Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Ganem D & Varmus HE (1987) The molecular biology of the hepatitis B viruses. Annu Rev Biochem 56, 651–693. PubMed

Nassal M & Schaller H (1993) Hepatitis B virus replication. Trends Microbiol 1, 221–228. PubMed

Ou JH (1997) Molecular biology of hepatitis B virus e antigen. J Gastroenterol Hepatol 12, S178–S187. PubMed

Chen M, Sällberg M, Hughes J, Jones J, Guidotti LG, Chisari FV, Billaud J‐N & Milich DR (2005) Immune tolerance split between hepatitis B virus precore and core proteins. J Virol 79, 3016–3027. PubMed PMC

Milich D & Liang TJ (2003) Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology 38, 1075–1086. PubMed

Milich DR, Chen MK, Hughes JL & Jones JE (1998) The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. J Immunol 160, 2013–2021. PubMed

Ou JH, Laub O & Rutter WJ (1986) Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci USA 83, 1578–1582. PubMed PMC

DiMattia MA, Watts NR, Stahl SJ, Grimes JM, Steven AC, Stuart DI & Wingfield PT (2013) Antigenic switching of hepatitis B virus by alternative dimerization of the capsid protein. Structure 21, 133–142. PubMed PMC

Nassal M & Rieger A (1993) An intramolecular disulfide bridge between Cys‐7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus. J Virol 67, 4307–4315. PubMed PMC

Selzer L, Katen SP & Zlotnick A (2014) The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability. Biochemistry 53, 5496–5504. PubMed PMC

Jean‐Jean O, Salhi S, Carlier D, Elie C, De Recondo AM & Rossignol JM (1989) Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site. J Virol 63, 5497–5500. PubMed PMC

Standring DN, Ou JH, Masiarz FR & Rutter WJ (1988) A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes. Proc Natl Acad Sci USA 85, 8405–8409. PubMed PMC

Ito K, Kim K‐H, Lok AS‐F & Tong S (2009) Characterization of genotype‐specific carboxyl‐terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme. J Virol 83, 3507–3517. PubMed PMC

Messageot F, Salhi S, Eon P & Rossignol J (2003) Proteolytic processing of the hepatitis B virus e antigen precursor. J Biol Chem 278, 891–895. PubMed

Chen MT, Billaud J‐N, Sällberg M, Guidotti LG, Chisari FV, Jones J, Hughes J & Milich DR (2004) A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci USA 101, 14913–14918. PubMed PMC

Riordan SM, Skinner N, Kurtovic J, Locarnini S & Visvanathan K (2006) Reduced expression of toll‐like receptor 2 on peripheral monocytes in patients with chronic hepatitis B. Clin Vaccine Immunol 13, 972–974. PubMed PMC

Visvanathan K, Skinner NA, Thompson AJV, Riordan SM, Sozzi V, Edwards R, Rodgers S, Kurtovic J, Chang J, Lewin S et al. (2007) Regulation of Toll‐like receptor‐2 expression in chronic hepatitis B by the precore protein. Hepatology 45, 102–110. PubMed

Lang T, Lo C, Skinner N, Locarnini S, Visvanathan K & Mansell A (2011) The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll‐like receptor signaling pathway. J Hepatol 55, 762–769. PubMed

Kimura T, Ohno N, Terada N, Rokuhara A, Matsumoto A, Yagi S, Tanaka E, Kiyosawa K, Ohno S & Maki N (2005) Hepatitis B virus DNA‐negative dane particles lack core protein but contain a 22‐kDa precore protein without C‐terminal arginine‐rich domain. J Biol Chem 280, 21713–21719. PubMed

Hong X, Luckenbaugh L, Perlman D, Revill PA, Wieland SF, Menne S & Hu J (2021) Characterization and application of precore/core‐related antigens in animal models of hepatitis B virus infection. Hepatology 74, 99–115. PubMed PMC

Hong X, Luckenbaugh L, Mendenhall M, Walsh R, Cabuang L, Soppe S, Revill PA, Burdette D, Feierbach B, Delaney W et al. (2021) Characterization of hepatitis B precore/core‐related antigens. J Virol 95, e01695‐20. PubMed PMC

Garcia PD, Ou JH, Rutter WJ & Walter P (1988) Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. J Cell Biol 106, 1093–1104. PubMed PMC

Ou JH, Yeh CT & Yen TS (1989) Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide. J Virol 63, 5238–5243. PubMed PMC

Duriez M, Rossigno JM & Sitterlin D (2008) The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER‐associated degradation pathway. J Biol Chem 283, 32352–32360. PubMed

Deroubaix A & Kramvis A (2021) In vitro expression of precore proteins of hepatitis B virus subgenotype A1 is affected by HBcAg, and can affect HBsAg secretion. Sci Rep 11, 8167. PubMed PMC

Scaglioni PP, Melegari M & Wands JR (1997) Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame. Virology 233, 374–381. PubMed

Locarnini S, Shaw T, Dean J, Colledge D, Thompson A, Li K, Lemon SM, Lau GGK & Beard MR (2005) Cellular response to conditional expression of the hepatitis B virus precore and core proteins in cultured hepatoma (Huh‐7) cells. J Clin Virol 32, 113–121. PubMed

Inoue J, Krueger EW, Chen J, Cao H, Ninomiya M & McNiven MA (2015) HBV secretion is regulated through the activation of endocytic and autophagic compartments mediated by Rab7 stimulation. J Cell Sci 128, 1696–1706. PubMed PMC

Liu D, Cui L, Wang Y, Yang G, He J, Hao R, Fan C, Qu M, Liu Z, Wang M et al. (2016) Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity. Hepatology 64, 390–404. PubMed

Mitra B, Wang J, Kim ES, Mao R, Dong M, Liu Y, Zhang J & Guo H (2019) Hepatitis B virus precore protein p22 inhibits alpha interferon signaling by blocking STAT nuclear translocation. J Virol 93, e00196‐19. PubMed PMC

Lang S, Pfeffer S, Lee PH, Cavalié A, Helms V, Förster F & Zimmermann R (2017) An update on Sec 61 channel functions, mechanisms, and related diseases. Front Physiol 8, 1–22. PubMed PMC

Alder NN, Shen Y, Brodsky JL, Hendershot LM & Johnson AE (2005) The molecular mechanisms underlying BiP‐mediated gating of the Sec61 translocon of the endoplasmic reticulum. J Cell Biol 168, 389–399. PubMed PMC

Hamman BD, Hendershot LM & Johnson AE (1998) BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92, 747–758. PubMed

Haßdenteufel S, Johnson N, Paton AW, Paton JC, High S & Zimmermann R (2018) Chaperone‐mediated Sec61 channel gating during ER import of small precursor proteins overcomes Sec61 inhibitor‐reinforced energy barrier. Cell Rep 23, 1373–1386. PubMed PMC

Fons RD, Bogert BA & Hegde RS (2003) Substrate‐specific function of the translocon‐associated protein complex during translocation across the ER membrane. J Cell Biol 160, 529–539. PubMed PMC

Pfeffer S, Burbaum L, Unverdorben P, Pech M, Chen Y, Zimmermann R, Beckmann R & Förster F (2015) Structure of the native Sec61 protein‐conducting channel. Nat Commun 6, 8403. PubMed PMC

Hartmann E, Görlich D, Kostka S, Otto A, Kraft R, Knespel S, Bürger E, Rapoport TA & Prehn S (1993) A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur J Biochem 214, 375–381. PubMed

Yang SQ, Walter M & Standring DN (1992) Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide. J Virol 66, 37–45. PubMed PMC

Lubyová B & Weber J (2020) Posttranslational modifications of HBV core protein. Acta Virol 64, 177–186. PubMed

Yeh CT & Ou JH (1991) Phosphorylation of hepatitis B virus precore and core proteins. J Virol 65, 2327–2331. PubMed PMC

Lainé S, Thouard A, Derancourt J, Kress M, Sitterlin D & Rossignol J‐M (2003) In vitro and in vivo interactions between the hepatitis B virus protein P22 and the cellular protein gC1qR. J Virol 77, 12875–12880. PubMed PMC

Heger‐Stevic J, Zimmermann P, Lecoq L, Böttcher B & Nassal M (2018) Hepatitis B virus core protein phosphorylation: identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure. PLoS Pathog 14, e1007488. PubMed PMC

Nguyen D, Stutz R, Schorr S, Lang S, Pfeffer S, Freeze HH, Förster F, Helms V, Dudek J & Zimmermann R (2018) Proteomics reveals signal peptide features determining the client specificity in human TRAP‐dependent ER protein import. Nat Commun 9, 3765. PubMed PMC

Ludgate L, Adams C & Hu J (2011) Phosphorylation state‐dependent interactions of Hepadnavirus core protein with host factors. PLoS One 6, e29566. PubMed PMC

Selzer L, Kant R, Wang JC‐Y, Bothner B & Zlotnick A (2015) Hepatitis B virus core protein phosphorylation sites affect capsid stability and transient exposure of the C‐terminal domain. J Biol Chem 290, 28584–28593. PubMed PMC

Wittkop L, Schwarz A, Cassany A, Grün‐Bernhard S, Delaleau M, Rabe B, Cazenave C, Gerlich W, Glebe D & Kann M (2010) Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation. Cell Microbiol 12, 962–975. PubMed

Liao W & Ou JH (1995) Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. J Virol 69, 1025–1029. PubMed PMC

Rabe B, Vlachou A, Pante N, Helenius A & Kann M (2003) Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci USA 100, 9849–9854. PubMed PMC

Lubyova B, Hodek J, Zabransky A, Prouzova H, Hubalek M, Hirsch I & Weber J (2017) PRMT5: a novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core. PLoS One 12, e0186982. PubMed PMC

Li HC, Huang EY, Su PY, Wu SY, Yang CC, Lin YS, Chang WC & Shih C (2010) Nuclear export and import of human hepatitis B virus capsid protein and particles. PLoS Pathog 6, e1001162. PubMed PMC

Yeh CT, Hong LH, Ou JH, Chu CM & Liaw YF (1996) Characterization of nuclear localization of a hepatitis B virus precore protein derivative P22. Arch Virol 141, 425–438. PubMed

Wasenauer G, Köck J & Schlicht HJ (1992) A cysteine and a hydrophobic sequence in the noncleaved portion of the pre‐C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus. J Virol 66, 5338–5346. PubMed PMC

Shaffer KL, Sharma A, Snapp EL & Hegde RS (2005) Regulation of protein compartmentalization expands the diversity of protein function. Dev Cell 9, 545–554. PubMed

Levine CG, Mitra D, Sharma A, Smith CL & Hegde RS (2005) The efficiency of protein compartmentalization into the secretory pathway. Mol Biol Cell 16, 279–291. PubMed PMC

Rapoport TA (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450, 663–669. PubMed

Bonardi F, Halza E, Walko M, Du Plessis F, Nouwen N, Feringa BL & Driessen AJM (2011) Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules. Proc Natl Acad Sci USA 108, 7775–7780. PubMed PMC

Arkowitz RA, Joly JC & Wickner W (1993) Translocation can drive the unfolding of a preprotein domain. EMBO J 12, 243–253. PubMed PMC

Hegde RS & Kang SW (2008) The concept of translocational regulation. J Cell Biol 182, 225–232. PubMed PMC

Kriegler T, Lang S, Notari L & Hessa T (2020) Prion protein translocation mechanism revealed by pulling force studies. J Mol Biol 432, 4447–4465. PubMed

Kriegler T, Kiburg G & Hessa T (2020) Translocon‐associated protein complex (TRAP) is crucial for co‐translational translocation of pre‐proinsulin. J Mol Biol 432, 166694. PubMed

Guo X, Chen P, Hou X, Xu W, Wang D, Wang TY, Zhang L, Zheng G, Gao ZL, He CY et al. (2016) The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely. Sci Rep 6, 25552. PubMed PMC

Holden P & Horton WA (2009) Crude subcellular fractionation of cultured mammalian cell lines. BMC Res Notes 2, 243. PubMed PMC

Langerová H, Lubyová B, Zábranský A, Hubálek M, Glendová K, Aillot L, Hodek J, Strunin D, Janovec V, Hirsch I et al. (2020) Hepatitis B core protein is post‐translationally modified through K29‐linked ubiquitination. Cells 9, 2547. PubMed PMC

Ni Y, Sonnabend J, Seitz S & Urban S (2010) The pre‐S2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L‐protein‐connected virus assembly. J Virol 84, 3879–3888. PubMed PMC

Perez‐Riverol Y, Csordas A, Bai J, Bernal‐Llinares M, Hewapathirana S, Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M et al. (2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res 47, D442–D450. PubMed PMC

Zobrazit více v PubMed

RefSeq
BAJ51641.1, Q64896, P0C6J2, P0C6J9