DNA Engineering and Hepatitis B Virus Replication.

Chun-Yang Gan,Ai-Long Huang,Yu-Wei Wang,Jie-Li Hu,Wen-Lu Zhang,Jing Cui

doi:10.3389/fmicb.2021.783040

Chun-Yang Gan, Ai-Long Huang + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2021.783040

Copy DOI

Abstract

Recombinant DNA technology is a vital method in human hepatitis B virus (HBV), producing reporter viruses or vectors for gene transferring. Researchers have engineered several genes into the HBV genome for different purposes; however, a systematic analysis of recombinant strategy is lacking. Here, using a 500-bp deletion strategy, we scanned the HBV genome and identified two regions, region I (from nt 2,118 to 2,814) and region II (from nt 99 to 1,198), suitable for engineering. Ten exogenous genes, including puromycin N-acetyl transferase gene (Pac), blasticidin S deaminase gene (BSD), Neomycin-resistance gene (Neo), Gaussia luciferase (Gluc), NanoLuc (Nluc), copGFP, mCherry, UnaG, eGFP, and tTA1, were inserted into these two regions and fused into the open reading frames of hepatitis B core protein (HBC) and hepatitis B surface protein (HBS) via T2A peptide. Recombination of 9 of the 10 genes at region 99–1198 and 5 of the 10 genes at region 2118–2814 supported the formation of relaxed circular (RC) DNA. HBV DNA and HBV RNA assays implied that exogenous genes potentially abrogate RC DNA by inducing the formation of adverse secondary structures. This hypothesis was supported because sequence optimization of the UnaG gene based on HBC sequence rescued RC DNA formation. Findings from this study provide an informative basis and a valuable method for further constructing and optimizing recombinant HBV and imply that DNA sequence might be intrinsically a potential source of selective pressure in the evolution of HBV.

Highlights

Infection with human hepatitis B virus (HBV) remains a public health problem around the world
The relaxed circular (RC) DNA genome of HBV is repaired after infection, converted into covalently closed circular DNA in the nucleus of hepatocyte, and transcribed to pregenomic mRNA, precore mRNA, preS1 mRNA, S mRNA, and X mRNA
HBV replication begins with the encapsidation of pregenomic mRNA (pgRNA), whereby Pol binds to the stem-loop structure, epsilon (ε), at the 5 end of pgRNA, triggering the assembly of hepatitis B core protein (HBC) and packaging of the ribonucleoprotein complex into an icosahedral nucleocapsid (Bartenschlager et al, 1990; Bartenschlager and Schaller, 1992; Pollack and Ganem, 1993; Shin et al, 2002)

Summary

Introduction

Infection with human hepatitis B virus (HBV) remains a public health problem around the world. PgRNA functions as a bicistronic mRNA directing the synthesis of hepatitis B core protein (HBC) and polymerase (Pol) and is the template for reverse transcription. PgRNA is reverse transcribed into minus-strand and simultaneously degraded by the RNase H domain of Pol, leaving the 11–16 oligonucleotides at 5 -terminal of pgRNA undigested (Figure 1C; Summers and Mason, 1982). These oligonucleotides function as primers for plus-strand synthesis. Most plus-strand primers translocate to DR2 and extend to the 5 end of the minus-strand, copying a 10-nt redundant sequence named 5 r (Figure 1E; Haines and Loeb, 2007). The nascent 3 end of plus-strand pairs with the 3 -proximal redundant region (3 r) of minus-strand, continuing elongation of plus-strand to form RC DNA (Figures 1F,G)

Methods

Results

Conclusion