Development of a Novel Transfection System to Study Regulation of Hepatitis Delta Virus RNAs by Hepatitis Delta Antigen

Abstract

Hepatitis delta virus (HDV) is a unique pathogen that co-infects with hepatitis B virus and increase the severity of acute and chronic liver disease. HDV is a negative stranded RNA virus and produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate, and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes (RNP) with HDAg. It was observed that HDAg:HDV RNA ratio was maintained constant by the virus in replicating cells. HDAg is known to be required for HDV replication. However, it is not known if the relative amounts of HDAg and HDV RNA are important for HDV replication, or whether HDAg levels are regulated by the virus. To investigate the regulation mechanism of HDV, we developed a novel transfection system in which HDV replication is initiated using in vitro synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. This result was consistent when tested with two other clones of HDV.

It is not known if HDAg has roles similar to N protein of negative stranded RNA viruses in mRNA and antigenomic RNA synthesis. From transfection experiments performed using HDV in vitro synthesized circular RNA, it was observed that low HDAg levels favored mRNA transcription. Antigenome RNA accumulation increased linearly with HDAg and dominated at high HDAg levels. Further, HDV mRNA synthesis was observed to require a minimum number of HDAg octamers bound to genomic RNA to form a functional genomic RNP. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.