Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation

PLoS One. 2013 Apr 2;8(4):e60306. doi: 10.1371/journal.pone.0060306. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Gene Expression Regulation, Viral
  • Gene Order
  • Gene Products, pol / genetics*
  • Gene Products, pol / metabolism*
  • Genes, Reporter
  • Genetic Vectors / genetics*
  • Genome, Viral
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / metabolism*
  • Humans
  • Protein Transport
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • Transgenes*
  • Virion / physiology
  • Virus Replication / genetics*

Substances

  • Gene Products, pol
  • P protein, Hepatitis B virus
  • RNA, Viral
  • RNA-Binding Proteins

Grant support

This study was supported by grants from the National Natural Science Foundation of China (81271843, 81201297) and the National Major Science and Technology Special Project for Infectious Diseases of China (2012ZX10004503-012). Initial experiments were in part supported by the Deutsche Forschungsgemeinschaft (DFG Na 154/7-3).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.