Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus

Yi Ni,Lisa Engelskircher,Stephan Urban,Thomas Tu,Zhenfeng Zhang,Georg Verch,Florian A Lempp

doi:10.1038/s41598-019-46493-1

Abstract

Human hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Approximately 15–25 million people are chronically infected with HDV. As a satellite virus of the human hepatitis B virus (HBV), HDV uses the HBV-encoded envelope proteins for egress from and de novo entry into hepatocytes. So far, in vitro production of HDV particles is restricted to co-transfection of cells with HDV/HBV encoding cDNAs. This approach has several limitations. In this study, we established HuH7-END cells, which continuously secrete infectious HDV virions. The cell line was generated through stepwise stable integration of the cDNA of the HDV antigenome, the genes for the HBV envelope proteins and the HBV/HDV receptor NTCP. We found that HuH7-END cells release infectious HDV particles up to 400 million copies/milliliter and support virus spread to co-cultured cells. Due to the expression of NTCP, HuH7-END cells are also susceptible to de novo HDV entry. Virus production is stable for >16 passages and can be scaled up for preparation of large HDV virus stocks. Finally, HuH7-END cells are suitable for screening of antiviral drugs targeting HDV replication. In summary, the HuH7-END cell line provides a novel tool to study HDV replication in vitro.

Highlights

Hepatitis delta virus (HDV), first described in 19771, is a viroid-like pathogen and belongs to the genus Deltavirus
The L-hepatitis delta antigen (HDAg) becomes C-terminally prenylated within this elongated sequence by a host farnesyl transferase[14], complexes with hepatitis delta virus (HDV) genomic RNA, and the new RNP complex is packaged into hepatitis B virus (HBV) envelope proteins, which is subsequently secreted by the infected cells
This result suggested that HepG2-HDV cells somehow down-regulates HDAg

Summary

Introduction

Hepatitis delta virus (HDV), first described in 19771, is a viroid-like pathogen and belongs to the genus Deltavirus. HDV encodes a single protein called hepatitis delta antigen (HDAg) that is expressed in a small and a large form (S-HDAg and L-HDAg) They are required for viral RNA replication and egress of particles respectively. Www.nature.com/scientificreports production of L-HDAg is regulated by a cellular RNA-specific adenosine deaminase ADAR1, which mutates the stop codon of the HDAg ORF on the anti-sense HDV RNA This mutation alters the stop codon into a tryptophan codon, thereby extending the S-HDAg into the 19aa-longer L-HDAg. The L-HDAg becomes C-terminally prenylated within this elongated sequence by a host farnesyl transferase[14], complexes with HDV genomic RNA, and the new RNP complex is packaged into HBV envelope proteins, which is subsequently secreted by the infected cells. While the HBV field has developed several virus-replicating cell lines (such as HepG2.2.1517 and HepAD3818, which have been used for virus production, investigation of the viral replication cycle and identification of antiviral drug candidates), there is currently no equivalent stable cell line that supports continuous HDV replication and secretion of infectious HDV particles

Methods

Results

Conclusion