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

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.

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

“…(3) The observation that pol becomes activated by binding to its RNA (Tavis & Ganem, 1996) supports the hypothesis that the supposed structural change of pol is essential for the viral life cycle. (4) The observation that in the absence of core, HBV pol is only poorly active, allowing priming and synthesis of a few nucleotides of the minus DNA strand (Chen et al, 2003;Wang et al, 2013) is also in agreement with the requirement of e-induced structural pol changes allowing core interactions. (5) The observation of Gazina et al (2000) showing that core protein phosphorylation is required for PG packaging can also be explained by our findings that phosphorylation is the main requirement for the pol-core interaction.…”

HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

“…Highly valuable for any such screening would be HBV reporter vectors that allow for non-invasive, sensitive and quantitative detection of cccDNA formation; for other viruses, including HCV, such reporter-bearing variants have been instrumental 117. However, manipulating the tiny, compactly organised HBV genome without disturbing its functionality has proven very difficult;118 119 hence, further research is highly warranted.…”

Visualization of hepatitis B virus entry – novel tools and approaches to directly follow virus entry into hepatocytes

“…Because of the tight organization and size limitation of the HBV genome (Fig. B), incorporation of a complete reporter gene (e.g.…”

“…B). Furthermore a size limitation of 3.5 kb , which restricts the direct incorporation of marker genes into the HBV genome, has been described. Secondly, the purification of HB virions is challenging because of the presence of a huge excess (up to 10 000‐fold) of so‐called subviral particles (SVPs), which do not contain HBV genomes.…”