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Fig. 1
A zebrafish model of liver-specific HCV sub-replicon. (A) Cartoon representation of the structures of the two HCV sub-replicon vectors. The HCV sub-replicon was composed of both vectors pmHNF-3N and pFLP-5B. pmHNF-rGC3N was constructed with a mouse HNF4a promoter sequence and a complementary cDNA sequence of gfp-IRES(EMCV)-core-5′UTR fused cDNA that was reversely inserted at the downstream of the mHNF promoter and followed by a sense HCV 3′UTR sequence. Vector pFLP-5BR was consisted with zebrafish liver-fab enhancer and human HL promoter sequence as the transcription regular elements, followed by HCV RNA polymerase cDNA (NS5B), IRES2 (EMCV), and RFP cDNA. (B) NS5B expression in the livers of zebrafish larvae injected with pFLP-5BR compared to uninjected wild type fish (WT). Four-days post fertilization larvae were detected using a white light, and fluorescent RFP filter (556 nm excitation, 586 nm emission) under a fluorescence microscopy. (C) Fluorescence image of a live zebrafish larva at 4 days post fertilization (dpf) showing liver-targeted expression of the HCV subreplicon. Photos taken under a fluorescence microscopy using a GFP filter (480 nm excitation, 505 nm emission) and a RFP filter (556 nm excitation, 586 nm emission). Green fluorescence indicates the HCV core protein and red fluorescence indicates the HCV NS5B protein. (D) Whole mount in situ hybridization was used to detect the HCV subgenome model. The probe core- (HCV core antisense RNA) identified the sense strand of HCV core RNA, representing a relative replication level of the HCV subreplicon; the core+ probe (HCV core sense RNA) identified the antisense strand of core RNA, representing a relative transcription level of the HCV subreplicon directed by the mHNF4a promoter; the ns5b+ probe identified antisense strand of ns5b RNA (as a negative control); the ns5b- probe identified sense strand of ns5b RNA transcribed by hHL promoter. Scale bars in D represent 150 μm. (E) RT-PCR for the HCV core+ replication and NS5B transcription in the HCV sub-replicon. core+, the sense strand of HCV core RNA, representing products of the HCV subreplicon replication. ns5b, ns5b transcription level. β-actin was used as a loading control. WT, wild type; both pmHNF-3N and pFLP-5B larvae were used as two negative controls for the HCV model, respectively. (F) Western blotting confirmed that NS5B protein was produced only in the larvae injected with the HCV model plasmids (co-injection of pmHNF-3N and pFLP-5B) and with NS5B expression vector.