Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids
Autoři:
Zhanying Hu aff001; Haiqun Ban aff001; Haiyan Zheng aff002; Mingliang Liu aff003; Jinhong Chang aff001; Ju-Tao Guo aff001
Působiště autorů:
Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
aff001; Biological mass spectrometry facility, Robert Wood Johnson Medical School and Rutgers, The State University of New Jersey. Piscataway, New Jersey, United States of America
aff002; Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tian-Tan Xi-Li, Beijing, China
aff003
Vyšlo v časopise:
Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids. PLoS Pathog 16(7): e1008669. doi:10.1371/journal.ppat.1008669
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.ppat.1008669
Souhrn
Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and β were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and β. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.
Klíčová slova:
Capsids – Gel electrophoresis – Immunoprecipitation – Nucleocapsids – Phosphorylation – Viral packaging – Encapsidation – Small interfering RNA
Zdroje
1. Mortality GBD, Causes of Death C. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;385(9963):117–71. doi: 10.1016/S0140-6736(14)61682-2 25530442; PubMed Central PMCID: PMC4340604.
2. Dienstag JL. Benefits and risks of nucleoside analog therapy for hepatitis B. Hepatology. 2009;49(5 Suppl):S112–21. Epub 2009/04/29. doi: 10.1002/hep.22920 19399795.
3. Perrillo R. Benefits and risks of interferon therapy for hepatitis B. Hepatology. 2009;49(5 Suppl):S103–11. Epub 2009/04/29. doi: 10.1002/hep.22956 19399806.
4. Chang J, Guo F, Zhao X, Guo JT. Therapeutic strategies for a functional cure of chronic hepatitis B virus infection. Acta pharmaceutica Sinica B. 2014;4(4):248–57. doi: 10.1016/j.apsb.2014.05.002 26579392; PubMed Central PMCID: PMC4629125.
5. Hu J, Cheng J, Tang L, Hu Z, Luo Y, Li Y, et al. Virological Basis for the Cure of Chronic Hepatitis B. ACS infectious diseases. 2019;5(5):659–74. doi: 10.1021/acsinfecdis.8b00081 29893548.
6. Alter H, Block T, Brown N, Brownstein A, Brosgart C, Chang KM, et al. A research agenda for curing chronic hepatitis B virus infection. Hepatology. 2018;67(3):1127–31. doi: 10.1002/hep.29509 28877549; PubMed Central PMCID: PMC5873273.
7. Seeger C, Mason WS. Molecular biology of hepatitis B virus infection. Virology. 2015;479–480:672–86. doi: 10.1016/j.virol.2015.02.031 25759099; PubMed Central PMCID: PMC4424072.
8. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife. 2012;1:e00049. doi: 10.7554/eLife.00049 23150796; PubMed Central PMCID: PMC3485615.
9. Guo JT, Guo H. Metabolism and function of hepatitis B virus cccDNA: Implications for the development of cccDNA-targeting antiviral therapeutics. Antiviral Res. 2015;122:91–100. doi: 10.1016/j.antiviral.2015.08.005 26272257; PubMed Central PMCID: PMC4586118.
10. Mason WS, Aldrich C, Summers J, Taylor JM. Asymmetric replication of duck hepatitis B virus DNA in liver cells: Free minus-strand DNA. Proc Natl Acad Sci U S A. 1982;79(13):3997–4001. doi: 10.1073/pnas.79.13.3997 6287459.
11. Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell. 1982;29:403–15. doi: 10.1016/0092-8674(82)90157-x 6180831
12. Venkatakrishnan B, Zlotnick A. The Structural Biology of Hepatitis B Virus: Form and Function. Annual review of virology. 2016;3(1):429–51. doi: 10.1146/annurev-virology-110615-042238 27482896.
13. Ning X, Nguyen D, Mentzer L, Adams C, Lee H, Ashley R, et al. Secretion of genome-free hepatitis B virus—single strand blocking model for virion morphogenesis of para-retrovirus. PLoS Pathog. 2011;7(9):e1002255. doi: 10.1371/journal.ppat.1002255 21966269; PubMed Central PMCID: PMC3178560.
14. Hu J, Liu K. Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses. 2017;9(3). doi: 10.3390/v9030056 28335554; PubMed Central PMCID: PMC5371811.
15. Perlman DH, Berg EA, O'Connor P B, Costello CE, Hu J. Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids. Proc Natl Acad Sci U S A. 2005;102(25):9020–5. doi: 10.1073/pnas.0502138102 15951426.
16. Basagoudanavar SH, Perlman DH, Hu J. Regulation of hepadnavirus reverse transcription by dynamic nucleocapsid phosphorylation. J Virol. 2007;81(4):1641–9. Epub 2006/12/01. JVI.01671-06 [pii] doi: 10.1128/JVI.01671-06 17135319.
17. Ning X, Basagoudanavar SH, Liu K, Luckenbaugh L, Wei D, Wang C, et al. Capsid Phosphorylation State and Hepadnavirus Virion Secretion. J Virol. 2017;91(9). Epub 2017/02/24. doi: 10.1128/jvi.00092-17 28228589; PubMed Central PMCID: PMC5391479.
18. Pugh J, Zweidler A, Summers J. Characterization of the major duck hepatitis B virus core particle protein. Journal of Virology. 1989;63(3):1371–6. 2915384
19. Roossinck MJ, Siddiqui A. In vivo phosphorylation and protein analysis of hepatitis B virus core antigen. J Virol. 1987;61(4):955–61. 3546728; PubMed Central PMCID: PMC254050.
20. Yeh CT, Ou JH. Phosphorylation of hepatitis B virus precore and core proteins. J Virol. 1991;65(5):2327–31. 1850014; PubMed Central PMCID: PMC240583.
21. Lewellyn EB, Loeb DD. Serine phosphoacceptor sites within the core protein of hepatitis B virus contribute to genome replication pleiotropically. PLoS One. 2011;6(2):e17202. doi: 10.1371/journal.pone.0017202 21358805; PubMed Central PMCID: PMC3039676.
22. Lan YT, Li J, Liao W, Ou J. Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication. Virology. 1999;259(2):342–8. Epub 1999/07/02. doi: 10.1006/viro.1999.9798 S0042682299997982 [pii]. 10388659.
23. Gazina EV, Fielding JE, Lin B, Anderson DA. Core protein phosphorylation modulates pregenomic RNA encapsidation to different extents in human and duck hepatitis B viruses. J Virol. 2000;74(10):4721–8. Epub 2000/04/25. doi: 10.1128/jvi.74.10.4721-4728.2000 10775610; PubMed Central PMCID: PMC111994.
24. Melegari M, Wolf SK, Schneider RJ. Hepatitis B virus DNA replication is coordinated by core protein serine phosphorylation and HBx expression. J Virol. 2005;79(15):9810–20. Epub 2005/07/15. 79/15/9810 [pii] doi: 10.1128/JVI.79.15.9810-9820.2005 16014942.
25. Zhao Q, Hu Z, Cheng J, Wu S, Luo Y, Chang J, et al. Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation. J Virol. 2018;92(13). doi: 10.1128/JVI.02139-17 29669831; PubMed Central PMCID: PMC6002726.
26. Heger-Stevic J, Zimmermann P, Lecoq L, Bottcher B, Nassal M. 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. 2018;14(12):e1007488. doi: 10.1371/journal.ppat.1007488 30566530; PubMed Central PMCID: PMC6317823.
27. Daub H, Blencke S, Habenberger P, Kurtenbach A, Dennenmoser J, Wissing J, et al. Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein. J Virol. 2002;76(16):8124–37. doi: 10.1128/jvi.76.16.8124-8137.2002 12134018; PubMed Central PMCID: PMC155132.
28. Okabe M, Enomoto M, Maeda H, Kuroki K, Ohtsuki K. Biochemical characterization of suramin as a selective inhibitor for the PKA-mediated phosphorylation of HBV core protein in vitro. Biol Pharm Bull. 2006;29(9):1810–4. doi: 10.1248/bpb.29.1810 16946490.
29. Kann M, Gerlich WH. Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. J Virol. 1994;68(12):7993–8000. 7966589
30. Wittkop L, Schwarz A, Cassany A, Grun-Bernhard S, Delaleau M, Rabe B, et al. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation. Cell Microbiol. 2010;12(7):962–75. Epub 2010/01/30. CMI1444 [pii] doi: 10.1111/j.1462-5822.2010.01444.x 20109160.
31. Diab A, Foca A, Fusil F, Lahlali T, Jalaguier P, Amirache F, et al. Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication. Hepatology. 2017;66(6):1750–65. doi: 10.1002/hep.29236 28445592; PubMed Central PMCID: PMC5658273.
32. Ludgate L, Ning X, Nguyen DH, Adams C, Mentzer L, Hu J. Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids. J Virol. 2012;86(22):12237–50. doi: 10.1128/JVI.01218-12 22951823; PubMed Central PMCID: PMC3486511.
33. Jones SA, Clark DN, Cao F, Tavis JE, Hu J. Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming. J Virol. 2014;88(3):1564–72. doi: 10.1128/JVI.02852-13 24227865; PubMed Central PMCID: PMC3911602.
34. Clark DN, Flanagan JM, Hu J. Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase. J Virol. 2017;91(3). doi: 10.1128/JVI.01785-16 27852858; PubMed Central PMCID: PMC5244320.
35. Wu S, Luo Y, Viswanathan U, Kulp J, Cheng J, Hu Z, et al. CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging. Antiviral Res. 2018;159:1–12. doi: 10.1016/j.antiviral.2018.09.001 30201396.
36. Swingle M, Ni L, Honkanen RE. Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol. 2007;365:23–38. doi: 10.1385/1-59745-267-X:23 17200551; PubMed Central PMCID: PMC2709456.
37. Chang KE, Wei BR, Madigan JP, Hall MD, Simpson RM, Zhuang Z, et al. The protein phosphatase 2A inhibitor LB100 sensitizes ovarian carcinoma cells to cisplatin-mediated cytotoxicity. Mol Cancer Ther. 2015;14(1):90–100. doi: 10.1158/1535-7163.MCT-14-0496 25376608; PubMed Central PMCID: PMC4557740.
38. Bollen M, Peti W, Ragusa MJ, Beullens M. The extended PP1 toolkit: designed to create specificity. Trends Biochem Sci. 2010;35(8):450–8. doi: 10.1016/j.tibs.2010.03.002 20399103; PubMed Central PMCID: PMC3131691.
39. Bertolotti A. The split protein phosphatase system. Biochem J. 2018;475(23):3707–23. doi: 10.1042/BCJ20170726 30523060; PubMed Central PMCID: PMC6282683.
40. Brautigan DL. Protein Ser/Thr phosphatases—the ugly ducklings of cell signalling. FEBS J. 2013;280(2):324–45. doi: 10.1111/j.1742-4658.2012.08609.x 22519956.
41. Tan Z, Pionek K, Unchwaniwala N, Maguire ML, Loeb DD, Zlotnick A. The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription. J Virol. 2015;89(6):3275–84. Epub 2015/01/09. doi: 10.1128/JVI.03545-14 25568211; PubMed Central PMCID: PMC4337549.
42. Zlotnick A, Venkatakrishnan B, Tan Z, Lewellyn E, Turner W, Francis S. Core protein: A pleiotropic keystone in the HBV lifecycle. Antiviral Res. 2015;121:82–93. Epub 2015/07/03. doi: 10.1016/j.antiviral.2015.06.020 26129969; PubMed Central PMCID: PMC4537649.
43. Wu S, Zhao Q, Zhang P, Kulp J, Hu L, Hwang N, et al. Discovery and mechanistic study of benzamide derivatives that modulate hepatitis B virus capsid assembly. J Virol. 2017;91(16):e0051917. doi: 10.1128/JVI.00519-17 28566379.
44. Bourne CR, Katen SP, Fulz MR, Packianathan C, Zlotnick A. A mutant hepatitis B virus core protein mimics inhibitors of icosahedral capsid self-assembly. Biochemistry. 2009;48(8):1736–42. doi: 10.1021/bi801814y 19196007; PubMed Central PMCID: PMC2880625.
45. Bartenschlager R, Junker-Niepmann M, Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990;64(11):5324–32. 2214019
46. Nassal M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 1992;66(7):4107–16. 1602535; PubMed Central PMCID: PMC241213.
47. Su PY, Yang CJ, Chu TH, Chang CH, Chiang C, Tang FM, et al. HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids. Sci Rep. 2016;6:38959. Epub 2016/12/14. doi: 10.1038/srep38959 27958343; PubMed Central PMCID: PMC5154190.
48. Chua PK, Tang FM, Huang JY, Suen CS, Shih C. Testing the balanced electrostatic interaction hypothesis of hepatitis B virus DNA synthesis by using an in vivo charge rebalance approach. J Virol. 2010;84(5):2340–51. Epub 2009/12/18. JVI.01666-09 [pii] doi: 10.1128/JVI.01666-09 20015989; PubMed Central PMCID: PMC2820918.
49. Luo J, Xi J, Gao L, Hu J. Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation. PLoS Pathog. 2020;16(3):e1008459. doi: 10.1371/journal.ppat.1008459 32226051; PubMed Central PMCID: PMC7145273.
50. Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT, et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother. 1997;41(8):1715–20. 9257747
51. Wu JC, Merlino G, Fausto N. Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha. Proc Natl Acad Sci U S A. 1994;91(2):674–8. Epub 1994/01/18. doi: 10.1073/pnas.91.2.674 7904757.
52. Zhu Q, Guo JT, Seeger C. Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virology. 2003;77(17):0000-.
53. Xu C, Guo TC, Mutoloki S, Haugland O, Marjara IS, Evensen O. Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro. J Virol. 2010;84(17):8903–12. Epub 2010/06/25. JVI.00851-10 [pii] doi: 10.1128/JVI.00851-10 20573808; PubMed Central PMCID: PMC2919011.
54. Guo F, Zhao Q, Sheraz M, Cheng J, Qi Y, Su Q, et al. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways. PLoS Pathog. 2017;13(9):e1006658. doi: 10.1371/journal.ppat.1006658 28945802.
55. Mao R, Zhang J, Jiang D, Cai D, Levy JM, Cuconati A, et al. Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells. J Virol. 2011;85(2):1048–57. Epub 2010/11/19. JVI.01998-10 [pii] doi: 10.1128/JVI.01998-10 21084489; PubMed Central PMCID: PMC3019998.
56. Summers J, Smith PM, Horwich AL. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. Journal of Virology. 1990;64(6):2819–24. 2335817
57. Zoulim F, Saputelli J, Seeger C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol. 1994;68(3):2026–30. 8107266
58. Ludgate L, Liu K, Luckenbaugh L, Streck N, Eng S, Voitenleitner C, et al. Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation. J Virol. 2016;90(12):5830–44. doi: 10.1128/JVI.00394-16 27076641; PubMed Central PMCID: PMC4886785.
59. Guo H, Jiang D, Zhou T, Cuconati A, Block TM, Guo JT. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J Virol. 2007;81(22):12472–84. Epub 2007/09/07. JVI.01123-07 [pii] doi: 10.1128/JVI.01123-07 17804499.
60. Xu C, Guo H, Pan XB, Mao R, Yu W, Xu X, et al. Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol. 2010;84(18):9332–40. doi: 10.1128/JVI.00918-10 20610715; PubMed Central PMCID: PMC2937652.
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