Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Highlights

• HBV full-length 5'RACE discriminates all viral transcripts including HBx.

• Viral particles contain pgRNA, pgRNA splice variants and different HBx RNAs.

• HBx RNAs in viral particles are both capped and uncapped.

• The composition of circulating viral RNA species can vary among patients.

Background & Aims

Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 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 an 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 pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 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

This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA.

Lay summary

Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.

Introduction

Hepatitis B remains a major public health problem worldwide despite the availability of prophylactic vaccination and antiviral treatments.¹ HBV persists as a covalently closed circular DNA (cccDNA) of approximately 3.2 kb that embeds into the chromatin of hepatocytes as an episomal entity.² The amount of cccDNA and its transcriptional activity vary along the natural course of infection and are a main determinant of viral persistence and reactivation. From 4 promoters, 6 major viral RNAs are expressed: preCore (preC) RNA, pregenomic (pg)RNA, large surface protein (preS1) RNA, middle surface protein (preS2) RNA, small surface protein (S) RNA, X protein RNA (HBx) (Fig. 1A). preC and pgRNA transcripts are characterized by a 100-base pair (bp) redundancy at their 3' ends and consequently contain 2 epsilon stem-loop structures at their 5' and 3' ends (Fig. 1A). Every viral RNA is translated into proteins that are essential for the viral life cycle. In this regard, the pgRNA is unique in the sense that apart from encoding the 2 viral proteins C (core) and P (polymerase), it serves as the template for the viral DNA synthesis. Genomic viral DNA synthesis is mediated by the reverse transcriptase activity of the viral P protein once pgRNA and the P protein are encapsidated into the nucleocapsids formed by the C protein.^3,4 preC RNA codes for the HBe protein that has a potential immunoregulatory function.⁵ preS1/2 and S mRNAs encode the 3 viral surface proteins L, M and S, respectively, that build up the viral envelope. The shortest of the 6 transcripts is the HBx transcript that gives rise to a 154 amino-acid long cytoplasmic and nuclear X protein.[6], [7], [8], [9], [10] X directly interacts with a multitude of host proteins explaining its diverse transactivating effects in host cells.[11], [12], [13], [14]

The organization of the HBV genome is highly condensed and all transcripts are to various degrees subsets of each other (Fig. 1A). Indeed, all HBV transcripts share the same 3' end and, thus, the HBx sequence constitutes the 3' end of every viral transcript (Fig. 1A). Therefore, most HBV RNAs are indistinguishable by quantitative real-time PCR (qPCR). At present, only the larger viral RNAs can be differentiated by northern blotting during the course of infection. Most probably because of low expression levels, HBx transcripts are undetectable by northern blotting under infection conditions. Accordingly, not much is known about HBx transcription during viral infection. Moreover, there is a knowledge gap about the viral RNA species, per se, found in cell culture, but also in viral particles and in chronically HBV-infected patients.

To this end, we set up a strategy called HBV full-length 5’RACE (rapid amplification of cDNA ends) with which we characterized all major intra- and extracellular HBV RNAs during viral infection of cultured hepatocytes. With the HBV full-length 5' RACE approach, we followed the expression of the different viral RNAs over the course of infection and additionally studied how they are modulated by various drug treatments. Furthermore, we identified the viral RNAs that are associated with viral particles produced by HepAD38 cells. Finally, we analyzed the full spectrum of circulating HBV RNAs in patients with chronic HBV infection.