The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

Department of Chemistry Washington University St Louis Missouri USA

Gilead Sciences Inc Foster City California USA

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

Komentář v

https://doi.org/10.1128/JVI.00248-19 PubMed

Zobrazit více v PubMed

Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. 2015. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet 386:1546–1555. doi:10.1016/S0140-6736(15)61412-X. PubMed DOI

Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, Andrews KG, Atkinson C, Baddour LM, Barker-Collo S, Bartels DH, Bell ML, Benjamin EJ, Bennett D, Bhalla K, Bikbov B, Bin Abdulhak A, Birbeck G, Blyth F, Bolliger I, Boufous S, Bucello C, Burch M, Burney P, Carapetis J, Chen H, Chou D, Chugh SS, Coffeng LE, Colan SD, Colquhoun S, Colson KE, Condon J, Connor MD, Cooper LT, Corriere M, Cortinovis M, de Vaccaro KC, Couser W, Cowie BC, Criqui MH, Cross M, Dabhadkar KC, et al. . 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380:2095–2128. doi:10.1016/S0140-6736(12)61728-0. PubMed DOI PMC

Kornyeyev D, Voitenleitner C, Livingston CM, Xing W, Hung M, Kwon HJ, Fletcher SP, Beran RK. 2019. Spatiotemporal analysis of hepatitis B virus X protein in primary human hepatocytes. J Virol 93:e00248-19. doi:10.1128/JVI.00248-19. PubMed DOI PMC

Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, Zoulim F, Hantz O, Protzer U. 2011. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol 55:996–1003. doi:10.1016/j.jhep.2011.02.015. PubMed DOI

Riviere L, Gerossier L, Ducroux A, Dion S, Deng Q, Michel ML, Buendia MA, Hantz O, Neuveut C. 2015. HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase. J Hepatol 63:1093–1102. doi:10.1016/j.jhep.2015.06.023. PubMed DOI

Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M, Raimondo G, Levrero M. 2009. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A 106:19975–19979. doi:10.1073/pnas.0908365106. PubMed DOI PMC

Decorsière A, Mueller H, van Breugel PC, Abdul F, Gerossier L, Beran RK, Livingston CM, Niu C, Fletcher SP, Hantz O, Strubin M. 2016. Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor. Nature 531:386–389. doi:10.1038/nature17170. PubMed DOI

Murphy CM, Xu Y, Li F, Nio K, Reszka-Blanco N, Li X, Wu Y, Yu Y, Xiong Y, Su L. 2016. Hepatitis B virus X protein promotes degradation of SMC5/6 to enhance HBV replication. Cell Rep 16:2846–2854. doi:10.1016/j.celrep.2016.08.026. PubMed DOI PMC

van Breugel PC, Robert EI, Mueller H, Decorsiere A, Zoulim F, Hantz O, Strubin M. 2012. Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates. Hepatology 56:2116–2124. doi:10.1002/hep.25928. PubMed DOI

Leupin O, Bontron S, Schaeffer C, Strubin M. 2005. Hepatitis B virus X protein stimulates viral genome replication via a DDB1-dependent pathway distinct from that leading to cell death. J Virol 79:4238–4245. doi:10.1128/JVI.79.7.4238-4245.2005. PubMed DOI PMC

Li T, Robert EI, van Breugel PC, Strubin M, Zheng N. 2010. A promiscuous alpha-helical motif anchors viral hijackers and substrate receptors to the CUL4-DDB1 ubiquitin ligase machinery. Nat Struct Mol Biol 17:105–111. doi:10.1038/nsmb.1719. PubMed DOI PMC

Lin-Marq N, Bontron S, Leupin O, Strubin M. 2001. Hepatitis B virus X protein interferes with cell viability through interaction with the p127-kDa UV-damaged DNA-binding protein. Virology 287:266–274. doi:10.1006/viro.2001.1036. PubMed DOI

Kumar V, Jayasuryan N, Kumar R. 1996. A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function. Proc Natl Acad Sci U S A 93:5647–5652. doi:10.1073/pnas.93.11.5647. PubMed DOI PMC

Hodgson AJ, Hyser JM, Keasler VV, Cang Y, Slagle BL. 2012. Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication. Virology 426:73–82. doi:10.1016/j.virol.2012.01.021. PubMed DOI PMC

Jiang T, Liu M, Wu J, Shi Y. 2016. Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx. Proc Natl Acad Sci U S A 113:2074–2079. doi:10.1073/pnas.1525616113. PubMed DOI PMC

Fischer ES, Bohm K, Lydeard JR, Yang H, Stadler MB, Cavadini S, Nagel J, Serluca F, Acker V, Lingaraju GM, Tichkule RB, Schebesta M, Forrester WC, Schirle M, Hassiepen U, Ottl J, Hild M, Beckwith RE, Harper JW, Jenkins JL, Thoma NH. 2014. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512:49–53. doi:10.1038/nature13527. PubMed DOI PMC

Li T, Chen X, Garbutt KC, Zhou P, Zheng N. 2006. Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell 124:105–117. doi:10.1016/j.cell.2005.10.033. PubMed DOI

Schwefel D, Groom HC, Boucherit VC, Christodoulou E, Walker PA, Stoye JP, Bishop KN, Taylor IA. 2014. Structural basis of lentiviral subversion of a cellular protein degradation pathway. Nature 505:234–238. doi:10.1038/nature12815. PubMed DOI PMC

Niu C, Livingston CM, Li L, Beran RK, Daffis S, Ramakrishnan D, Burdette D, Peiser L, Salas E, Ramos H, Yu M, Cheng G, Strubin M, Delaney WE IV, Fletcher SP. 2017. The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection. PLoS One 12:e0169648. doi:10.1371/journal.pone.0169648. PubMed DOI PMC

Abdul F, Filleton F, Gerossier L, Paturel A, Hall J, Strubin M, Etienne L. 2018. Smc5/6 antagonism by HBx is an evolutionarily conserved function of hepatitis B virus infection in mammals. J Virol 92:e00769-18. doi:10.1128/JVI.00769-18. PubMed DOI PMC

Urban S, Hildt E, Eckerskorn C, Sirma H, Kekule A, Hofschneider PH. 1997. Isolation and molecular characterization of hepatitis B virus X-protein from a baculovirus expression system. Hepatology 26:1045–1053. doi:10.1002/hep.510260437. PubMed DOI

Sidhu K, Kumar S, Reddy VS, Kumar V. 2014. Mass spectrometric determination of disulfide bonds in the biologically active recombinant HBx protein of hepatitis B virus. Biochemistry 53:4685–4695. doi:10.1021/bi500140t. PubMed DOI

Feige MJ, Hendershot LM. 2011. Disulfide bonds in ER protein folding and homeostasis. Curr Opin Cell Biol 23:167–175. doi:10.1016/j.ceb.2010.10.012. PubMed DOI PMC

Nakatani T, Tawaramoto M, Opare Kennedy D, Kojima A, Matsui-Yuasa I. 2000. Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes. Chem Biol Interact 125:151–163. doi:10.1016/S0009-2797(99)00166-0. PubMed DOI

Hashemi M, Ghavami S, Eshraghi M, Booy EP, Los M. 2007. Cytotoxic effects of intra and extracellular zinc chelation on human breast cancer cells. Eur J Pharmacol 557:9–19. doi:10.1016/j.ejphar.2006.11.010. PubMed DOI

Grant K, Grant L, Tong L, Boutell C. 2012. Depletion of intracellular zinc inhibits the ubiquitin ligase activity of viral regulatory protein ICP0 and restricts herpes simplex virus 1 replication in cell culture. J Virol 86:4029–4033. doi:10.1128/JVI.06962-11. PubMed DOI PMC

Guo Y, Dong L, Qiu X, Wang Y, Zhang B, Liu H, Yu Y, Zang Y, Yang M, Huang Z. 2014. Structural basis for hijacking CBF-beta and CUL5 E3 ligase complex by HIV-1 Vif. Nature 505:229–233. doi:10.1038/nature12884. PubMed DOI

Becker SA, Lee TH, Butel JS, Slagle BL. 1998. Hepatitis B virus X protein interferes with cellular DNA repair. J Virol 72:266–272. PubMed PMC

Lee SH, Cha EJ, Lim JE, Kwon SH, Kim DH, Cho H, Han KH. 2012. Structural characterization of an intrinsically unfolded mini-HBX protein from hepatitis B virus. Mol Cells 34:165–169. doi:10.1007/s10059-012-0060-z. PubMed DOI PMC

Tu T, Budzinska MA, Shackel NA, Urban S. 2017. HBV DNA integration: molecular mechanisms and clinical implications. Viruses 9:E75. doi:10.3390/v9040075. PubMed DOI PMC

Liu XH, Lin J, Zhang SH, Zhang SM, Feitelson MA, Gao HJ, Zhu MH. 2008. COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma. World J Gastroenterol 14:1346–1352. doi:10.3748/wjg.14.1346. PubMed DOI PMC

Tu H, Bonura C, Giannini C, Mouly H, Soussan P, Kew M, Paterlini-Bréchot P, Bréchot C, Kremsdorf D. 2001. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues. Cancer Res 61:7803–7810. PubMed

Ma NF, Lau SH, Hu L, Xie D, Wu J, Yang J, Wang Y, Wu MC, Fung J, Bai X, Tzang CH, Fu L, Yang M, Su YA, Guan XY. 2008. COOH-terminal truncated HBV X protein plays key role in hepatocarcinogenesis. Clin Cancer Res 14:5061–5068. doi:10.1158/1078-0432.CCR-07-5082. PubMed DOI

Arii M, Takada S, Koike K. 1992. Identification of three essential regions of hepatitis B virus X protein for trans-activation function. Oncogene 7:397–403. PubMed

Renner M, Haniel A, Burgelt E, Hofschneider PH, Koch W. 1995. Transactivating function and expression of the x gene of hepatitis B virus. J Hepatol 23:53–65. doi:10.1016/0168-8278(95)80311-4. PubMed DOI

Kim YH, Kang SK, Lee YI. 1993. Functional analysis of hepatitis B virus transactivator X: implication of the leucine zipper-like region and C-terminal seven conserved amino acids in functional regions. Biochem Biophys Res Commun 197:894–903. doi:10.1006/bbrc.1993.2563. PubMed DOI

Takada S, Koike K. 1990. Trans-activation function of a 3′ truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc Natl Acad Sci U S A 87:5628–5632. doi:10.1073/pnas.87.15.5628. PubMed DOI PMC

Wooddell CI, Yuen MF, Chan HL, Gish RG, Locarnini SA, Chavez D, Ferrari C, Given BD, Hamilton J, Kanner SB, Lai CL, Lau JYN, Schluep T, Xu Z, Lanford RE, Lewis DL. 2017. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med 9:eaan0241. doi:10.1126/scitranslmed.aan0241. PubMed DOI PMC

Levrero M, Zucman-Rossi J. 2016. Mechanisms of HBV-induced hepatocellular carcinoma. J Hepatol 64:S84–S101. doi:10.1016/j.jhep.2016.02.021. PubMed DOI

Ringelhan M, Protzer U. 2015. Oncogenic potential of hepatitis B virus encoded proteins. Curr Opin Virol 14:109–115. doi:10.1016/j.coviro.2015.08.015. PubMed DOI

Livingston CM, Ramakrishnan D, Strubin M, Fletcher SP, Beran RK. 2017. Identifying and characterizing interplay between hepatitis B virus X protein and Smc5/6. Viruses 9:E69. doi:10.3390/v9040069. PubMed DOI PMC

McNaughton AL, D’Arienzo V, Ansari MA, Lumley SF, Littlejohn M, Revill P, McKeating JA, Matthews PC. 2019. Insights from deep sequencing of the HBV genome—unique, tiny, and misunderstood. Gastroenterology 156:384–399. doi:10.1053/j.gastro.2018.07.058. PubMed DOI PMC

Murakami S, Cheong JH, Kaneko S. 1994. Human hepatitis virus X gene encodes a regulatory domain that represses transactivation of X protein. J Biol Chem 269:15118–15123. PubMed

Misra KP, Mukherji A, Kumar V. 2004. The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function. Virus Res 105:157–165. doi:10.1016/j.virusres.2004.05.006. PubMed DOI

Altinel K, Hashimoto K, Wei Y, Neuveut C, Gupta I, Suzuki AM, Dos Santos A, Moreau P, Xia T, Kojima S, Kato S, Takikawa Y, Hidaka I, Shimizu M, Matsuura T, Tsubota A, Ikeda H, Nagoshi S, Suzuki H, Michel ML, Samuel D, Buendia MA, Faivre J, Carninci P. 2016. Single-nucleotide resolution mapping of hepatitis B virus promoters in infected human livers and hepatocellular carcinoma. J Virol 90:10811–10822. doi:10.1128/JVI.01625-16. PubMed DOI PMC

Kwee L, Lucito R, Aufiero B, Schneider RJ. 1992. Alternate translation initiation on hepatitis B virus X mRNA produces multiple polypeptides that differentially transactivate class II and III promoters. J Virol 66:4382–4389. PubMed PMC

Yan Y, Grant GA, Gross ML. 2015. Hydrogen-deuterium exchange mass spectrometry reveals unique conformational and chemical transformations occurring upon [4Fe-4S] cluster binding in the type 2 L-serine dehydratase from Legionella pneumophila. Biochemistry 54:5322–5328. doi:10.1021/acs.biochem.5b00761. PubMed DOI PMC

Chalmers MJ, Busby SA, Pascal BD, Southern MR, Griffin PR. 2007. A two-stage differential hydrogen deuterium exchange method for the rapid characterization of protein/ligand interactions. J Biomol Tech 18:194–204. PubMed PMC

Pascal BD, Willis S, Lauer JL, Landgraf RR, West GM, Marciano D, Novick S, Goswami D, Chalmers MJ, Griffin PR. 2012. HDX workbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom 23:1512–1521. doi:10.1007/s13361-012-0419-6. PubMed DOI PMC

Zhang Z, Smith DL. 1993. Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci 2:522–531. doi:10.1002/pro.5560020404. PubMed DOI PMC

Yang L, Adhikari J, Gross ML, Li L. 2017. Kinetic isotope effects and hydrogen/deuterium exchange reveal large conformational changes during the catalysis of the Clostridium acetobutylicum spore photoproduct lyase. Photochem Photobiol 93:331–342. doi:10.1111/php.12697. PubMed DOI PMC

Cheng M, Zhang B, Cui W, Gross ML. 2017. Laser-initiated radical trifluoromethylation of peptides and proteins: application to mass-spectrometry-based protein footprinting. Angew Chem Int Ed Engl 56:14007–14010. doi:10.1002/anie.201706697. PubMed DOI PMC

Hayer J, Jadeau F, Deleage G, Kay A, Zoulim F, Combet C. 2013. HBVdb: a knowledge database for hepatitis B virus. Nucleic Acids Res 41:D566–D570. doi:10.1093/nar/gks1022. PubMed DOI PMC

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser (Oxf) 41:95–98.

Transcriptional regulators of human oncoviruses: structural and functional implications for anticancer therapy