Naturally occurring core protein mutations compensate for the reduced replication fitness of a lamivudine-resistant HBV isolate
Introduction
Hepatitis B virus (HBV) is a medically important human pathogen which causes chronic infection in approximately 257 million people worldwide, leading to a series of life-threatening complications, including cirrhosis, liver failure and hepatocellular carcinoma (Alter et al., 2018). HBV is a partially double stranded DNA virus that replicates its genome through reverse transcription of a pregenomic (pg) RNA (Block et al., 2007). HBV replication can be suppressed effectively by nucleos(t)ide analogue (NA) reverse transcriptase (RT) inhibitors, which target the viral polymerase (pol) and cease DNA chain elongation. Unfortunately, the emergence of resistant viral mutants significantly limits the effectiveness of some NAs (Gish et al., 2012; Lampertico and Liaw, 2012). Usually, the NA-resistance mutations in the catalytic YMDD motif of pol lead to a decreased viral replication due to reduced enzymatic activity of the mutant pol (Allen et al., 1998; Sheldon et al., 2006). However, compensatory mutations have been reported to partially restore the viral replication capacity, including rtL80V/I, rtL82M, rtV173L and rtV207I, mostly in the RT region of pol (Delaney et al., 2003; Ono et al., 2001; Pichoud et al., 1999). In addition, a NA-resistant mutant HBV with increased replication has also been found in chronic hepatitis B (CHB) patients with progressive liver disease (Zoulim and Locarnini, 2009). We have previously identified a highly replicative lamivudine (LMV)-resistant HBV isolate from CHB patients experiencing fulminant hepatitis, which, in addition to the YMDD mutation, possesses multiple mutations in pol, core, X, and surface genes (Zhang et al., 2005) (Fig. 1).
HBV core protein or hepatitis B core antigen (HBcAg) plays a critical role in HBV life cycle, which self-assembles into the capsid of virus and encapsidates viral pgRNA and pol to form the cytoplasmic nucleocapsid (Bartenschlager and Schaller, 1992; Birnbaum and Nassal, 1990), inside of which pol synthesizes HBV DNA by reverse transcribing pgRNA (Summers and Mason, 1982). HBV core protein is composed of 183 amino acids, of which the first N-terminal 149 residues are characterized as the assembly domain that mediates the capsid formation (Hu and Liu, 2017). HBcAg possesses a four-helix bundle structure, two core monomers bind together to form a dimer first, followed by the construction of viral capsid from 90 or 120 copies of dimer (Birnbaum and Nassal, 1990; Bottcher et al., 1997; Wynne et al., 1999). Naturally-occurring mutations within the N-terminus of HBcAg have been reported to affect capsid assembly, uncoating, or virion secretion (Cui et al., 2015; Ning et al., 2018; Pairan and Bruss, 2009). Among those core mutations with high frequencies in CHB patients, the switch of phenylalanine (F) or isoleucine (I) to leucine (L) at residue 97 results in an enhanced immature secretion phenotype, in which the virion mainly contains single-stranded (SS) HBV DNA rather than the mature relaxed circular (rc) DNA (Ceres et al., 2004; Suk et al., 2002; Yuan et al., 1999a, 1999b). Codon 5 is another hot spot of naturally occurring mutation of HBcAg. Previous studies have shown that the substitution of proline (P) with threonine (T) at codon 5 of HBcAg caused lower levels of virion secretion, which, would revert to the wild type secretion phenotype when it coexisted with F97L mutation (Chua et al., 2003; Le Pogam et al., 2000). Moreover, it has also been suggested that P5T/A mutation is an independent factor for acute-on-chronic liver failure (ACLF) (Yan et al., 2011; Zhang et al., 2013). However, the mechanism of P5T-associated liver disease progression remains unclear.
To further investigate the potential role of core protein mutations in hepatitis B exacerbation, we cloned the full-length HBV isolates from patients prior to treatment and after emergence of LMV-resistance with exacerbation, and assessed their replication fitness in vitro. Our study revealed that the mutant core, predominantly P5T, boosts the levels of HBV capsid formation and pgRNA encapsidation, and subsequently enhances the viral replication competency, which may contribute to disease progression during LAM treatment. Therefore, the core mutation-mediated enhancement of HBV replication fitness represents an important viral strategy to surmount the antiviral drug pressure due to suppression of polymerase function by NAs.
Section snippets
Construction of replication-competent recombinant HBV DNA
Paired serum samples from the patient before treatment (wild type, WT, GenBank Accession No. AY220698; genotype B, serotype adw) and after the lamivudine drug-resistance exacerbation (mutant type, isolate GYF634, GenBank Accession No. AY220697; genotype B, serotype adw) were collected previously (Zhang et al., 2005). The 1.0mer replication-competent HBV genomes were PCR amplified and cloned into vector pUC19 at the SacI restriction site to generate pHBV-WT and pHBV-GYF as previously described (
Replication fitness of wildtype and LMV-resistant HBV isolates
We have previously identified a clinical HBV isolate that exhibited resistance to LMV from a CHB patient undergoing LMV treatment with remarkably high viremia. The patient was initially infected with the wildtype genotype B HBV before the treatment, the drug-resistant mutant virus then became dominant during LMV therapy, resulting in acute exacerbations (Zhang et al., 2005). In the current study, we firstly assessed the replication fitness of this mutant virus, termed GYF, in vitro in HepG2
Discussion
In clinical practice, the development of HBV drug resistance in CHB patients treated with low barrier NAs, such as lamivudine, is commonly accompanied by virological breakthrough, exacerbation of hepatitis, and even liver failure in severe cases (Zoulim and Locarnini, 2009). The resistance mutations within viral reverse transcriptase region (RT) are selected by NAs, which would however decrease the viral replication capacity (Melegari et al., 1998). Therefore, the underlying mechanism of
Acknowledgments
We thank Dr. William Mason (Fox Chase Cancer Center), Dr. Xiaodong Xu (Pharmabridge Inc), and the Research Department at Arbutus Biopharma for providing experimental compounds. Dr. Andrea Cuconati is thanked for critical reading of the manuscript. This study is supported by the Natural Science Foundation of Shanghai (18ZR1405600 to YZ), the Research Foundation of Huashan Hospital (North Hospital) (HSBY2016016 to YZ), the National Natural Science Foundation of China (81800529 to YZ, 81672009 to
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These authors contribute equally to this study.