Concentration of Na+-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus

Andreas Oswald,Ulrike Protzer,Anindita Chakraborty,Yi Ni,Stephan Urban,Jochen M Wettengel

doi:10.1038/s41598-021-99263-3

Abstract

Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.

Highlights

Introduction of NTCP inHeLa, HEK293, A549 and U2OS did not allow productive hepatitis B virus (HBV) infection, even when NTCP was expressed at high levels supporting previous findings[46,47]
One day post transfection efficiency was determined by flow cytometry, showing 90% HepG2 and 95% HEK293 NTCP-tdTomato positive cells when transfected with IVT mRNA
We here established the transfection of various cell lines with IVT mRNA as a suitable tool to study whether NTCP expression suffices to render non-hepatic cells permissive for HBV and how NTCP concentration determines the efficiency of HBV infection

Summary

Introduction

HeLa, HEK293, A549 and U2OS did not allow productive HBV infection, even when NTCP was expressed at high levels supporting previous findings[46,47]. Transfection of IVT mRNA resulted in surface expression of functional NTCP, which was confirmed by bile acid uptake and MyrB binding, this was not sufficient to render these cells permissive for HBV indicating that other host factors necessary to support HBV infection are m issing[47]. One should repeat HDV infection experiment in NTCP transfected non-hepatic cells to determine if this would render them permissive for HDV. Yang et al reported that infection of HEK293 cells with HBV requires the introduction of NTCP and at least three additional factors (HNF4a, PPARa and RxRa)[48] while this remains unknown for other, harder to transfect cell types. Using IVT mRNA may help to overcome this problem because it allows highly efficient introduction of multiple proteins simultaneously[26] or consecutively

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Conclusion