Characterization of genotype-specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme

Abstract

Hepatitis B e antigen (HBeAg) is a secreted version of hepatitis B virus (HBV) core protein that promotes immune tolerance and persistent infection. It is derived from a translation product of the precore/core gene by two proteolytic cleavage events: removal of the amino-terminal signal peptide and removal of the carboxyl-terminal arginine-rich sequence. Four RXXR motifs are present at the carboxyl terminus of the HBeAg precursor, with the first two fused as (151)RRGRSPR(157). Genotype A possesses two extra amino acids at the first motif ((151)RRDRGRSPR(159)), which weakens the first motif and separates it from the second one. Western blot analysis of patient sera revealed a single HBeAg form for genotypes B to D but two additional forms of larger sizes for genotype A. Site-directed mutagenesis and transfection experiments with human hepatoma cell lines indicated that HBeAg of genotype B is derived from cleavage at the first ((151)RRGR(154)) motif. The major HBeAg form of genotype A corresponds to cleavage at the second ((156)RSPR(159)) motif, and the other two forms are cleavage products of the first ((151)RRDR(154)) and third ((166)RRRR(169)) motifs, respectively. Only the cleavage product of the third motif of genotype A was observed in furin-deficient LoVo cells, and an inhibitor of furin-like proprotein convertases blocked cleavage of the first and second motifs in human hepatoma cells. In conclusion, our study reveals genotypic differences in HBeAg processing and implicates furin as the major enzyme involved in the cleavage of the first and second RXXR motifs.

FIG. 1.

Carboxyl-terminal sequences of e antigen precursors and sequential steps involved in HBeAg biosynthesis. (A) Schematic representation of the molecular events of HBeAg production. The number of residues in the core protein (183 aa) is based on non-A genotypes. (B) Comparison of the C-terminal sequences of p22 between genotype A and non-A genotypes. The four RXXR motifs are underlined, and the DR insertion in genotype A is boxed. (C) C-terminal sequence comparison of the e antigen precursors among WHV, WMHBV, and different HBV genotypes.

FIG. 2.

IP-Western blot analysis of HBeAg present in patient serum samples. HBeAg secreted from Huh7 cells transfected with genotype A (A2) and D (D1) constructs served as controls. Culture sup, culture supernatant.

FIG. 3.

Molecular forms of HBeAg secreted from human liver and kidney cell lines following transfection with precore/core constructs under the CMV promoter. (A) C-terminal sequences of P22 from 10 expression constructs, with numbering based on genotype A. 154Tr is an artificial construct with protein translation terminated after R154. (B) IP-Western blot analysis of HBeAg secreted from transfected HEK 293, Huh7, and HepG2 cells, respectively. The different size forms of HBeAg are labeled 1 to 4, from small to large. Mock, mock transfected.

FIG. 4.

Impact of mutating the first and second RXXR motifs on HBeAg production by a genotype B clone. (A) Amino acid sequences of clone B2 and its site-directed mutants, with the four RXXR motifs numbered. Cleavage efficiency in transfected Huh7 cells is indicated by the size of the triangle. (B) IP-Western blot analysis of HBeAg secreted from Huh7 cells. The numbers shown to the right correspond to the RXXR motifs shown in panel A. Mock, mock transfected.

FIG. 5.

Mutations capable of converting the multiple HBeAg forms of genotype A into a single-size form. (A) Amino acid sequences of three site-directed mutants of clone A2 and one mutant of clone B2. The triangle size indicates the efficiency of cleavage in Huh7 cells. (B and C) HBeAg expression from Huh7 and HepG2 cells, respectively. Numbers shown to the right indicate the motifs recognized. Mock, mock transfected.

FIG. 6.

Assignment of the multiple HBeAg species from wild-type genotype A to three RXXR motifs. (A) Amino acid sequences of clones A2 and A3, site-directed mutants of A2, as well as truncation mutants 154Tr and 169Tr, which serve as size markers. The two variant forms of these deletion mutants, R151G/R154G/154Tr and R167G/R169G/169Tr, are resistant to C-terminal trimming by basic carboxypeptidases. The size of the triangle indicates the abundance of the corresponding product in Huh7 cells. (B) HBeAg expression in Huh7 cells (top) and HepG2 cells (bottom). The numbers shown to the side correspond to motifs shown in panel A under A2. (C) Parallel analysis of HBeAg forms produced in HepG2 and Huh7 cells and impact of mutations that prevent C-terminal trimming of the Tr154 and Tr169 mutants. Mock, mock transfected.

FIG. 7.

Furin confers cleavage at the first and second motifs of genotype A. The furin-deficient LoVo cells were transfected with the A2 or A3 construct and subsequently infected with the adenovirus vector or recombinant adenovirus expressing chicken furin. 154Tr served as a size marker. (A) Schematic representation of cleavage in the absence or presence of chicken furin. (B) Quantification of secreted HBeAg by a commercial kit and IP-Western blot analysis of the various HBeAg forms. Mock, mock transfected.

FIG. 8.

Effects of a PC inhibitor and human furin overexpression on HBeAg processing in HEK 293, Huh7, and HepG2 cells. Cells were transfected in duplicate with HBeAg expression constructs together with an empty plasmid or a plasmid encoding human furin. One set of samples was treated with dec-RVKR-cmk. HBeAg was detected by IP-Western blot analysis. Mock, mock transfected.