Deep sequencing identifies hepatitis B virus core protein signatures in chronic hepatitis B patients

Highlights

• The HBV core protein is a promising target for the design of new antiviral treatments.

• We identified amino acid changes in HBV core protein associated with HBeAg status and response to antiviral treatment.

• HBc Amino acid variation was significantly higher in HBeAg negative patients, and negatively correlated with HBV DNA levels.

Abstract

Background

We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients.

Methods

Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg-positive and -negative patients, and between patients with combined response and non-response to peginterferon/adefovir combination therapy.

Results

We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg-positive and -negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non-response. The fraction non-consensus sequence on selected positions was significantly higher in HBeAg-negative patients, and was negatively correlated with HBV DNA and HBsAg levels.

Conclusions

Sequence Harmony identified a number of amino acid changes associated with HBeAg-status and response to peginterferon/adefovir combination therapy.

Introduction

Hepatitis B virus (HBV) infection is considered as a major public health issue with an estimated 240 million people worldwide being chronically infected. In chronic hepatitis B (CHB) patients, the immune systems fails to clear the virus and patients are at increased risk for developing liver-related complications such as liver cirrhosis and hepatocellular carcinoma (Dienstag, 2008). Two types of drugs are approved for the treatment of CHB patients: nucleos(t)ide analogues (NUCs) and pegylated interferon (peg-IFN). Although NUCs efficiently block viral replication, treatment rarely leads to a functional cure (defined as hepatitis B surface antigen (HBsAg) loss with or without the formation of anti-HBs antibodies) (Chevaliez et al., 2013). In CHB patients treated with peg-IFN a functional cure is only achieved in 3–7% of patients (Janssen et al., 2005; Marcellin et al., 2004). Improved understanding of the replication cycle of HBV resulted in the development of new antiviral drugs that directly inhibit viral replication (Durantel and Zoulim, 2016; Tong and Revill, 2016).

The HBV core protein (HBc) is a promising antiviral target since it is involved in almost all steps of the HBV replication cycle (Summers and Mason, 1982; Tong and Revill, 2016). HBc consists of an assembly domain (residues 1 to 149), and a C-terminal domain (CTD) (residues 150 to 183 or 185, depending on HBV genotype), which are connected by a linker region (residues 141 to 149) (Birnbaum and Nassal, 1990; Watts et al., 2002; Yu et al., 2013; Zlotnick et al., 1997). HBc self-assembles to form viral capsids, predominantly consisting of 120 HBc homodimers (Zlotnick et al., 1996), wherein pregenomic RNA (pgRNA) is reverse transcribed to relaxed circular DNA (rcDNA). Viral capsids containing rcDNA are either encapsulated by the viral envelope proteins and secreted as virions, or they are shuttled back to the nucleus where the rcDNA is released and converted to covalently closed circular DNA (cccDNA) (Summers and Mason, 1982). The cccDNA is the template for transcription of viral mRNAs utilizing host DNA-dependent RNA polymerase. Next to virus assembly, HBc is involved in regulation of cccDNA and host-gene expression in the nucleus of infected hepatocytes (Guo et al., 2011; Li et al., 2010).

HBV replication through an RNA intermediate causes a high mutation rate during chronic HBV infection (Akarca and Lok, 1995; Chuang et al., 1993). Mutations in the basal core promotor (BCP) and precore (PC) regions are most extensively studied, and abrogate the production of hepatitis B e antigen (HBeAg) (Alexopoulou et al., 1997; Buckwold et al., 1996; Carman et al., 1989; Lok et al., 1994), and were shown to influence peg-IFN based treatment response in CHB patients (Erhardt et al., 2000; Jansen et al., 2017; Marrone et al., 2003; Sonneveld et al., 2012). Previously, it was postulated that mutations in HBc could induce secretion of virions containing an immature viral genome, affect envelopment of viral capsids, and may evade immune recognition (Akarca and Lok, 1995; Hosono et al., 1995; Koschel et al., 2000; Yuan et al., 1999a, 1999b). However, most previous studies analysing HBc mutants used population based sequencing techniques which limits the detection of mutations with low frequencies. Importantly, data on the effect of minority variants of HBc and their effect on HBeAg status and treatment response are lacking. In our cohort of HBeAg-positive and -negative CHB patients treated with a combination of peg-IFN and adefovir for 48 weeks, a high rate of HBsAg loss was observed (11–17% at year 2 of treatment-free follow-up) (Takkenberg et al., 2013). Here, we studied amino acid differences in HBc in our cohort of CHB patients using deep sequencing. We aimed to identify amino acids differences in HBc between HBeAg positive and negative patients. Furthermore, we aimed to identify HBc signatures associated with treatment response in CHB patients.