HBsAg mRNA degradation induced by a dihydroquinolizinone compound depends on the HBV posttranscriptional regulatory element
Introduction
Chronic hepatitis B virus (HBV) infection affects more than 250 million people worldwide and is the major etiology of severe liver diseases such as fulminant hepatitis, hepatic cirrhosis and liver cancer (Liang et al., 2015). Although interferon alpha (IFNα) and nucleos(t)ide analogues (NA) are approved for the management of chronic HBV infection, a cure for the disease remains difficult to achieve (Lok et al., 2016, Terrault et al., 2016). Regarding IFNα treatment, sustained virologic response is only seen in 30%–40% selected patients, and is quiet often associated with severe side effects (Janssen et al., 2005, Perrillo, 2009). On the other hand, NAs are potent in the inhibition of HBV replication and are generally well tolerated. However, treatment with NAs may last lifelong and development of drug resistance could occur. Both IFNα and NAs are known for their low efficiency to induce hepatitis B virus surface antigen (HBsAg) seroconversion even after long-term treatment. In the blood, HBsAg exists as small lipid vesicles with an approximate size of 22 nm, and outnumbers virion particles 1000 to 100,000 times. The role of HBsAg in the maintenance of HBV infection is not clear. It is speculated that HBsAg may suppress immune reactions against virus or virus infected cells. High level of HBsAg is thought to be responsible for T cell exhaustion and depletion (Bertoletti and Ferrari, 2012, Boni et al., 2007). Reduction of HBsAg in blood quite often predicts better prognosis for patients receiving IFNα treatment (Cornberg et al., 2017). Ideally, the disappearance of HBsAg followed by the emergence of anti-HBsAg antibodies would result in a sustained virological response to HBV, which is regarded as a sign of a functional cure (Hoofnagle et al., 2007, McMahon et al., 2016). Therefore, down regulation of HBsAg, which is both an essential viral protein and an immune suppressor, is thought to be an important therapeutic target (Chen et al., 2012).
HBsAg is composed of large, middle and small surface proteins. The large (L) surface protein is translated from the 2.4 kb PreS1 transcript. The middle (M) and small (S) surface proteins are translated from the 2.1 kb PreS2/S transcript. The 2.4 and 2.1 kb mRNAs share in frame the S ORF and the remaining 3′ UTR (Seeger and Mason, 2000). HBsAg mRNA is intronless but contains cryptic splicing donor and acceptor sites (Hass et al., 2005). In mammalian cells, the regulation of RNA export is tightly linked with splicing processes and RNA maturation (Wagner and Lykke-Andersen, 2002). In order to avoid the disruption of HBsAg synthesis due to aberrant RNA splicing HBV has managed to guarantee its intronless RNA to be transported out of the nucleus. Different to HIV, whose spliced RNA transportation depends on the interaction of Rev and RRE RNA sequence, HBV uses a cis-element at the 3′ region of the transcript called HBV posttranscriptional regulatory element (HPRE) for RNA transportation (Huang and Liang, 1993, Huang and Yen, 1994, Visootsat et al., 2015). Without the help of HPRE, HBsAg mRNA is found to be degraded in the nucleus. Moreover, HPRE mediated intronless RNA nuclear export does not require the involvement of any viral protein (Huang and Yen, 1995). HBV PRE contains approximately 450 nucleotides encompassing nt 1151 to 1582 and is divided into 3 sub-elements HPREα (nt 1151–1346), HPREβ1 (nt 1347–1457) and HPREβ2 (nt 1458–1582) (Schwalbe et al., 2008, Smith et al., 1998). Each sub-element is able to independently but partially support mRNA transportation. The full export capacity needs the three sub-elements to work synergistically to transport HBsAg mRNA to the cytoplasm (Schwalbe et al., 2008). There are not many host proteins that have been found to interact with HPRE. La protein is found to bind the HPREα region and plays a role to maintain pgRNA and HBsAg mRNA stability (Heise et al., 1999b). In addition, polypyrimidine tract binding protein (PTB1) is reported to be associated with HPREβ2 region and is important to HBsAg mRNA nuclear export (Zang et al., 2001). Peculiarly, although GAPDH is regarded as a cytoplasmic protein it is found to be bound with HPREβ2 sub-element in the nucleus with an unknown function (Zang et al., 1998). Recent bioinformatics analysis of 6495 mammalian hepadnaviruses has revealed that the HPRE sequence contains two conserved stem-loops: SLα and SLβ, located in HPREα and HPREβ1 sub-elements, respectively. Homologs of these two conserved sequences have not been found in remotely related hepadnaviruses such as avian hepatitis B viruses, or in human mRNA (Lim and Brown, 2016). It is therefore of great interest to explore whether these special HBV PRE structures can be targeted by small molecule compounds (Chen et al., 2014).
In the current study, we report that a small molecule dihydroquinolizinone, 6-R2-10-methoxy-9-R1-2-oxo-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]-isoquinoline-3-carboxylicacid (DHQ-1), is able to induce HBV RNA degradation in tissue culture and mouse models. The molecule does not affect replication of a range of viruses including HCV, RSV, HIV, CMV, HSV, and HRV and thus is not believed to activate the innate antiviral response pathway through which HBV RNA could be affected. Degradation of HBV pgRNA and HBsAg mRNA occurs in the hepatocyte nucleus and requires de novo synthesis of host proteins. Mutagenesis analysis of the HBsAg expressing vector revealed that a 109-nucleotide region of the HBV PRE alpha sub-element, which contains the conserved La binding site and the stem-loop SLα is required for DHQ-1 antiviral activity.
Section snippets
Small molecules
6-R2-10-methoxy-9-R1-2-oxo-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]-isoquinoline-3-carboxylic acid is referred to as only DHQ-1. Its enantiomer is DHQ-2 (Fig. 1A). DHQ-1 and DHQ-2 were both provided by Arbutus Biopharma and were referred to in patent application WO 2015/113990A1 (Han et al., 2015).
Cells, HBV transfections and infections
The HBV producing cell line HepG2.2.15 (Sells et al., 1987) was cultured in DMEM/F12 containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). HepG2,
The dihydroquinolizinone (DHQ-1) molecule specifically inhibited HBV replication in tissue culture
DHQ-1 was first evaluated for its ability to reduce HBV related products, including virion, HBsAg and e antigen (HBeAg) in the supernatants of HepG2.2.15 cells. Confluent HepG2.2.15 cells were incubated with varying concentrations of DHQ-1 for 5 days, after which the levels of viral particle, HBsAg and HBeAg in the culture media were determined. As shown in Fig. 1B, dose-dependent inhibition of three viral markers were determined with EC50 of 0.3 nM, 2 nM and 9 nM for viral particle, HBsAg and
Discussion
A small dihydroquinolizinone molecule, called DHQ-1, appeared to cause repression of HBsAg in the culture medium, as well as viral replicative intermediate DNA produced from integrated genomes (Han et al., 2015). We further found that DHQ-1 acted at the level of RNA by accelerating the nuclear degradation of viral transcripts, and confirmed that the compound also reduced liver HBV RNA and serum HBsAg in an HBV mouse model when administered orally.
DHQ-1 mediated reduction of HBV transcripts
Acknowledgements
We thank Dr. Shuping Tong for providing HBV plasmids of genotypes A, B and C, Dr. Koichi Watashi for the HepG2-NTCPc4 cell line. This work was supported by grants from the Commonwealth of Pennsylvania, the Hepatitis B Foundation and Arbutus Biopharma.
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