Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 21, 2019; 25(39): 5961-5972
Published online Oct 21, 2019. doi: 10.3748/wjg.v25.i39.5961
Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines
Jie Ruan, Cai-Yan Ping, Shuo Sun, Xin Cheng, Peng-Yu Han, Dian-Xing Sun, Yin-Ge Zhang
Jie Ruan, Cai-Yan Ping, Shuo Sun, Xin Cheng, Peng-Yu Han, Yin-Ge Zhang, Dian-Xing Sun, The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Shijiazhuang 050082, Hebei Province, China
Jie Ruan, Department of Infection and Liver Disease, Shannxi University of Chinese Medicine Affiliated Hospital, Xianyang 712000, Shannxi Province, China
Author contributions: Sun DX developed the methodology and provided funding; Ruan J, Sun S, Cheng X, and Sun DX designed and coordinated the research; Ruan J, Ping CY, Sun S, Han PY, and Zhang YG performed the majority of the experiments and analyzed the data; Ruan J and Sun DX wrote the manuscript; Ruan J, Ping CY, and Sun S contributed equally to this work.
Supported by the National Natural Science Foundation of China, No. 81672041; and the National Major Science and Technology Special Project for Infectious Diseases of China, No. 2012ZX10004503-012.
Institutional review board statement: Because no animals and patients were involved in this study, the IRB chose to waive this requirement.
Institutional animal care and use committee statement: Because no animals were involved in this study, the Institutional Animal Care and Use Committee chose to waive this requirement.
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and because no animals were involved in this study, the ARRIVE guidelines are not applicable.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Dian-Xing Sun, PhD, Doctor, Professor, The Liver Disease Center of Chinese People’s Liberation Army, the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force, Zhongshanxi Street, Shijiazhuang 050082, Hebei Province, China. sundianxing@hotmail.com
Telephone: +86-13081100156
Received: May 20, 2019
Peer-review started: May 20, 2019
First decision: July 21, 2019
Revised: August 8, 2019
Accepted: September 13, 2019
Article in press: September 13, 2019
Published online: October 21, 2019
Processing time: 154 Days and 22.7 Hours
Abstract
BACKGROUND

Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research.

AIM

To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.

METHODS

We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.

RESULTS

The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity.

CONCLUSION

Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

Keywords: Hepatitis B virus; Replication-competent hepatitis B virus vector; Secreted luciferase gene; Hepatitis B virus cell line

Core tip: In this research, we constructed a replication-competent hepatitis B virus (HBV) vector carrying a secreted luciferase transgene and established HBV replication and expression cell lines that could stably secrete secretory Nanoluc Luciferase recombinant viral particles. This vector is a convenient and quantifiable tool for monitoring HBV replication, transcription, and expression. It is safe to assume that this new HBV replication system ought to be used in HBV molecular biology research, such as for discovering inhibitors that affect virus infection, entry, transcription, translation, reverse transcription, and replication.