actindependent manner

• EGFR is required for internalization of HCV at the tight junction