Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation

Zihua Wang,Caiyan Ping,Michael Nassal,Fubiao Kang,Xin Cheng,Baosheng Li,Li Wu,Junping Wang,Shizhu Liu,Haijun Li,Huan Xia,Dianxing Sun

doi:10.1371/journal.pone.0060306

Abstract

Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.

Highlights

Chronic infection with hepatitis B virus (HBV) affects up to 400 million people worldwide, putting them at an increased risk to develop liver fibrosis, cirrhosis and hepatocellular carcinoma [1]
As yet there are no reports on the translation initiation efficiency of the RNA-binding motif protein 3 (Rbm3) internal ribosome entry site (IRES) in human Huh7 and HepG2 hepatoma cells
We included constructs in which the reporter gene was directly fused to the authentic Pol start to test for background (i.e. Rbm3 IRESindependent) translation of the transgene via potentially present endogenous translation control signals in the preceding core open reading frames (ORFs) region

Summary

Introduction

Chronic infection with hepatitis B virus (HBV) affects up to 400 million people worldwide, putting them at an increased risk to develop liver fibrosis, cirrhosis and hepatocellular carcinoma [1]. Finding new treatment strategies is hampered by experimental limitations [3]; due to HBV’s liver tropism and narrow host range, restricted to humans and the Great Apes, primary hepatocytes from humans and (for poorly understood reasons) from tupaias [4] have long remained the only cell culture infection system; more recently, a single human hepatoma cell line, HepaRG, has shown to be susceptible to HBV infection upon differentiation [5]. The early steps of infection are still poorly understood, including the identity of the cellular receptors. In contrast, is known in considerable detail from genetic studies in transfected cells and from biochemical reconstitution of some key replication steps (for reviews: [6,7]). Overall these data revealed an intricate interplay between the few viral gene products and numerous cis-elements, streamlined to warrant function of the tiny (3.2 kb) and extremely compactly organized HBV genome which is exquisitely sensitive to sequence manipulations

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Conclusion