Full-length 5’RACE identifies all major Hepatitis B Virus transcripts during infection in cultured hepatocytes and in patients

Background and aims: Covalently closed circular DNA (cccDNA) is the episomal form of the Hepatitis B virus (HBV) genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of six major viral RNAs, i.e. preC- pg-, preS1/2-, S- and HBx-RNA. All viral transcripts share the same 3’ end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab.Methods: We set-up the HBV full-length 5’RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. Results: In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that Interferon β treatment leads to a strong reduction of preC- and pg-RNAs but only has a moderate effect on the other viral transcripts. We found pgRNA, one spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx.Conclusions: The HBV full-length 5’RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may inform the development of novel therapies aimed at targeting cccDNA.