Characterization of genotype-specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme.
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.
Ito
Kiyoaki
K
Liver Research Center, Island Hospital and Warren Alpert School of Medicine, Brown University, Providence, Rhode Island, USA.
Kim
Kyun-Hwan
KH
Lok
Anna Suk-Fong
AS
Tong
Shuping
S
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D045744:Cell Line, Tumor; D045683:Furin / Q000172:deficiency / Q000378:metabolism*; D005838:Genotype; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D014162:Transfection
D006513:Hepatitis B e Antigens; C468731:FURIN protein, human; D045683:Furin