DNA Engineering and Hepatitis B Virus Replication

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.

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Safety and Immunogenicity of a Purified Hepatitis B Virus Vaccine Prepared by Using Recombinant DNA Technology

The Journal of Infectious Diseases ◽

10.1093/infdis/155.5.903 ◽

1987 ◽

Vol 155 (5) ◽

pp. 903-908 ◽

Cited By ~ 12

Author(s):

J. M. Zahradnik ◽

R. B. Couch ◽

J. L. Gerin

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Virus Vaccine ◽

Recombinant Dna ◽

Recombinant Dna Technology ◽

B Virus ◽

Dna Technology ◽

Hepatitis B Virus Vaccine

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Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene

Journal of Virology ◽

10.1128/jvi.01263-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1368-1378 ◽

Cited By ~ 2

Author(s):

Feng Cao ◽

Catherine A. Scougall ◽

Allison R. Jilbert ◽

John E. Tavis

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cultured Cells ◽

Core Protein ◽

Open Reading Frames ◽

Duck Hepatitis B Virus ◽

Reading Frame ◽

The Core ◽

Duck Hepatitis ◽

B Virus

ABSTRACT The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms “pre-P” by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.

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