The N-Terminus Makes the Difference: Impact of Genotype-Specific Disparities in the N-Terminal Part of The Hepatitis B Virus Large Surface Protein on Morphogenesis of Viral and Subviral Particles

Abstract

The N-terminus of the hepatitis B virus (HBV) large surface protein (LHB) differs with respect to genotypes. Compared to the amino terminus of genotype (Gt)D, in GtA, GtB and GtC, an additional identical 11 amino acids (aa) are found, while GtE and GtG share another similar 10 aa. Variants of GtB and GtC affecting this N-terminal part are associated with hepatoma formation. Deletion of these amino-terminal 11 aa in GtA reduces the amount of LHBs and changes subcellular accumulation (GtA-like pattern) to a dispersed distribution (GtD-like pattern). Vice versa, the fusion of the GtA-derived N-terminal 11 aa to GtD causes a GtA-like phenotype. However, insertion of the corresponding GtE-derived 10 aa to GtD has no effect. Deletion of these 11aa decreases filament size while neither the number of released viral genomes nor virion size and infectivity are affected. A negative regulatory element (aa 2-8) and a dominant positive regulatory element (aa 9-11) affecting the amount of LHBs were identified. The fusion of this motif to eGFP revealed that the effect on protein amount and subcellular distribution is not restricted to LHBs. These data identify a novel region in the N-terminus of LHBs affecting the amount and subcellular distribution of LHBs and identify release-promoting and -inhibiting aa residues within this motive.

Keywords: HBV filaments; HBV genotypes; HBV surface protein; PreS1 deletion; morphogenesis.

Figure 1

Sequence comparison of amino acids 1–18 in the pres1 of different genotypes and the derivatives.

Figure 2

The presence of GtA-derived 11 aa is accompanied by higher amounts of intracellular and extracellular large surface proteins (LHBs). (A) Serums from patients infected with GtA variants containing PreS1 start codon deletion were analyzed by Western blot using a PreS1-specific serum (MA18/7). Purified wildtype GtA particles were used as control. (B) Supernatants and lysates from Huh7.5 cells transfected with genomes GtA, GtD and the derivatives were analyzed by Western blot using MA18/7 and a core-specific serum (K46). β-actin was used as loading control. (C–F) The signals of LHBs and core in the Western blot were quantified by Image studio lite from LI-COR Biosciences. (G,H) Supernatants from Huh7.5 cells transfected with genomes GtA, GtD and the derivatives were analyzed by core ELISA. (I,J) Supernatants and lysates from Huh7.5 cells transfected with genomes GtA, GtD and the derivatives were analyzed by HBsAg ELISA. The signal from GtA or GtD was standardized as 1. Ls, lysate; sp, supernatant. These quantitative data are mean values from three independent experiments. *, p < 0.05; **, p < 0.01.

Figure 3

The presence of GtA-derived 11 aa causes significant subcellular accumulation of LHBs. Huh7.5 cells overexpressing genome GtA, GtD and the derivatives were analyzed by confocal laser scanning microscopy (CLSM) using a PreS1-specific monoclonal MA18/7 and a core-specific polyclonal antiserum (Dako). Nuclei were stained with DAPI (blue). The merge of the fluorescences were shown in the bottom row. The figures showed a representative result; comparable data were obtained for the analysis of 10 different cells.

Figure 4

The presence of GtA-derived 11 aa promotes the generation of abundant longer filaments. (A,B) Polyethylene glycol (PEG)-concentrated supernatants from Huh7.5 cells overexpressing the genome GtA, GtD and the derivatives were analyzed by 10–30% (w/w) discontinuous sucrose gradient ultracentrifugation. A total of 24 fractions were harvested and fractions 10 to 24 were analyzed by refractive index, as well as Western blot using the PreS1-domain-specific monoclonal MA18/7. The signal of LHBs was measured by Image studio lite and the signal from fraction 24 was standardized as 1. (C,D) Filament-rich fractions (F20 in GtA, F17 in GtA_∆11aa, F15 in GtD, F21 in GtD_(+)11aa(GtA) and F18 in GtD_(+)10aa(GtE)) were analyzed by electron microscopy (EM). The size (diameter and length) of more than 80 filaments (101 in GtA, 136 in GtA_∆11aa, 87 in GtD, 108 in GtD_(+)11aa(GtA) and 81 in GtD_(+)10aa(GtE)) was measured by an EM-specific software Radius (EMSIS). (E–H) HBsAg-containing particles from Huh7.5 cells overexpressing GtA, GtD and the derivatives were precipitated by 10% PEG and then purified by sucrose ultracentrifugation followed by qPCR (E,F) and EM (G,H). (G) The diameter of more than 20 virions (47 in GtA, 92 in GtA_∆11aa, 90 in GtD, 42 in GtD_(+)11aa(GtA) and 22 in GtD_(+)10aa(GtE)) was measured by an EM-specific software Radius (EMSIS). *, p < 0.05; **, p < 0.01; ***, p < 0.001, **** p < 0.0001.

Figure 5

Deletion of 11 aa in the N-terminal part of GtA PreS1 has no impact on viral infectivity. (A–C) Differentiated HepaRG were infected with HBV from GtA and GtA_∆11aa at multiplexity of infection (MOI) 1600. Culture supernatants collected at indicated time points were analyzed by HBsAg ELISA (5A) and HBeAg ELISA (5B). The lowest signals (5 days post-infection) were standardized as 1. The cells were fixed and analyzed by CLSM using the MA18/7. (D,E) Differentiated HepaRG were infected with HBV from GtD, GtD_(+)10aa(GtE) and GtD_(+)11aa(GtA) at MOI 200 and 1600. Culture supernatants collected at 5- and 11-day post-infection were analyzed by HBsAg ELISA. These quantitative data are mean values from three independent experiments.

Figure 6

The GtA-derived 11 aa comprise a positive and a negative region regulating LHBs production and secretion. (A) Supernatants and lysates from Huh7.5 cells transfected with genomes GtA and the derivatives were analyzed by Western blot using a PreS1-specific serum (MA18/7) and a core-specific serum (K46). β-actin was used as loading control. (B–E) The signals of LHBs and core in the Western blot were quantified by Image studio lite from LI-COR Biosciences. (F) Supernatants from Huh7.5 cells transfected with genomes GtA and the derivatives were analyzed by core ELISA. (G,H) Supernatants and lysates from Huh7.5 cells transfected with genomes GtA and the derivatives were analyzed by HBsAg ELISA. The signal from GtA was standardized as 1. Ls, lysate; sp, supernatant. These quantitative data are mean values from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7

C-terminal deletions and substitution in 11 aa change the pattern of subcellular LHBs distribution. Huh7.5 cells overexpressing genome GtA and the derivatives were analyzed by confocal laser scanning microscopy (CLSM) using the LHB-specific monoclonal MA18/7 and core-specific polyclonal antiserum (Dako). Nuclei were stained with DAPI (blue). The merge of the fluorescences were shown in the right column. The figures showed a representative result; comparable data were obtained for the analysis of 10 different cells.

Figure 8

N-terminal 10-aa-deletion in GtE PreS1 domain increases dramatically the production and release of LHBs. (A) Supernatants and lysates from Huh7.5 cells transfected with genomes GtE and the derivatives were analyzed by Western blot using the PreS1-specific serum (MA18/7) and the core-specific serum (K46). β-actin was used as loading control. (B–D) The signals of LHBs and core in the Western blot were quantified by Image studio lite from LI-COR Biosciences. (E,F) Supernatants from Huh7.5 cells transfected with genomes GtE and the derivatives were analyzed by core ELISA and HBsAg ELISA. pUC18-transfected cells were used as negative control. The signal from GtE was standardized as 1. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 9

The impact of the N-terminal 11aa and 10aa on amount and distribution is independent from the LHBs context. (A) Schematic illustration of the start sites of eGFP and its fusion protein containing either 11 aa or 10 aa derived from N-terminal PreS1 of GtA or GtE. (B) eGFP level in Huh7.5 cells overexpressing genomes eGFP and its derivatives were analyzed by Western blot using an anti-eGFP specific serum and by microplate reader from Tecan by exciting the protein at 490 nm. As control, actin was used in Western blot, and in the microplate reader assay, nuclei were stained with DAPI and excited at 358 nm. (C) Huh7.5 cells overexpressing genomes eGFP and its derivatives were analyzed by Operetta high-content imaging system from PerkinElmer. Nuclei were stained with DAPI in blue. The signal from eGFP was standardized as 1. Ls, lysate; sp, supernatant. These quantitative data are mean values from three independent experiments. (D) CLSM analysis of Huh7.5 cells expressing these mutants. *, p < 0.05; **, p < 0.01.