Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP

Grace Naswa Makokha,C Nelson Hayes,Takuro Uchida,Rie Akiyama,Masataka Tsuge,Hiromi Abe-Chayama,Tadahiko Yoshima,Kazuaki Chayama,Sajeda Chowdhury,Yuji Ishida,Yizhou Zhang,Chise Tateno

doi:10.1038/s41598-019-44934-5

Grace Naswa Makokha, C Nelson Hayes + Show 10 more

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https://doi.org/10.1038/s41598-019-44934-5

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Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein’s broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

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Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma
The 3.5-kb mRNA, which consists of precore and pregenomic mRNAs together with total HBV mRNAs were remarkably reduced in trans-active response DNA binding protein (TARDBP) depleted cells (Fig. 1f)
In this study we demonstrate for the first time that TARDBP binds both to HBV DNA as well as RNA and functions as a transcriptional activator through binding to the core promoter and as a splicing suppressor through binding to pgRNA

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Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identifying cellular host factors that are required to support the HBV life cycle will be a major step forward in the process of developing new anti-HBV drugs. To further understand the molecular mechanisms behind the control of HBV replication and pathogenesis, we identified novel host factors that could play a role in the viral lifecycle using primary human hepatocytes derived from human hepatocyte transplanted chimeric mice, an infection system that we recently established[10]. TARDBP is known to be frequently mutated in sporadic and familial amyotrophic lateral sclerosis (ALS), as well as in patients with frontotemporal lobar degeneration (FTLD), providing evidence of a direct link between TARDBP abnormalities and neurodegeneration[15] The role of this protein in HBV pathogenesis remains unreported

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