Structural basis of the hepatitis B virus X protein in complex with DDB1

Abstract

A cure for chronic hepatitis B requires eliminating or permanently silencing covalently closed circular DNA (cccDNA). A pivotal target of this approach is the hepatitis B virus (HBV) X protein (HBx), which is a key factor that promotes transcription from cccDNA. However, the HBx structure remains unsolved. Here, we present the cryoelectron microscopy structure of HBx in complex with DDB1, which is an essential complex for cccDNA transcription. In this structure, hydrophobic interactions within HBx were identified, and mutational analysis highlighted their importance in the HBV life cycle. Our biochemical analysis revealed that the HBx-DDB1 complex directly interacts simultaneously with NSE3, which is a component of the SMC5/6 complex, and Spindlin1. Additionally, HBx-DDB1 complex dynamics were explored via high-speed atomic force microscopy. These findings provide comprehensive insights into the structure and function of HBx in HBV replication.

Keywords: HBx; cryo-EM structure; hepatitis B virus; high-speed AFM.

Fig. 1.

Structure of the HBx–DDB1 complex. (A) Functional analysis of HBx fused to DDB1 in SMC5/6 degradation. Western blotting analysis revealed the effects of the HBx–DDB1 and HBx–DDB1(4M) fusions on the degradation of the SMC5/6 complex in Expi293 cells. The reproducibility of this assay was confirmed by two independent experiments. (B) Cryo-EM density map of the HBx–DDB1(4M)–fused complex. The HBx–DDB1(4M)-fused complex was overexpressed and purified from Expi293 cells (SI Appendix, Fig. S1 D–F). HBx and DDB1 are colored orange and gray, respectively. (C) Cartoon representation of the atomic model fitted to the cryo-EM map (B), with the same color scheme as in (B). (D) Schematic linear representation of the HBx–DDB1 fusion protein. The helix–loop–helix of HBx that was observed in the cryo-EM structure (B and C) is labeled as HLH. The three beta-propeller domains of DDB1 are labeled BPA, BPB, and BPC.

Fig. 2.

Crucial roles of hydrophobic interactions within HBx in HBV replication. (A) Cryo-EM density map of the HBx–DDB1(4M)-fused complex shown in Fig. 1B. HBx and DDB1 are colored orange and gray, respectively. The regions containing the binding interface of HBx and DDB1 (B) and the hydrophobic interactions within HBx (C) are indicated by the blue and red dashed boxes, respectively. (B) The binding interface of HBx and DDB1. Arg96 of HBx (orange) and the main chain of DDB1 (gray) are shown as sticks. The dotted lines indicate intermolecular hydrogen bonds. Leu98 of HBx (orange) and Leu328, Pro358, Ala381, and Phe382 of DDB1 (gray) are shown as spheres. (C) Hydrophobic interactions within the HBx molecule. The hydrophobic residues Leu89, Leu93, Leu108, and Phe112 of HBx (orange) are shown as spheres. (D–F) Impact of the hydrophobic residues Leu89, Leu93, Leu108, and Phe112 of HBx on the ability of HBx to promote the degradation of the SMC5/6 complex. HEK293T cells were transduced with lentivirus for the expression of wild-type HA-HBx and the HBx mutants HA-HBx (R96E), HA-HBx (L89S, L93S), and HA-HBx (L108S, F112S). Western blotting analysis revealed the effects of HBx mutants on SMC6 degradation in HEK293T cells (D). The relative protein levels of SMC6 (E) and HA-HBx (F) normalized to those of α-tubulin are shown as the means ± SDs of three independent experiments. (G) Coimmunoprecipitation assays were performed with HEK293T cells transfected with StrepII-HBx mutants. The abundances of immunoprecipitated StrepII-HBx and coimmunoprecipitated endogenous DDB1 were assessed via western blotting. The reproducibility of this assay was confirmed by two independent experiments. (H–K) The impact of the hydrophobic residues Leu89, Leu93, Leu108, and Phe112 of HBx on viral replication was determined. HepG2-NTCP cells that were infected with HBx-deficient HBV (HBVΔX) were transcomplemented with HA-tagged wild-type HBx, HBx (L89S, L93S), HBx (L108S, F112S), or HBx (R96E). Western blotting analysis revealed the effects of HBx mutants on SMC6 degradation in HepG2-NTCP cells (H). The relative protein levels of SMC6 (I) and HA-HBx (J) normalized to those of α-tubulin are shown as the means ± SDs of three independent experiments. The levels of HBV RNA [pregenomic and precore (HBV3.5 kb)] and all RNA except for HBx (HBV3.5/2.4/2.1 kb) were measured via RT–qPCR and normalized to that of the Rhot2 gene. The means ± SDs from three independent experiments are shown (K).

Fig. 3.

HBx in complex with DDB1 directly binds to Spindlin1 and NSE3. (A) Interaction between the HBx–DDB1 fusion protein and Spindlin1. StrepII pull-down assays were performed with the StrepII–HBx–DDB1 and Spindlin1 proteins. (B and C) Interactions of the HBx–DDB1 fusion with SMC5/6 complex components. StrepII pull-down assays were performed with the StrepII–HBx–DDB1 and NSE1 (B), NSE3 (B), NSE4 (B), and NSE2 (C) proteins. (D and E) GST pull-down assays were performed with the GST-Spindlin1, StrepII–HBx–DDB1, and NSE3 proteins. The molar ratio of GST-Spindlin1 to StrepII–HBx–DDB1 was maintained at 1:1, while the amount of NSE3 was progressively increased to 1, 2, and 3 times the molar ratio (E). (F) Schematic representation of the binding of the HBx–DDB1 complex to NSE3 and Spindlin1. (G and H) Endogenous Spindlin1 interacts with the SMC5/6 complex in HeLa (G) and HepG2 (H) cells. Endogenous Spindlin1 was immunoprecipitated from HeLa (G) or HepG2 (H) cell lysates treated with Benzonase. The abundances of immunoprecipitated Spindlin1 and the coimmunoprecipitated SMC5/6 complex were evaluated by western blotting. The asterisk indicates the antibody light chain from the immunoprecipitation assay. All the experiments were independently repeated at least twice, and the results were reproduced.

Fig. 4.

HS-AFM analysis of HBx complexed with DDB1. (A) Cryo-EM density map of HBx complexed with DDB1 (without covalent fusion). HBx complexed with DDB1 without fusion was overexpressed and purified from Expi293 cells (SI Appendix, Fig. S9C). HBx and DDB1 are colored orange and gray, respectively. (B) Schematic linear representation of HBx complexed with DDB1 (without covalent fusion). The helix–loop–helix of HBx that was observed in the cryo-EM structure (A) is labeled as HLH. The three beta-propeller domains of DDB1 are labeled BPA, BPB, and BPC. (C and D) Sequential HS-AFM images of the HBx molecule complexed with DDB1 (C) and the HBx molecule dissociating from DDB1 (D) on the mica surface. The color gradient from black to white indicates the variation in height. (Scale bar, 10 nm.) (E) Calculated surface area (nm²) of the DDB1 molecule (n = 29) and the free HBx molecule dissociated from DDB1 (n = 9). (F) HS-AFM image of HBx in complex with DDB1 (upper panel). The line indicates the cross-sectional position used for the height distribution analysis shown in the lower panel. The arrows indicate the boundaries of the HBx molecule used to acquire the length data shown in (G). (G) The length of the HBx molecule protruding from the complex with DDB1 was determined from cross-sectional profiles acquired along the line shown in (F). The median length was calculated and plotted on the basis of measurements from fifteen representative HBx molecules in complex with DDB1. (H) Angular differences in DDB1 positioning. Angular differences from the initial position of the DDB1 molecule were measured at 0.3 s intervals (n = 5). (I) Sequential HS-AFM images of the HBx complex with both DDB1 and Spindlin1. The color gradient from black to white indicates the variation in height. (Scale bar, 10 nm.)