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Abstract Abstract：Sodium taurcholate cotransport peptide (NTCP) has been identified as a functional receptor with high affinity for hepatitis B virus (HBV). Nevertheless, even in NTCP-reconstituted cells, the low-efficiency of in vitro HBV infection and spread remains a key challenge for the study of the post-entry events in HBV life cycle. In the present study, we developed a recombinant (r) HBV replicon in order to visualize virus-infected living cells via reporter gene expression. We first constructed an HBV replicon vector, i.e. HBV1.1-ΔHBc113, with partial deletion of the sequence encoding HBV core (HBc). The vector demonstrated a competent viral DNA replication in transfected cells, only if HBc was complemented in tans. However, the replication efficiency of ΔHBc113 vector was greatly impaired by insertion of full-length reporter genes being tested. Taking advantage of the property of intein-mediated protein splicing, we chose enhanced-GFP (EGFP) and super folder GFP (sfGFP) as exteins, and developed EGFPN1-8/EGFPC9-11 and sfGFPN1-10/sfGFPC11 split system, respectively. We further constructed EGFPC9-11 or sfGFPC11 recombinant HBV replicons based on the ΔHBc113 vector, the latter of which demonstrated an HBc-rescued, functional intracellular replication, and produced progeny virions in the culture medium. In cells co-expressing EGFPN or sfGFPN, rHBV replicon-expressed EGFPC9-11 or sfGFPC11 were capable of working with their counterparts, respectively, forming intact and functional GFP through intein-mediated protein splicing. We thus successfully developed a fluorescent rHBV replicon system, which can be not only used for cloning HBV-susceptible cell lines, but also have an extensive application prospect for high-throughput antiviral screening.
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Received: 29 January 2021
Published: 01 January 2021
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Corresponding Authors:
Qiang DENG
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