Establishment of HBx responsive quantitative cell culture reporter system for monitoring HBV infection
Muhammad Atif Zahoor, Adrian Kuipery, Alexander I. Mosa, Joshua B. Feld, Adam J. Gehring and Jordan J. Feld*
Toronto Center for Liver Disease, Toronto General Hospital Research Institute, University Health Network, University of Toronto, Canada. *Correspondence jordan.feld@uhn.ca

ABSTRACT
Background: Recombinant engineered viruses or cell-based reporter assays are helpful tools in studies related to virus replication and drug susceptibility testing. As such, a few engineered HBV reporter viruses are available, however their utility in monitoring HBV infection is limited and they cannot use serum-derived virus, thus development of a cell-based quantifiable reporter system is highly desirable.
Purpose: Among the proteins encoded by HBV, HBx is an essential multifunctional regulator of virus replication in vivo and is thought to contribute to hepatocarcinogenesis. It is involved in modulating various transcription factors, notably cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). Here, we coupled CRE/CREB promoter with a nano-Luciferase and generated a HBx responsive HepG2-NTCP stable cell line.
Methods: A stable HepG2-NTCP cell line expressing nano luciferase under the promoter of CRE was generated (designated as HepG2-NTCP-CRE-nL) and its responsiveness to HBx was confirmed utilizing wild type or HBx-defective molecular clones. Different antiviral compounds were tested to further exploit the utility of this reporter system in vitro. Serum samples obtained from HBV infected patients were used to infect the reporter cell line and luciferase activities were measured.
Results: Through PCR amplifications and cloning, we generated a lentiviral construct expressing a nano luciferase (nL) under the promoter of CRE (Figure 1A) and was sequence confirmed. HEK293T cells transfected with pHBV1.3mer molecular clone or pHBx alone showed luciferase activity whereas the effect was completely abrogated with pHBV1.2*7-ΔX (Figure 1B) confirming the responsiveness of generated construct towards HBx. Next, we generated a HepG2-NTCP-CRE-nL stable cell line expressing CRE-nL through lentivirus mediated transduction and infected it with HBV which permitted us to quantitatively measure the HBV infection (Figure 2A). Treatment of HBV infected HepG2-NTCP-CRE-nL cell line with CREB inhibitor 666-15 reduced the luciferase activity in a dose dependent manner (Figure 2B), whereas TDF or IFNα2 treatment yielded no results suggesting that HBx transcript is a non-target of these drugs (Figure 2C). In addition, HepG2-NTCP-CRE-nL cell line allowed us to quantify the magnitude of infection using the serum samples obtained directly from HBV infected patients (not shown).
Conclusion: Given that binding of HBx to DDB1 is crucial in degradation of Smc5/6 complex and in promoting HBV transcription from cccDNA, this newly established CREB driven cell culture reporter system will be helpful in screening new therapeutic agents targeting HBx and a step toward a functional cure of HBV.