A hepatitis B virus RNA-sensing and RNA-editing-dependent reporter system

TL;DR

Scientists developed a new reporter system for hepatitis B virus that detects viral RNA and allows for studying virus-host interactions and drug screening.

Contribution

The HBV-RADARS system is a novel RNA-sensing and RNA-editing-dependent reporter platform that enables replication-competent HBV studies in live cells.

Findings

HBV-RADARS activates reporter gene expression in a sequence-specific and RNA-abundance-dependent manner.

The system works in multiple HBV cell culture settings, including authentic HBV infections.

It allows phenotypic selection of HBV-infected cells and supports high-throughput screening for antiviral compounds.

Abstract

The compact genomic organization of hepatitis B virus (HBV) has long hindered
the development of reporter viruses that do not compromise viral gene
expression and replication. Leveraging the advantage of an RNA-sensing and
ADAR-editing-dependent cellular reporter system termed
reprogrammable adenosine
deaminase acting on
RNA sensors (RADARS), we
developed an HBV-RADARS reporter. In this system, the expression of the
reporter gene is activated in trans by HBV RNA-guided,
cellular adenosine deaminase acting on RNA 1 (ADAR1)-dependent reporter RNA
editing. Using a luciferase reporter, we systematically scanned all ADAR1
targetable sites present in HBV RNAs and selected an optimal sensor
sequence. As anticipated, the activation of reporter mRNA translation is HBV
RNA sequence-specific and quantitatively correlates with the abundance of
HBV RNA and cellular ADAR1 deaminase activity. The optimized HBV-RADARS
system can be used with versatile reporter proteins in multiple HBV cell
culture settings, including HBV replicon plasmid-transfected cells, cell
lines expressing HBV RNAs from integrated transgenes, and cells infected
with HBV. Particularly, using an antibiotic resistance gene as the reporter
for HBV-RADARS allows for the phenotypic selection of HBV-infected HepG2
cells expressing human sodium taurocholate cotransporting polypeptide.
Therefore, this HBV RNA-sensing reporter system is compatible with authentic
HBV infection and can serve as a versatile platform for high-throughput
screening of compounds that inhibit HBV infection and genome-wide genetic
screens to identify cellular factors required for HBV infection of
hepatocytes.

Traditional recombinant hepatitis B virus (HBV) reporter viruses are
compromised in replication fitness and restricted to a single round of
infection. HBV-RADARS, in principle, does not interfere with the viral
life cycle as it acts through sensing the presence of HBV RNA and thus
reports de novo HBV infection as well as the
replication of transfected HBV replicons. The HBV-RADARS system
represents a significant advancement in HBV research tool development
and offers a replication-competent and highly adaptable reporter
platform in live cells for genome-wide genetic and chemical screens.
Hence, it opens new avenues for dissecting HBV-hepatocyte interactions
and holds promises for the identification of host-encoded antiviral
targets, thereby advancing efforts toward a functional cure for chronic
HBV infections.

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