In the present study, we developed a recombinant hepatitis B virus (rHBV) replicon in order to visualize virus-infected living cells via reporter gene expression. We first constructed an HBV replicon vector 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 provided in trans. However, the replication efficiency of ΔHBc113 vector was greatly impaired by insertion of a large foreign DNA fragment. Taking advantage of the property of intein-mediated protein splicing, we chose enhanced-green fluorescent protein (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 and sfGFPC11 rHBV replicons based on the ΔHBc113 vector, the latter of which was proved to be competent for 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 was 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 HBV infection mechanism study, but also have an extensive application prospect for high-throughput antiviral screening.
TIAN Qingyou
,
ZHU Yuanfei
,
CHANG Hao
,
YU Lin
,
DENG Qiang
. Establishment of a recombinant hepatitis B virus replicon expressing a split green fluorescent protein[J]. Journal of Microbes and Infections, 2021
, 16(2)
: 79
-87
.
DOI: 10.3969/j.issn.1673-6184.2021.02.002