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, Yin-Ge Zhang, Dian-Xing Sun
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
ARTICLE HIGHLIGHTS
Research background

Replication-competent viral vectors carrying additional genetic information have become invaluable tools for virus molecular biology research and anti-viral drug screening, such as those for infections with HCV, HIV, and influenza virus. Due to the extremely compact organization of the hepatitis B virus (HBV) genome, HBV-based vectors had met with very limited success. In our previously study, via inserting two 22 nt Rbm3 IRES sequences and transgene in between the overlap region of Core and Polymerase genes, the replication-competent HBV vectors can be successfully constructed, which allowed production of HBV vectors carrying at least around 400 bp and possibly up to 720 bp of foreign genetic information yet maintaining replication competence and even infectivity.

Research motivation

Regarding the replication-competent HBV vectors, the pCH-BsdR carries blasticidin resistance gene (399 bp), the replication efficiency is higher, but it is tedious to use. The pCH-hrGFP carries humanized renilla green fluorescent protein gene (720 bp), but the replication efficiency is poor and could not be quantified. Hence, we tried to use the secreted luciferase (secNLuc) report gene (597bp) as foreign genetic inserted to the replication-competent HBV vector, which is convenient and quantifiable for the further research.

Research objectives

The secNLuc report gene can express luciferase protein that is secreted in culture supernatant, which is beneficial for monitoring transcriptional activation of target gene. We utilized this report gene to construct the other replication-competent HBV vector which can generate secNLuc recombinant HBV particles, and to establish quantifiable and standardized HBV replication cell lines that can stably secret recombinant HBV particles.

Research methods

We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with 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 that can stably secret HBV particles carrying secNluc report gene.

Research results

We successfully constructed a replication-competent HBV vector carrying SecNluc reporter gene, pCH-sNLuc and pTRE-sNLuc, and successfully obtained quantifiable and standardized HBV replication cell lines, HBV-NLuc-35 cells. The former could produce all major viral RNAs and full set of envelope proteins, and achieve high level expression of secreted luciferase. The latter could secret secNLuc recombinant viruses that are sensitive for existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity.

Research conclusions

Despite that the organization of HBV genomes is extremely compact, the replication-competent HBV vector can be successfully constructed by redesigning HBV genomes to fully maintain the HBV genetic information and creating transgene insertion site to have a minimal impact on HBV replication competence. The recombinant HBV pgRNA carrying small-to-medium-sized transgenes can be packaged. By carefully redesigning its intricate genome organization, HBV can be harnessed into a replication-competent infectious vector bearing substantial additional genetic information. HBV genomes can be reformed to have a minimal impact on HBV replication competence and expression. Numerous available reporter and effector genes meet the apparent size limit of 500-700 bp. In addition, viral-based vectors could be highly used for drug screening. With the increase in transgene size, the replication efficiency of HBV vectors gradually decreases compared with that of wild-type HBV. Some medium-sized transgenes about 500 bp are compatible with replication competence. The replication-competent HBV vectors carrying appropriate transgenes can be expected to find numerous applications, from further unraveling the molecular mechanism of HBV infections, including the involved host factors, to the identification of infectable cells and new antiviral drugs.

Research perspectives

Given strict hepatocyte tropism, the convenient HBV infection model has met with very limited success. The replication-competent HBV vectors carrying transgenes will provide a helpful tool for this research. The replication-competent HBV vectors carrying transgenes could be utilized to establish animal models of HBV infection. HBV genomes could be reformed to overcome species specificity.