An HBV susceptibility variant of KNG1 modulates the therapeutic effects of interferons α and λ1 in HBV infection by promoting MAVS lysosomal degradation

Abstract

Background: Hepatitis B virus (HBV) infection is one of the main causes of hepatocellular carcinoma (HCC). The relationship between HBV infection and the host genome as well as their underlying mechanisms remain largely unknown.

Methods: In this study, we performed a whole-genome exon sequencing analysis of 300 sib-pairs of Chinese HBV-infected families with the goal of identifying variants and genes involved in HBV infection. A site-direct mutant plasmid was used to investigate the function of SNP rs76438938 in KNG1. The functional and mechanical studies of KNG1 were conducted with in vitro liver cell lines and a hydrodynamic injection model in vivo. The impact of KNG1 on HBV infection therapy was determined in hepatocytes treated with IFN-α/λ1.

Findings: Our whole-exon association study of 300 families with hepatitis B infection found that SNP rs76438938 in KNG1 significantly increased the risk for HBV infection, and the rs76438938-T allele was found to promote HBV replication by increasing the stability of KNG1 mRNA. By competitively binding HSP90A with MAVS, KNG1 can inhibit the expression of types I and III IFNs by promoting MAVS lysosomal degradation. Such suppression of IFN expression and promotion of HBV replication by Kng1 were further demonstrated with an animal model in vivo. Lastly, we showed that the rs76438938-C allele can improve the therapeutic effect of IFN-α and -λ1 in HBV infection.

Interpretation: This study identified a SNP, rs76438938, in a newly discovered host gene, KNG1, for its involvement in HBV infection and treatment effect through modulating the cellular antiviral process.

Funding: This study was supported in part by the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of the First Affiliated Hospital of Zhejiang University, the China Precision Medicine Initiative (2016YFC0906300), and the Research Center for Air Pollution and Health of Zhejiang University.

Keywords: Functional SNP; HBV infection; Interferon; KNG1; Treatment.

Fig. 1

Characterization of a functional variant rs76438938 in KNG1 affecting HBV infection. (a) Participants carrying the T allele and their sibs in family samples. (b) Schematic diagram of KNG1 alternative splicing. The red arrow marks rs76438938. (c) Both wild-type and mutant LMWK were overexpressed in HepG2.2.15 cells. (d) Levels of HBV DNA, HBV pgRNA, HBeAg, and HBsAg in HepG2.2.15 cells transfected with wild-type or mutant LMWK plasmid. (e) qRT-PCR analysis of KNG1 in L02 cells with CC and TT genotypes. (f) Effect of rs76438938 on the KNG1 mRNA stability. Actinomycin D (5 μg/ml) was used to treat cells, which were collected at 0, 2, 4 and 6 h after treatment. The mRNA was detected by qRT-PCR normalized to GAPDH. (g) Western blotting analysis of KNG1 in L02 cells with CC and TT genotypes. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 2

Determination of KNG1 in promoting HBV replication. (a) The difference of KNG1 expression level in PHHs before and after HBV infection for 40 h. The expression data was obtained from the GEO database (GSE69580). (b and c) Changes of KNG1 mRNA and protein levels in HepG2-NTCP cells before and after HBV infection for 48 h. (d) Western blotting analysis of KNG1 overexpression in HepG2 cells. Cells were transfected with pHBV and KNG1-Flag or vector plasmids for 48 h. (e) Levels of HBV DNA, HBV pgRNA, HBeAg, and HBsAg in HepG2 cells with KNG1 overexpression. (f) KNG1 knockdown in HepG2 cells transfected with pHBV plasmid and si-CTRL or si-KNG1 for 48 h. (g) Levels of HBV DNA, HBV pgRNA, HBeAg, and HBsAg in HepG2 cells with KNG1 knockdown. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 3

Downregulation of IFNs by KNG1 through MAVS inhibition. (a) Effect of KNG1 overexpression on types I and III IFNs in HepG2 cells. (b and c) Effect of KNG1 overexpression on MAVS in HepG2 cells. HepG2 cells were transfected with pHBV and KNG1-Flag or vector plasmids for 48 h. The MAVS mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively. (d) Effect of KNG1 knockdown on types I and III IFNs in HepG2 cells. (e and f) Effect of KNG1 knockdown on MAVS in HepG2 cells. HepG2 cells were transfected with pHBV plasmid and si-Ctrl or si-KNG1 for 48 h. The MAVS mRNA and protein levels were determined by qRT-PCR and Western blotting. (g and h) Role of MAVS in KNG1 regulating types I and III IFNs in HepG2.2.15 cells. HepG2.2.15 cells were transfected with control or MAVS-specific siRNAs together with KNG1 overexpression. (i) Levels of HBV DNA and HBV pgRNA in HepG2.2.15 cells. The mRNA and protein levels were determined by qRT-PCR and Western blotting. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 4

KNG1 decreases IFNs through MAVS inhibition in HSP90A-dependent manner. (a) Effect of KNG1 on MAVS lysosomal degradation in HepG2.2.15 cells. HepG2.2.15 cells were treated with 20 mM NH₄Cl or 10 μM MG132 for 6 h after KNG1-Flag plasmids were transfected. (b) Effect of 17-AAG on the regulation of KNG1 on MAVS protein. HepG2.2.15 cells were treated with 0.5 μM 17-AAG for 24 h after KNG1 overexpression. (c) Effect of 17-AAG on the regulation of KNG1 on types I and III IFNs. Levels of IFNs were determined by qRT-PCR normalized to GAPDH. (d and e) KNG1 and MAVS interact with HSP90A in HepG2.2.15 cells. Cells were co-transfected with HSP90A-His along with KNG1-Flag (d) or MAVS-Flag plasmids (e), then immunoprecipitated and immunoblotted with indicated antibodies. (f) Colocalization of KNG1, MAVS, and HSP90A in HepG2.2.15 cells. Cells were co-transfected with HSP90A-His along with KNG1-Flag or MAVS-Flag plasmids. Nuclei were stained with DAPI. Scale bar = 5 μm. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 5

KNG1 competitively blocks the binding of MAVS to HSP90A. (a) Effect of 17-AAG on viability in HepG2 cells. Cells were treated with different concentrations of 17-AAG for 24 h, and viability assays were performed by Cell Counting Kit-8. (b) Effect of 17-AAG on the binding of MAVS to HSP90A in HepG2 cells. Cells were treated with 0.5 μM 17-AAG for 24 h before immunoprecipitation of MAVS. (c) Effect of 17-AAG on MAVS protein in HepG2 cells. Cells were treated with increased concentrations of 17-AAG for 24 h. (d) Effect of 17-AAG on MAVS lysosomal degradation in HepG2 cells. Cells were treated with or without NH₄Cl (20 mM) before the addition of 17-AAG (0.5 μM). (e) Comparison of MAVS protein stability in HepG2 cells in cycloheximide (CHX) chase assay. Cells were treated with CHX (100 μg/μl) for increasing times (0–2 h) in the presence or absence of 17-AAG (0.5 μM). (f and g) Effect of KNG1 on the binding of MAVS and HSP90A in HepG2 cells. Vector or KNG1-Flag plasmids were co-transfected into HepG2 cells with pHBV and MAVS-His or HSP90A-His plasmids. Cell lysates were incubated with anti-His antibody and Protein A + G agarose. (h) Identification of HSP90A domain responsible for binding to MAVS and KNG1. HepG2 cells were transfected with HSP90A truncation mutants (top). Cell lysates were incubated with anti-His agarose, and immunoprecipitated proteins were immunoblotted with indicated antibodies. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 6

Kng1 promotes HBV replication by inhibiting Mavs-mediated IFNs expression in vivo. (a) C57BL/6 mice (n = 4) received HDI with pHBV (10 μg) along with vector (20 μg) or Kng1-Flag (20 μg) plasmids for 2, 4 and 6 days. (b and c) Levels of HBV DNA and HBV pgRNA in mice samples. (d and e) Western blotting analysis and qRT-PCR analysis of Mavs in mice liver samples. C57BL/6 mice (n = 9) received HDI with pHBV (10 μg) along with vector (20 μg) or Kng1-Flag (20 μg) plasmids for 4 days. (f) qRT-PCR analysis of Ifnl2/3 expression in mice liver samples. (g) ELISA assay analysis of IFN-β in mice serum samples. (h) Levels of HBV DNA, HBV pgRNA, and HBeAg in mice liver samples. (i) The immunohistochemical staining of Mavs and HBx in mice liver. The data were the average of three independent experiments. Scale bar = 50 μm. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 7

KNG1 overexpression reduces the therapeutic effect of IFN-α and IFN-λ1 in HBV infection. (a) KNG1 overexpression in HepG2-NTCP cells under IFN-α treatment. HepG2-NTCP cells were transfected with vector or KNG1-Flag plasmid for 24 h before HBV infection and then treated with 10 ng/ml IFN-α for 48 h after infection. (b–d) qRT-PCR analysis of IFNs, ISGs and HBV DNA in HepG2-NTCP cells after IFN-α treatment. (e) KNG1 overexpression in HepG2-NTCP cells under IFN-λ1 treatment. HepG2-NTCP cells were transfected with vector or KNG1-Flag plasmid for 24 h before HBV infection and then treated with 100 ng/ml IFN-λ1 for 48 h after infection. (f–h) qRT-PCR analysis of IFNs, ISGs and HBV DNA in HepG2-NTCP cells after IFN-λ1 treatment. Error bars indicate SEM. P-value was determined using a two-tailed unpaired t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Fig. 8

Working model of rs76438938 in KNG1 regulation of HBV infection. Under normal circumstances, both KNG1 and MAVS bind to the N-terminal domain of molecular chaperone HSP90A, which can stabilize MAVS protein. After HBV infection, MAVS receives and transmits signals and mediates the expression of types I and III IFNs. When carrying the risk allele T, increased mRNA stability of KNG1 leads to an increase in that protein, which competes with MAVS for binding to HSP90A. MAVS dissociated from HSP90A undergoes lysosomal degradation and cannot mediate the expression of types I and III IFNs, resulting in enhanced HBV replication, thereby increasing the risk of HBV infection.