Antagonizing NSDHL Mediates Innate Immunity Suppression through IFNAR1 Degradation and Facilitates Hepatitis B Virus Replication

乙型肝炎病毒通過抑制NSDHL表達從而促進病毒複製的機制研究

Student thesis: Doctoral Thesis

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Award date7 May 2021

Abstract

Hepatitis virus B (HBV) is a hepatotropic, enveloped, coated, double-stranded DNA virus that causes both acute and chronic hepatitis. Chronic HBV infection is one of the most important etiological factors for liver cirrhosis and hepatocellular carcinoma (HCC), which causes more than 750 million HBV-related deaths each year. Despite routine vaccines, which consist of nucleoside or nucleotide drugs (NUCs) together with interferon-α (IFNα), current treatment cannot eradicate the virus from the host’s body sustainably. Instead, we can prevent its replication, although this is only partly successful in delaying and/or reducing rates of liver cirrhosis and HCC. There is still considerable value in exploring the mechanism by which the disease progresses and suggesting novel antiviral therapies.

Host factors play key roles in establishing successful viral infection. One major challenge is to reinvigorate the exhausted immune response within the liver microenvironment. Previous work on OxICAT/MS in our lab has identified NSDHL as a redox protein in HBV infections. In this study, we firstly discovered that NSDHL was downregulated in HBV infections. The downregulation of NSDHL could be induced in HepG2 and Huh7 cells by transfection with viral proteins, including HBcAg, HBXAg, and HBsAg. Secondly, we constructed stable NSDHL knockdown and overexpression cell lines. By loss- and gain-of-function experiments, we showed that NSDHL played crucial roles in facilitating HBV infection. Both cytoplasm and supernatant viral DNA were increased when we knocked down the expression level of NSDHL. Both Western blotting and ELISA revealed that HBV protein expression increased in NSDHL-silenced cell lines. These data demonstrated the importance of NSDHL in HBV infections. Downregulation of NSDHL increased the reproduction of HBV, while upregulation inhibited the reproduction of the virus.

There are very few studies on the functions of NSDHL. We performed an RNA sequencing assay to reveal the transcriptome regulated by NSDHL. The RNA-sequence data suggested that NSDHL was involved in the innate immune response by modulating the expression of interferon alpha and beta receptor subunit 1 (IFNAR1). We confirmed that the levels of both IFNAR1 mRNA and protein IFNAR1 declined in HepG2 and Huh7 cell lines with NSDHL knockdown, compared with the control. In addition, we found that downregulation of NSDHL functioned as a negative regulator of the type I interferon (IFN) response. Phosphorylation of Janus tyrosine kinase (JAK) 1, tyrosine kinase 2 (TYK2), signal transducer, and activator of transcription 1/2 (STAT1/2) was decreased after silencing NSDHL. Downstream genes in the IFN pathway, such as IFN-α, IFN-β, IRF7, OAS3, CXCL10, and IL-1β, were reduced accordingly. When we overexpressed NSDHL, the protein level of IFNAR1 was increased and the related genes in the downstream pathway were upregulated. The phosphorylation of JAK1, TYK2, and STAT1/2 respectively was elevated in NSDHL-overexpressed cells compared with the control. However, NSDHL overexpression did not change the mRNA level of IFNAR1. These results support NSDHL as an innate immunity regulator.

We further demonstrated that NSDHL downregulation induced the degradation of IFNAR1 via activation of autophagy response. As a result of the knockdown of NSDHL, the degradation of IFNAR1 was increased, and the degradation could be partially rescued by lysosome inhibitor. We detected the expression levels of p62, LC3I/II, HSC70 and LAMP2A protein and found that autophagy response was induced when NSDHL was inhibited. Opposite results were observed in NSDHL overexpressed cells.

Last but not the least, with the help of GO and KEGG pathway enrichment analysis, we confirmed with Western blotting and revealed that downregulation of NSDHL abolished the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, inducing the autophagy response for IFNAR1 degradation, in turn facilitating HBV infections. Moreover, oil red staining showed that downregulation of NSDHL caused lipid droplets accumulation through precursors in cholesterol synthesis pathway, such as lanosterol, FF-MAS and T-MAS, which in turn might induce autophagy response.