Isolation of HCV-JFH1-tau substrains permissive to CLDN1-defective S7-A cells
Previously, we demonstrated that the HCV-JFH1-tau substrain having K74T/I414T mutation has higher infectivity and shows cytopathic effects (CPE) on Huh7.5.1-8 cells, compared to the wild-type HCV-JFH116. We repeatedly passaged HCV-JFH1-tau substrains with Huh7.5.1-8 cells and stocked them at each passage. Surprisingly, we found that a multi-passaged HCV-JFH1-tau lot, designated as HCV-JFH1-tau-S, showed CPE on CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1 and HCV-JFH1-tau infection9,16. Cell viability using a tetrazolium-based colorimetric assay (MTT assay) was performed to confirm this effect (Fig. 1a). When Huh7.5.1-8 cells and S7-A cells were infected with original (parental) HCV-JFH1-tau, the viability of Huh7.5.1-8 cells was decreased in the viral dose-dependent manner; however, the viability of S7-A cells was not affected even at the maximum viral load. Conversely, when Huh7.5.1-8 cells and S7-A cells were infected with HCV-JFH1-tau-S, the viability of Huh7.5.1-8 cells and S7-A cells was similarly decreased in a viral dose-dependent manner. Then we checked the CPE of all passaged viral stocks on S7-A cells and got the first lot showing CPE on S7-A cells, designated as HCV-JFH1-tau Lot B. The lot just before HCV-JFH1-tau Lot B was named HCV-JFH1-tau Lot A (Fig. 1b), showing no CPE on S7-A cells. Since there was a possibility that HCV-JFH1-tau Lot B consists of various substrains containing original HCV-JFH1-tau, HCV-JFH1-tau Lot B was passaged four times with S7-A cells to purify a population permissive to S7-A cells. The passaged lot was named HCV-JFH1-tau Lot B1 (Fig. 1b). We then examined whether HCV-JFH1-tau Lot B1 is infectious to S7-A cells (Fig. 1C and Supplementary Fig. S1). Huh7.5.1-8, S7-A, and CD81-defective 751r cells were incubated with parental HCV-JFH1-tau or HCV-JFH1-tau Lot B1, and then HCV RNA contents in the cells (Fig. 1c) and supernatants (Supplementary Figs. S1a and S1b) were determined 1–3 days postinfection (p.i.). Huh7.5.1-8 cells were permissive to both HCV-JFH1-tau and HCV-JFH1-tau Lot B1 infection, and 751r cells were non-permissive to both virus infections, suggesting that HCV-JFH1 Lot B1 infection depends on CD81. In contrast, S7-A cells were non-permissive to HCV-JFH1-tau infection but permissive to HCV-JFH1-tau Lot B1 infection. Consistent with these results, immunoblot analyses using antibodies against HCV core and NS3 proteins (Supplementary Figs. S1c and S1d) and immunofluorescent microscopic analyses using an antibody against HCV core protein (Supplementary Figs. S1e and S1f.) showed that HCV-JFH1-tau Lot B1 could infect CLDN1-defective S7-A cells. We also determined viral titers of HCV-JFH1-tau Lot B1 using Huh7.5.1-8 and S7-A cells. The viral titer of HCV-JFH1-tau Lot B1 for S7-A cells was similar to those of HCV-JFH1-tau Lot B1 and parental HCV-JFH1-tau for Huh7.5.1-8 cells (Supplementary Table S1). These results indicated that HCV-JFH1-tau Lot B1 could infect cells without using CLDN1. We further verified the dependence of HCV-JFH1-tau Lot B1 infection on CLDN1 using previously developed anti-CLDN1 monoclonal antibodies (mAbs)9. When Huh7.5.1-8 cells were infected with HCV-JFH1-tau or HCV-JFH1-tau Lot B1 in the presence of anti-CLDN1 mAbs (clone 2C1 or 3A2), genomic RNA and protein productions of HCV-JFH1-tau were severely blocked by anti-CLDN1 mAbs, but those of HCV-JFH1-tau Lot B1 were not (Supplementary Fig. S2). From these results, we confirmed that HCV-JFH1-tau Lot B1 could infect cells, not via CLDN1.
We next evaluated the dependence of HCV-JFH1-tau Lot B1 on OCLN, another tight junction protein reported to be essential for infection with various HCV genotypes10,11,12. Huh7.5.1-8, S7-A, and OKH-4 cells, Huh7.5.1-8-derived OCLN-knockout cells11, were incubated with HCV-JFH1-tau or HCV-JFH1-tau Lot B1, and then cellular HCV RNA contents 4 days p.i. were determined by qRT-PCR. As previously reported16, HCV-JFH1-tau RNA was only detected in Huh7.5.1-8 cells. HCV-JFH1-tau Lot B1 RNA was detected in both Huh7.5.1-8 and S7-A cells but not in OKH-4 cells (Supplementary Fig. S3a). Data from immunoblot analyses of cellular HCV core and NS3 proteins were consistent with these results (Supplementary Fig. S3b). These results indicated that HCV-JFH1-tau Lot B1 infection as well as HCV-JFH1-tau infection depend on OCLN.
Determination of envelope amino acid residues in HCV-JFH1-tau Lot B1 involved in its infection of CLDN1-defective S7-A cells
Owing to the viral genomic mutations, HCV-JFH1-tau Lot B1 should acquire the CLDN1-independent infection (entry) phenotype. Thus, we first determined the nucleotide sequences for the envelope protein region of HCV-JFH1-tau Lot B1 and HCV-JFH1-tau Lot A, which is involved in viral entry steps. HCV-JFH1-tau, showing higher infectivity, has two amino acid mutations (K74T in the core protein and I414T in the E2 protein)16, compared to the original clinical isolate HCV-JFH1. HCV-JFH1-tau Lot A, exhibiting the same phenotype as parental HCV-JFH1-tau, had four additional nonsynonymous amino acid mutations: N234D and V293A in the E1 protein, as well as V402A and Y443H in the E2 protein (Table 1). HCV-JFH1-tau Lot B1 had additional eleven nonsynonymous amino acid mutations: A218G, T260A, Q296R, H316N, T331S, A351D, and I374V in the E1 protein, and V392A, A464D, M706L, and V724I in the E2 protein compared to HCV-JFH1-tau Lot A (Table 1).
Then we tried to identify the residues among eleven mutated amino acids in HCV-JFH1-tau Lot B1 responsible for the CLDN1-independent infection phenotype using an infection system where infection assays are performed by HCV-JFH1-tau encapsidated with exogenously supplemented HCV envelope proteins with mutations (HCVee) (Supplementary Fig. S4a).
We first evaluated whether the HCVee infection system functions well in our experimental conditions. Huh7.5.1-8 cells were infected with HCV-JFH1-tau at a multiplicity of infection (MOI) of 1 and cultured for 2 days. And then, infected cells were transfected with plasmids encoding envelope proteins from HCV-JFH1 or HCV-JFH1-tau Lot B1. After 3 days, the supernatant that may contain HCVee, produced from these infected cells, were collected and infected naive Huh7.5.1-8, S7-A, and 751r cells. HCVee infectivity was determined by immunohistochemistry using anti-HCV core protein mAb. HCV particles in the culture supernatant from the infected cells expressing HCV-JFH1-tau envelope did not infect S7-A cells. However, those in the culture supernatant from the infected cells expressing HCV-JFH1-tau Lot B1 envelope infected S7-A cells and Huh7.5.1-8 cells (Supplementary Fig. S4b). These results show that HCVee infection system is working well.
Next, to identify mutation sites involved in the CLDN1-independent infection by HCV-JFH1-tau Lot B1, we tested the infectivity of HCVee having Lot B1-type envelope proteins with each wild-type point mutation. When these eleven kinds of HCVee inoculated S7-A cells, Lot B1-based HCVee carrying wild-type point mutation: A218, T260, Q296, H316, T331, A351, T374, A464, or V724 could infect S7-A cells, but those carrying V392 or M706 reverting mutation were not (Supplementary Fig. S5 and Table 2). Furthermore, all HCVee tested were permissive to Huh7.5.1-8 cells but not to 751r cells (Supplementary Fig. S5). These results suggested that V392A and M706L mutations in HCV-JFH1-tau Lot B1 were involved in its CLDN1-independent infection.
Introduction of an M706L point mutation into HCV-JFH1 leads to the acquisition of CLDN1-independent infection phenotype
Conversely, we then tested whether introducing V392A and/or M706L mutations into HCV-JFH1 leads to the acquisition of infectivity to S7-A cells. HCV-JFH1-based HCVee with M706L or V392A/M706L mutation and Lot B1-based HCVee can infect S7-A cells, but HCV-JFH1-based HCVee with or without V392A mutation cannot (Fig. 2). These results revealed that the CLDN1-independent infection phenotype could be acquired by only M706L mutation in HCV-JFH1. To confirm these, we further examined the characteristics of V392A and M706L mutant viruses using the HCV pseudoparticle (HCVpp) system. HCV-JFH1-based HCVpps with M706L or V392A/M706L mutation could infect S7-A cells, whereas those with no mutation (wild-type) or V392A mutation could not; although all HCVpps tested can infect Huh7.5.1-8 cells (Fig. 3a). Moreover, infection of Huh7.5.1-8 cells with wild-type HCV-JFH1-based HCVpp was strongly inhibited by anti-CLDN1 mAb, whereas infection of Huh7.5.1-8 cells with HCV-JFH1-based HCVpp having M706L was not (Fig. 3b). These results using HCVpps were consistent with those using the HCVee infection system. Altogether we concluded that M706L mutation in HCV-JFH1 is essential for its CLDN1-independent infection phenotype.
Additionally, infection with HCV-JFH1-based HCVpp having M706L mutation, similar to that with wild-type HCV-JFH1-based HCVpp, showed a CD81-, SRBI-, and OCLN-dependent manner (Supplementary Fig. S6).
M706L mutation in HCV-JFH1 expands entry receptor dependency from CLDN1 to other CLDN family proteins: CLDN6 and CLDN9
What receptors, other than CLDN1, does HCV-JFH1-based HCVpp having M706L mutation use in the entry step? We had a hint through our trial experiments using M706L-carrying HCV-JFH1-tau Lot B1 and hepatic CLDN1-defective S7-A cells. When S7-A cells were infected with HCV-JFH1-tau Lot B1 with or without a broad CLDN binder, i.e., C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), which can bind several CLDNs such as CLDN3, CLDN6, CLDN7, and CLDN917,18, infection was completely blocked by C-CPE (Supplementary Fig. S7). These results strongly suggested that some CLDN family proteins other than CLDN1 are involved in its CLDN1-independent infection.
We then explored the dependency of HCVpp with M706L mutation on various CLDN family proteins. Human embryonic kidney 293 T (HEK293T) cells stably expressing either CLDN1, CLDN2, CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, or CLDN9 were infected with HCV-JFH1-based HCVpp without mutation (wild-type) or with M706L mutation. In Fig. 4, wild-type HCVpp can infect only CLDN1-expressing HEK293T cells, while HCVpp with M706L mutation can infect CLDN6- and CLDN9-expressing HEK293T cells as well as CLDN1-expressing cells. These results exhibited that HCVpp with M706L mutation can use CLDN6 and CLDN9 as an additional receptor.
In Huh7.5.1-8 cells, CLDN6 is expressed at the approximately 20% levels of CLDN1, but CLDN9 is expressed much less (< 1/10,000) than CLDN1 (Supplementary Table S2). CLDN1-defective S7-A cells have similar levels of CLDN6 and CLDN9 as Huh7.5.1-8 cells (Supplementary Table S2). Thus, CLDN6 is more likely than CLDN9 to contribute to M706L-carrying HCV-JFH1 infection in hepatic Huh7-derived cells. Indeed, infection with HCV-JFH1-based HCVpp having M706L mutation was prevented by more than 75% when CLDN6 was knocked down in S7-A cells by two types of CLDN6 siRNAs (Figs. 5a and 5b). Consistently, the treatment of S7-A cells with anti-CLDN6 antibodies resulted in more than 80% inhibition of infection with HCV-JFH1-based HCVpp with M706L mutation (Fig. 5c). In other hepatic cell line HUH-6 cells, CLDN6 is expressed more than tenfold higher than CLDN1, and the level of CLDN1 in HUH-6 cells is more than tenfold lower than that in Huh7.5.1-8 cells (Supplementary Table S2). Cell surface expression patterns of these CLDNs were also confirmed by flow cytometry analysis (Supplementary Fig. S8a). After infection with HCV-JFH1-tau or HCV-JFH1-tau Lot B1, viral production levels of both strains in Huh7.5.1-8 cells and the culture supernatants were similar, as already shown above in Supplementary Figs. S1a and S1b (white bars). Conversely, viral production levels of HCV-JFH1-tau Lot B1 in HUH-6 cells and the culture supernatants were significantly higher than those of HCV-JFH1-tau (Supplementary Figs. S8b and S8c). These results also agree with the finding that CLDN6 is involved in M706L-carrying HCV-JFH1 infection.
M706L-carrying HCV-JFH1 can infect non-hepatic iPS cells
CLDN6 is expressed mainly in stem cells, such as induced pluripotent stem (iPS) cells19,20. We also verified that 253G1 cell, a human iPS cell line established from adult dermal fibroblast, had a very high level of CLDN6 protein, compared with Huh7.5.1-8 cells (Fig. 6a). In contrast, 253G1 cells lacked the expression of CLDN1 protein, essential for general HCV infection. Other host proteins involved in HCV infection: SRBI, EGFR, low-density lipoprotein receptor (LDLR), OCLN, and CD81 were found in 253G1 cells, even though the expression levels of SRBI, EGRF, and LDLR were significantly lower than Huh7.5.1-8 cells (Fig. 6a).
We then addressed whether HCV-JFH1-tau Lot B1 with M706L mutation can infect iPS cells. HCV-JFH1-tau or HCV-JFH1-tau Lot B1 were inoculated into 253G1 cells, Huh7.5.1-8 cells, and OKH-4 cells, and cellular HCV RNA contents at 0.5–2 days p.i. were determined by qRT-PCR. Both HCV-JFH1-tau and HCV-JFH1-tau Lot B1 contents were time-dependently increased in the positive control Huh7.5.1-8 cells but not in the negative control OKH-4 cells (Fig. 6b). Interestingly, the HCV-JFH1-tau contents were background levels in 253G1 cells, but HCV-JFH1-tau Lot B1 contents in 253G1 cells were significantly high compared with those in the negative control OKH-4 cells, although the viral RNA levels in 253G1 cells were much lower than in Huh7.5.1-8 cells (Fig. 6b and supplementary Fig. S9). To further investigate whether HCV-JFH1-tau Lot B1 replicates in 253G1 cells, we examined the effect of HCV replication inhibitor, sofosbuvir, on HCV-tretaed 253G1 cells. As shown in Fig. 6c, HCV-JFH1-tau Lot B1 RNA contents in cells and culture supernatants were significantly reduced by sofosbuvir treatment in HCV-treated 253G1 cells as well as in HCV-tretated Huh7.5.1-8 cells. These results indicated that that HCV-JFH1-tau Lot B1 can infect and replicate in iPS cells. Consistent with these results, HCV-JFH1-based HCV pseudovesicles (HCVpv) with M706L mutation could infect iPS cells, but HCV-JFH1-based HCVpv with wild-type envelopes could not (Supplementary Fig. S10). Furthermore, infection of 253G1 cells with HCV-JFH1-tau Lot B1 having M706L mutation was blocked by anti-CLDN6 antibody as well as anti-CD81 and anti-OCLN antibodies (Fig. 6d). These results demonstrated that non-hepatic iPS cells could be infected with HCV-JFH1 substrain with M706L mutation via a CLDN6-dependent manner.