CHB is one of the most common human infectious diseases worldwide. Infected individuals are at a high risk of developing liver cirrhosis and HCC
. Treatment of CHB is aimed at suppressing HBV replication, at reducing the accompanying histological inflammation, and at decreasing the risk of cirrhosis and HCC
. Therefore, antiviral therapies with NAs or interferon are currently the key treatments for CHB.
However, drug resistance is the major challenge associated with long-term therapy with NAs. Drug resistant HBV mutant strains will gradually become dominant, which leads to disease deterioration and other serious consequences. Many studies showed that treatment of CHB patients with LAM, ADV, or LdT could result in the emergence of the HBV rtA181T mutant
[5, 16–18]. Clinically rtA181T is a common resistance mutation to ADV. This mutation not only induces a decreased susceptibility to ADV, LAM, and tenofovir
, but also encodes S gene mutation. It is reported that the nonsence mutation at position 172 in the S gene leads to a 55 amino acids truncation of HBsAg. This mutation is therefore also called HBV rtA181T/sW172*, which may affect the biological characteristics and pathogenesis of HBV
. However, the biological features of this HBV mutant in vivo still remain unclear.
Most of recent studies on HBV mutant were done in cultured cells. Transgenic mice
[19, 20] are useful models to study HBV. However, establishing specific transgenic mice is time and cost consuming. The HBV replication mouse model that we established with a hydrodynamic-based procedure is a rapid and convenient alternative, especially for the characterization of HBV mutations. We have constructed the rtA181T/sW172* mutant strain of HBV and investigated its biological characteristics in vivo with our HBV replication mouse model.
Firstly, we found that serum HBsAg level of the rtA181T/sW172* mutant was very low (OD<0.4) from day 1 to day 15 after in vivo transfection (Figure
1A), and that the serum HBV DNA level of the mutant was lower than that of wild type (Figure
1C). This may be explained by the fact that the rtA181T/sW172* mutation causing the production of truncated HBsAg, which cannot be secreted into the serum. HBsAg plays an important role in the packaging and secretion of virion
. The complete HBV virion consists of an icosahedral nucleocapsid core and an outer lipid envelope, in which the three envelope proteins [small (S), medium (M) and large (L)] are anchored as transmembrane proteins playing a major role in HBV morphogenesis and infectivity. And the mature nucleocapsid enveloped by the surface proteins can be released to serum
. As the S gene overlaps the P gene completely, the A→T mutation at position 181 of the P gene in the RT domain replaces the amino acid tryptophan 172 of the S gene with a termination codon, resulting in the truncation of the last 55 amino acids. All of the HBV surface proteins (S protein, M protein, L protein) decrease approximately 6 kDa in molecular weight. These truncated surface proteins may affect the virus assembly and secretion. In this study, the mostly HBV virions and HBsAg assembled by these truncated surface proteins may not be secreted into serum and thus became accumulated in liver. It seems that the function of the truncated 55 amino acids in the C-terminal is critical for HBV assembly. The rtA181T/sW172* mutation thus leads to defects in the assembly and secretion of HBsAg and virion, which results in decreased levels of serum HBsAg and HBV DNA.
Detection of HBsAg is achieved by antibody-based assays targeting the ‘a’ determinant, the highly homologous region within HBsAg, which is also used as the main target by hepatitis B vaccines
. Recognition of the ‘a’ determinant by antibody against HBsAg (anti-HBs) depends on its 3D conformation, which also relies on the amino acid sequence of the regions flanking the ‘a’ determinant
. However, mutants of HBsAg continue to evolve as a result of vaccine escape, immune selection, and an error prone reverse transcriptase
. Because the sW172* mutation of HBsAg gene is not inside the ‘a’ determinant, currently commercially antibody for truncated HBsAg is effective. In addition, the immunohistochemistry results of HBsAg in liver tissue also supported the effectiveness of conventional antibody for truncated HBsAg detection. So the low serum HBsAg in mutant HBV mice model should be due to the intracelluar retention of truncated HBsAg. As detection of HBV DNA in serum is qPCR-based assays, the HBV DNA can be observed even if it is very little. Compared with the wild mice, the level of HBV DNA in mutant mice serum decreased significantly, and these finds were consistent with the conditions of patients who had the HBV rtA181T/sW172* mutation.
However, the synthesis and secretion of HBeAg, a secretory antigen, is regulated differently from HBsAg, and is regulated by HBV C open reading frame. Serum HBeAg of two groups showed similar patterns.
Secondly, our study revealed that the expression levels of HBsAg and HBcAg in liver of mice infected with the mutant HBV were higher than those of wild type, especially on day 5 and day 7 (Figure
3). HBsAg, the envelope protein, is normally secreted into serum following infection. HBcAg, the nucleocapsid protein, is synthesized in infected cells and is required for HBV viral replication. It seems that the mutant HBV has defects in the secretion of virions and HBsAg, leading to the accumulation of a large number of viral particles and HBsAg in liver.
The expression levels of HBV RNA and DNA of the mutant HBV were higher and lasted longer in liver than wild type (Figure
3), which may be due to the increased capability of mutant HBV transcription and replication . To classify this issue, it is necessary to study the promoter activity of the mutant HBV in the future research. As compared to wild type HBV strain, the transcription and replication levels of the HBV rtA181T/sW172* mutant strain in vivo delayed in reaching the peak level and lasted significantly longer, and the mutation also was demonstrated HBsAg and HBcAg continued retention in liver. And these antigens in the liver tissue may stimulate prolonged immune response. Previous evidences showed that the adaptive immune response should be responsible for viral clearance and disease pathogenesis during HBV infection
. Though HBV is not directly hepatotoxic
[27, 28], recent studies of HBV insertions in HBV-related HCCs revealed that HBV integration can target the telomerase reverse transcriptase gene, suggesting a potential role for viral insertion in HBV-related carcinogenesis
[29–31]. The cytotoxic T lymphocyte (CTL) not only kills the virus, but also kills the infected hepatocyte, leading to liver damage. Persistent infection of HBV is characterized by chronic liver cell injury, regeneration, inflammation, widespread DNA damage, cirrhosis of the liver, and hepatocellular carcinoma
. Further studies are ongoing to investigate where and why the secretion of the mutant HBsAg and HBV virions is impaired, and to explore the mechanism of mutant HBV accumulation in liver. The related research will be reported in the future.
Thirdly, cell culture studies have showed that the single amino acid substitution at position rt181 in HBV is the primary resistance mutation to ADV
 and it also causes cross resistance to LAM and LdT
. The concentrations of LAM and LdT for 50% inhibition of HBV replication (IC50) are 0.023μM and 0.335μM, respectively. Our study found that the folds of decrease in the sensitivity of the mutant to LAM, ADV, and LdT are different. However, this study utilized 0.022 μM of LAM, 0.0051 μM of ETV and 0.15 μM of LdT in our mouse models. The corresponding concentration of LAM in human would be 16.67 times the normal oral dose, that of ETV would be equivalent to the normal dose, and that of LdT would have reached 20 times the normal dose. Oral ADV reduced HBV DNA in both serum and liver significantly better than LAM, and also worked well at concentrations as low as 1.0 mg/kg/day
. So in this study, the antiviral effect to HBV rtA181T/sW172* is ETV>ADV>LAM>LdT. It is related to the establishment method of the mouse model, individual difference of mice and the drug concentration.
Currently most drug resistance monitoring in clinical practice is achieved by detecting viral load, not by direct sequencing. Serum viral load would not be as high as expected for the kind of variant. This makes us to focus on the HBV rtA181T/sW172* mutation. If the patient carries the variation and we failed to change drug in time, the disease would deteriorate, once rtA181T/sW172* become the dominant strains. However, the mouse model used in this study is a transient HBV infection model. The chronic influence of the mutant virus to mice requires further study.
In conclusion, our results indicated that the rtA181T/sW172* mutation might impair serum HBsAg secretion and reduce serum HBV DNA level. However, the transcription and replication levels of the mutant strain in liver tissues were increased, which may be explained by the accumulation of viral core particles in liver or the enhanced replication ability of this mutant strain. The clinical consequences of infection by these S gene mutants demand further clarification. Judicious selection of antiviral agents and vigilant monitoring of viral mutants during the course of therapy are advised.