Fig 1.
MSX1 reduces HBx protein level in vitro and in vivo.
Huh7 cells were transfected with pHBx-Flag (derived from HBV genotype B) and an increasing amount of pMSX1-Flag (A) or pshMSX1 (B) at a transfection ratio of 1:0, 1:0.5, 1:1, 1:2, 1:3 and 1:0, 1:0.5, 1:1, 1:2, respectively. (C) BALB/c mice were hydrodynamically injected (HDI) with 5 μg of pHBx-Flag and 5 μg of pMSX1-Flag or control plasmid (pCtrl). (D) Huh7, HepG2 and HEK293T cells were transfected with pHBx-Flag of indicated HBV genotype and pMSX1-Flag at a transfection ratio of 1:1. At 3 days post transfection or injection, HBx and exogenous/endogenous MSX1 in cultured cells or mouse livers were determined in Western blot. Protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1.
Fig 2.
MSX1 promotes ubiquitin-independent proteasomal degradation of HBx.
Huh7 cells were transfected with pHBx-Flag plus pMSX1-Flag or pCtrl (A), or plus pshMSX1 or its control plasmid (pshCtrl) (B) at a transfection ratio of 1:1. At 3 days post transfection, cells were treated with CHX (50 μg/ml) for indicated time. (C-E) Huh7 cells were transfected with pHBx-Flag and pMSX1-Flag at a transfection ratio of 1:1. At 60 h post transfection, cells were treated with chloroquine (10 μM) (C), different concentrations of MG132 (D) or PYR41 (E), or left untreated for additional 12 h. HBx and exogenous MSX1 were determined in Western blot using Flag antibody, while endogenous MSX1 and LC3B determined using MSX1 and LC3B antibodies respectively. Protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1. Quantification of HBx (A and B, right panels) at each time point was normalized to the respective β-actin and then to the HBx level at 0 h. (F) The effects of MSX1 on HBx/Kless expression in Huh7 cells were examined in Western blot (left). Huh7 cells were transfected with HA-tagged ubiquitin-expressing plasmid (pUb-HA) and pHBx-Flag or pHBx-Flag/Kless. At 60 h post transfection, cells were treated with MG132 for additional 12 h and then subjected to co-immunoprecipitation (Co-IP) assay to compare the ubiquitylation levels between wild type (WT) HBx and HBx/Kless (right panel). (G) Schematic presentation of the expression cassettes for HBx-fused NanoLuciferase (HBx-Nluc) plasmid and its serial deletion mutants. The HBx-Nluc contains the sequence of HBx, Nluc, and a flexible glycine–serine linker (G4S)3. (G4S)3 is a three-time repeat composed of four glycine (G) and one serine (S) residues. Deleted region within HBx is represented by dashed lines. Numbers denote amino acid positions. (H) Huh7 cultured in 24-well plates were transfected with 0.3 μg of HBx-NLuc or its derived mutants, plus 0.3 μg of pMSX1-Flag or pCtrl. Secreted Nluc was determined using Nluc reporter assays. Group means and SEMs of normalized data were presented and significances calculated using unpaired two-tailed t test. **, P < 0.01; ***, P < 0.001.
Fig 3.
Screening and identification of MSX1’s downstream effector genes associated with promoting HBx degradation.
(A) Huh7 cells were transfected with pMSX1-Flag and HA-tagged HBx-expressing plasmid (pHBx-HA) or its control plasmid at a transfection ratio of 1:1. At 60 h post transfection, cells were treated with MG132 for additional 12 h. Interaction between MSX1 and HBx was analyzed using Co-IP assay. (B) Schematic representation of pMSX1-Flag and its serial deletion mutants. Deleted region within MSX1 is represented by dashed lines, and numbers denote amino acid positions. (C) Western blot was performed to analyze the effects of MSX1 and its deletion mutants on HBx expression in Huh7 cells. (D) Huh7 cells were transfected with pMSX1-Flag or pCtrl in triplicates and then subjected to transcriptome sequencing analysis. Volcano plot (left) and heat map (right) were presented with top 10 upregulated DEGs indicated. (E) RT-qrtPCR assay was performed on pMSX-Flag- or pCtrl-transfected Huh7 cells to determine the mRNA expression levels of indicated genes. (F) 5 of the 7 confirmed MSX1-upregulated genes belong to heat shock protein (HSP) families. (G) Huh7 cells were transfected with pHBx-Flag and an increasing amount of Flag-tagged MSX1, HSPA6, DNAJA4, DNAJB1, HSPA1B, VGF, CRYAB, or RRAD overexpression plasmid at a transection ratio of 1:0, 1:0.5, 1:1, 1:2. Western blot was utilized to analyze the effects of exogenously expressed genes on HBx. (C and H) HBx protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1. Group means and SEMs of normalized data were presented and significances calculated using unpaired two-tailed t test. **, P < 0.01; ***, P < 0.001.
Fig 4.
MSX1 promotes HBx degradation by transcriptionally activating DNAJA4 and CRYAB expression.
Huh7 cells cultured in 12-well plates were transfected with 0.5 µg of pHBx-Flag and 0.5 µg of pMSX1-Flag or pCtrl, plus 0.5 µg of pshDNAJA4 or pshCRYAB or both (A). Huh7 cells were transfected with pHBx-Flag and pDNAJA4-Flag (B and E), or pCRYAB-Flag (C and F) or pCtrl at a transfection ratio of 1:1. At 60 h post transfection, cells were treated with MG132 (B and C) or PYR41 (E and F) for additional 12 h. At 3 days post transfection, HBx and exogenous/endogenous host proteins were determined in Western blot. (D) Huh 7 cells were transfected with pHBx-Flag and pDNAJA4-Flag, or pCRYAB-Flag or pCtrl at a transfection ratio of 1:1. At 3 days post transfection, cells were subjected to RT-qrtPCR to analyze HBx mRNA levels. The effects of exogenous DNAJA4 (G) and CRYAB (H) on HBx/Kless expression in Huh7 cells were examined in Western blot. (I) Huh7 cultured in 24-well plates were transfected with 0.3 μg of HBx-NLuc or its serial deletion mutants, plus 0.3 μg of pDNAJA4-Flag, pCRYAB-Flag or pCtrl. Secreted Nluc was determined using Nluc reporter assays. Protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1. Huh7 cells cultured in 24-well plates were transfected with 0.3 µg of DNAJA4 promoter reporter plasmid (J and K) or CRYAB promoter reporter plasmid (L), 0.1 µg of Renilla luciferase reporter plasmid (pRL-TK), plus 0. 3 µg of pMSX1-Flag or pCtrl, or 0.3, 0.6, 0.9 µg of pdel218–260. At 2 days post transfection, wild type and mutant MSX1 were determined in Western blot (J) while DNAJA4 and CRYAB promoter activities determined using dual-luciferase reporter assay (K and L). Huh7 cells cultured in 6-cm dishes were transfected with 2 µg of pMSX1-Flag or 6 µg of pdel218–260. At 3 days post transfection, cells were subjected to ChIP assay, and sonicated DNA immunoprecipitated by Flag antibody or mouse control IgG was quantitated in qrtPCR using specific primers targeting DNAJA4 promoter (M) and CRYAB promoter (N), and presented as percentage of input. Immunoprecipitated wild type and mutant MSX1 were determined using Western blot (O). Group means and SEMs of normalized values were presented and significances calculated using unpaired two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 5.
DNAJA4 and CRYAB promote HBx degradation through an active J domain and association with HBx C-terminal domain, respectively.
Huh7 cells were transfected with pDNAJA4-Flag, pCRYAB-Flag or pCtrl, together with or without pHBx-HA co-transfection. At 60 h post transfection, cells were treated with MG132 for additional 12 h. Interaction between DNAJA4/CRYAB and HBx was determined via Co-IP assay using anti-Flag (A) or anti-HA (B) beads. (C) The interaction between endogenous CRYAB and HBx was determined in pHBx-HA transfected Huh7 cells using Co-IP assay. (D) Schematic presentation of plasmids expressing EGFP, EGFP-HBx or EGFP-HBx(delC). Deleted region within HBx is represented by dashed lines. Numbers denote amino acid positions. (E) Huh7 cells were transfected with pEGFP, pEGFP-HBx or pEGFP-HBx(delC), plus pDNAJA4-Flag, pCRYAB-Flag or pCtrl at a transfection ratio of 1:1. At 3 days post transfection, proteins were analyzed via Western blot using anti-EGFP or anti-Flag antibody. (F) The interaction between EGFP-HBx/EGFP-HBx(delC) and exogenous CRYAB was analyzed using Co-IP assay as similarly done in A. (G) Huh7 cells were co-transfected with pCRYAB-Flag and pEGFP-HBx or pEGFP-HBx(delC). At 2 days post transfection, intracellular distribution of exogenous CRYAB and EGFP-HBx or EGFP-HBx(delC) was analyzed in immunofluorescence assay. (H) Comparison of the effects of wild type DNAJA4 and its mutant with H34Q substitution on HBx expression in Huh7 cells. Protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1.
Fig 6.
DNAJA4 and CRYAB HBx-dependently inhibit HBV gene expression and genome replication in vitro.
Huh7 cells cultured in 6-well plates were transfected with 1 µg of prcccDNA (A and B) or prcccDNA/Xnull (C) and 1 µg of pCre, plus 1 µg of pMSX1-Flag, pDNAJA4-Flag, pCRYAB-Flag or pCtrl (A and C), or plus 1 µg of pshMSX1, pshDNAJA4, pshCRYAB or pshCtrl (B). At 3 days post transfection, intracellular HBV replication, HBx and exogenous/endogenous host proteins were analyzed using Southern and Western blots, respectively. Secreted HBV antigens (HBsAg and HBeAg) and supernatant HBV DNA were assayed using ELISA and qrtPCR, respectively. Viral replication and protein levels were quantified using densitometry scanning and normalized using respective control as 1. Group means and SEMs of normalized values were presented and significances calculated using unpaired two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 7.
Comparison of expression of MSX1 and its effector genes in CHB patients at IT phase vs.
IA phase. Liver biopsy samples collected from patients in IT and IA phases were subjected to RNA microarray analysis in our previous study [33] and intrahepatic MSX1 (A), DNAJA4 (B), CRYAB (C) and DNAJB1 (D) RNA levels were analyzed with group size indicated (n). Biopsy samples from 20 IT and IA patients (S2 Table) were assayed for protein expression of above genes using Western blot (I). Protein levels were quantified using densitometry scanning (E-H). Group means and SEMs were presented and significances calculated using unpaired two-tailed t-test. *, P < 0.05; **, P < 0.01.
Fig 8.
MSX1 restricts HBV gene expression and replication in a humanized liver mouse model of HBV infection.
(A) Schematic description of the experiments in human liver-chimeric mice. Mice were injected with AAV-MSX1-Flag or AAV-Ctrl via tail vein and each group contained 3 mice. At 3 weeks post AAV injection, mice were injected with HBV, and 3 weeks later serum and liver tissue samples collected. Serum human albumin (B), ALT (C), HBsAg (D) and HBeAg (E) were analyzed in commercial quantitative assays. (F) H&E staining was performed on liver tissues. HBV cccDNA (G), 3.5 kb RNA (H) and total viral RNA (I) in livers were analyzed using qrtPCR. (J) Intracellular HBV replication, endogenous DNAJA4/CRYAB and exogenous MSX1 were analyzed using Southern and Western blots, respectively. Capsid levels were determined using native agarose gel electrophoresis followed by Western blot. Group means and SEMs of normalized values were presented and significances calculated using unpaired two-tailed t test. *, P < 0.05; **, P < 0.01. Scale bars: 200 μm.