Glutathione contributes to efficient post-Golgi trafficking of incoming HPV16 genome

Autoři: Shuaizhi Li aff001;  Matthew P. Bronnimann aff001;  Spencer J. Williams aff002;  Samuel K. Campos aff001
Působiště autorů: Department of Immunobiology, University of Arizona, Tucson, AZ, United States of America aff001;  Department of Molecular & Cellular Biology, University of Arizona, Tucson, AZ, United States of America aff002;  Cancer Biology Graduate Interdisciplinary Program, University of Arizona, Tucson, AZ, United States of America aff003;  BIO5 Institute, University of Arizona, Tucson, AZ, United States of America aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225496


Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cellular glutathione (GSH) and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.

Klíčová slova:

Cell staining – Glutathione – HPV-16 – Human papillomavirus infection – Small interfering RNAs – Viral packaging – Virions – Membrane trafficking


1. Braaten K. P., and Laufer M. R. (2008) Human Papillomavirus (HPV), HPV-Related Disease, and the HPV Vaccine. Reviews in Obstetrics and Gynecology 1, 2–10 18701931

2. Van Doorslaer K., Li Z., Xirasagar S., Maes P., Kaminsky D., Liou D., Sun Q., Kaur R., Huyen Y., and McBride A. A. (2017) The Papillomavirus Episteme: a major update to the papillomavirus sequence database. Nucleic acids research 45, D499–D506 doi: 10.1093/nar/gkw879 28053164

3. Doorbar J., Quint W., Banks L., Bravo I. G., Stoler M., Broker T. R., and Stanley M. A. (2012) The biology and life-cycle of human papillomaviruses. Vaccine 30 Suppl 5, F55–70

4. Doorbar J. (2016) Model systems of human papillomavirus-associated disease. J Pathol 238, 166–179 doi: 10.1002/path.4656 26456009

5. Walboomers J. M., Jacobs M. V., Manos M. M., Bosch F. X., Kummer J. A., Shah K. V., Snijders P. J., Peto J., Meijer C. J., and Muñoz N. (1999) Human papillomavirus ia a necessary cause of invasive cervical cancer worldwide. J Pathol 189, 12–19 doi: 10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F 10451482

6. zur Hausen H. (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2, 342–350 doi: 10.1038/nrc798 12044010

7. Forman D., de Martel C., Lacey C. J., Soerjomataram I., Lortet-Tieulent J., Bruni L., Vignat J., Ferlay J., Bray F., Plummer M., and Franceschi S. (2012) Global burden of human papillomavirus and related diseases. Vaccine 30 Suppl 5, F12–23

8. Padmanabhan S., Amin T., Sampat B., Cook-Deegan R., and Chandrasekharan S. (2010) Intellectual property, technology transfer and manufacture of low-cost HPV vaccines in India. Nature biotechnology 28, 671–678 doi: 10.1038/nbt0710-671 20622834

9. Buck C. B., Day P. M., and Trus B. L. (2013) The papillomavirus major capsid protein L1. Virology 445, 169–174 doi: 10.1016/j.virol.2013.05.038 23800545

10. Buck C. B., Cheng N., Thompson C. D., Lowy D. R., Steven A. C., Schiller J. T., and Trus B. L. (2008) Arrangement of L2 within the papillomavirus capsid. J Virol 82, 5190–5197 doi: 10.1128/JVI.02726-07 18367526

11. Wang J. W., and Roden R. B. (2013) L2, the minor capsid protein of papillomavirus. Virology 445, 175–186 doi: 10.1016/j.virol.2013.04.017 23689062

12. Cerqueira C., Samperio Ventayol P., Vogeley C., and Schelhaas M. (2015) Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells. J Virol 89, 7038–7052 doi: 10.1128/JVI.00234-15 25926655

13. Richards R. M., Lowy D. R., Schiller J. T., and Day P. M. (2006) Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci U S A 103, 1522–1527 doi: 10.1073/pnas.0508815103 16432208

14. Mikulicic S., and Florin L. (2019) The endocytic trafficking pathway of oncogenic papillomaviruses. Papillomavirus research 7, 135–137 doi: 10.1016/j.pvr.2019.03.004 30946955

15. Ozbun M. A. (2019) Extracellular events impacting human papillomavirus infections: Epithelial wounding to cell signaling involved in virus entry. Papillomavirus research 7, 188–192 doi: 10.1016/j.pvr.2019.04.009 30981651

16. Schelhaas M., Shah B., Holzer M., Blattmann P., Kühling L., Day P. M., Schiller J. T., and Helenius A. (2012) Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis. PLoS Pathogens 8, e1002657 doi: 10.1371/journal.ppat.1002657 22536154

17. Becker M., Greune L., Schmidt M. A., and Schelhaas M. (2018) Extracellular conformational changes in the capsid of human papillomaviruses contribute to asynchronous uptake into host cells. J Virol

18. Inoue T., Zhang P., Zhang W., Goodner-Bingham K., Dupzyk A., DiMaio D., and Tsai B. (2018) γ-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection. J Cell Biol 217, 3545–3559 doi: 10.1083/jcb.201804171 30006461

19. Zhang W., Kazakov T., Popa A., and DiMaio D. (2014) Vesicular trafficking of incoming human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-secretase activity. mBio 5, e01777–01714 doi: 10.1128/mBio.01777-14 25227470

20. Popa A., Zhang W., Harrison M. S., Goodner K., Kazakov T., Goodwin E. C., Lipovsky A., Burd C. G., and DiMaio D. (2015) Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection. PLoS Pathog 11, e1004699 doi: 10.1371/journal.ppat.1004699 25693203

21. Bergant Marusic M., Ozbun M. A., Campos S. K., Myers M. P., and Banks L. (2012) Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17. Traffic 13, 455–467 doi: 10.1111/j.1600-0854.2011.01320.x 22151726

22. Campos S. K. (2017) Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2. Viruses 9

23. Zhang P., Monteiro da Silva G., Deatherage C., Burd C., and DiMaio D. (2018) Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking. Cell 174, 1465–1476 e1413 doi: 10.1016/j.cell.2018.07.031 30122350

24. Day P. M., Thompson C. D., Schowalter R. M., Lowy D. R., and Schiller J. T. (2013) Identification of a role for the trans-Golgi network in human papillomavirus 16 pseudovirus infection. J Virol 87, 3862–3870 doi: 10.1128/JVI.03222-12 23345514

25. Lipovsky A., Popa A., Pimienta G., Wyler M., Bhan A., Kuruvilla L., Guie M.-A., Poffenberger A. C., Nelson C. D. S., Atwood W. J., and DiMaio D. (2013) Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus. Proceedings of the National Academy of Sciences of the United States of America 110, 7452–7457 doi: 10.1073/pnas.1302164110 23569269

26. Aydin I. A, Villalonga-Planells R. A., Greune L., Bronnimann M. P., Calton C. M., Becker M. A., Lai K. Y., Campos S. K., Schmidt M. A., and Schelhaas M. (2017) A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry.

27. Calton C. M., Bronnimann M. P., Manson A. R., Li S., Chapman J. A., Suarez-Berumen M., Williamson T. R., Molugu S. K., Bernal R. A., and Campos S. K. (2017) Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis.

28. Day P. M., Baker C. C., Lowy D. R., and Schiller J. T. (2004) Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression. Proc Natl Acad Sci U S A 101, 14252–14257 doi: 10.1073/pnas.0404229101 15383670

29. Guion L., Bienkowska-Haba M., DiGiuseppe S., Florin L., and Sapp M. (2019) PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery. PLoS Pathog 15, e1007590 doi: 10.1371/journal.ppat.1007590 30802273

30. Longworth M. S., and Laimins L. A. (2004) Pathogenesis of human papillomaviruses in differentiating epithelia. Microbiol Mol Biol Rev 68, 362–372 doi: 10.1128/MMBR.68.2.362-372.2004 15187189

31. Schiffman M., Doorbar J., Wentzensen N., de Sanjose S., Fakhry C., Monk B. J., Stanley M. A., and Franceschi S. (2016) Carcinogenic human papillomavirus infection. Nature reviews. Disease primers 2, 16086 doi: 10.1038/nrdp.2016.86 27905473

32. Buck C. B., Thompson C. D., Pang Y. Y., Lowy D. R., and Schiller J. T. (2005) Maturation of papillomavirus capsids. J Virol 79, 2839–2846 doi: 10.1128/JVI.79.5.2839-2846.2005 15709003

33. Cardone G., Moyer A. L., Cheng N., Thompson C. D., Dvoretzky I., Lowy D. R., Schiller J. T., Steven A. C., Buck C. B., and Trus B. L. (2014) Maturation of the human papillomavirus 16 capsid. mBio 5, e01104–01114 doi: 10.1128/mBio.01104-14 25096873

34. Biryukov J., and Meyers C. (2015) Papillomavirus Infectious Pathways: A Comparison of Systems. Viruses 7, 4303–4325 doi: 10.3390/v7082823 26247955

35. Buck C. B., Pastrana D. V., Lowy D. R., and Schiller J. T. (2005) Generation of HPV pseudovirions using transfection and their use in neutralization assays. Methods in molecular medicine 119, 445–462 doi: 10.1385/1-59259-982-6:445 16350417

36. Pyeon D., Lambert P. F., and Ahlquist P. (2005) Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation. Proc Natl Acad Sci U S A 102, 9311–9316 doi: 10.1073/pnas.0504020102 15958530

37. Conway M. J., Alam S., Ryndock E. J., Cruz L., Christensen N. D., Roden R. B., and Meyers C. (2009) Tissue-spanning redox gradient-dependent assembly of native human papillomavirus type 16 virions. J Virol 83, 10515–10526 doi: 10.1128/JVI.00731-09 19656879

38. Meister A. (1995) Glutathione metabolism. Methods in enzymology 251, 3–7 doi: 10.1016/0076-6879(95)51106-7 7651209

39. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., and Fusenig N. E. (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106, 761–771 doi: 10.1083/jcb.106.3.761 2450098

40. Campos S. K., and Ozbun M. A. (2009) Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes. PLoS One 4, e4463 doi: 10.1371/journal.pone.0004463 19214230

41. Schneider C. A., Rasband W. S., and Eliceiri K. W. (2012) NIH Image to ImageJ: 25 years of image analysis. Nature methods 9, 671–675 doi: 10.1038/nmeth.2089 22930834

42. Bronnimann M. P., Calton C. M., Chiquette S. F., Li S., Lu M., Chapman J. A., Bratton K. N., Schlegel A. M., and Campos S. K. (2016) Furin Cleavage of L2 During Papillomavirus Infection: Minimal Dependence on Cyclophilins. J Virol

43. Manders E. M. M., Verbeek F. J., and Aten J. A. (1993) Measurement of co-localization of objects in dual-colour confocal images. Journal of microscopy 169, 375–382

44. Bolte S., and Cordelieres F. P. (2006) A guided tour into subcellular colocalization analysis in light microscopy. Journal of microscopy 224, 213–232 doi: 10.1111/j.1365-2818.2006.01706.x 17210054

45. McDonald J. H., and Dunn K. W. (2013) Statistical tests for measures of colocalization in biological microscopy. Journal of microscopy 252, 295–302 doi: 10.1111/jmi.12093 24117417

46. Meister A. (1988) Glutathione metabolism and its selective modification. The Journal of biological chemistry 263, 17205–17208 3053703

47. Wild A. C., and Mulcahy R. T. (2000) Regulation of gamma-glutamylcysteine synthetase subunit gene expression: insights into transcriptional control of antioxidant defenses. Free radical research 32, 281–301 doi: 10.1080/10715760000300291 10741850

48. Njalsson R., and Norgren S. (2005) Physiological and pathological aspects of GSH metabolism. Acta paediatrica (Oslo, Norway: 1992) 94, 132–137

49. Arthur J. R. (2000) The glutathione peroxidases. Cellular and molecular life sciences: CMLS 57, 1825–1835 doi: 10.1007/pl00000664 11215509

50. Xiao Z., La Fontaine S., Bush A. I., and Wedd A. G. (2019) Molecular Mechanisms of Glutaredoxin Enzymes: Versatile Hubs for Thiol-Disulfide Exchange between Protein Thiols and Glutathione. Journal of molecular biology 431, 158–177 doi: 10.1016/j.jmb.2018.12.006 30552876

51. Zhao Y., Seefeldt T., Chen W., Wang X., Matthees D., Hu Y., and Guan X. (2009) Effects of glutathione reductase inhibition on cellular thiol redox state and related systems. Arch Biochem Biophys 485, 56–62 doi: 10.1016/ 19272349

52. Franklin C. C., Backos D. S., Mohar I., White C. C., Forman H. J., and Kavanagh T. J. (2009) Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase. Molecular aspects of medicine 30, 86–98 doi: 10.1016/j.mam.2008.08.009 18812186

53. Lu S. C. (2013) Glutathione synthesis. Biochimica et biophysica acta 1830, 3143–3153 doi: 10.1016/j.bbagen.2012.09.008 22995213

54. Grant C. M., MacIver F. H., and Dawes I. W. (1997) Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine. Molecular Biology of the Cell 8, 1699–1707 doi: 10.1091/mbc.8.9.1699 9307967

55. Ristoff E., Hebert C., Njalsson R., Norgren S., Rooyackers O., and Larsson A. (2002) Glutathione synthetase deficiency: is gamma-glutamylcysteine accumulation a way to cope with oxidative stress in cells with insufficient levels of glutathione? Journal of inherited metabolic disease 25, 577–584 doi: 10.1023/a:1022095324407 12638941

56. Griffith O. W. (1982) Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. The Journal of biological chemistry 257, 13704–13712 6128339

57. Smith A. D., and Dawson H. (2006) Glutathione is required for efficient production of infectious picornavirus virions. Virology 353, 258–267 doi: 10.1016/j.virol.2006.06.012 16860836

58. Markovic J., Mora N. J., Broseta A. M., Gimeno A., de-la-Concepción N., Viña J., and Pallardó F. V. (2009) The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts. PLoS ONE 4, e6413 doi: 10.1371/journal.pone.0006413 19641610

59. Vahrmeijer A. L., van Dierendonck J. H., Schutrups J., van de Velde C. J., and Mulder G. J. (1999) Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells. Biochemical pharmacology 58, 655–664 doi: 10.1016/s0006-2952(99)00130-6 10413303

60. Tomoda H., Kishimoto Y., and Lee Y. C. (1989) Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages. The Journal of biological chemistry 264, 15445–15450 2768271

61. Calton C. M., Schlegel A. M., Chapman J. A., and Campos S. K. (2013) Human papillomavirus type 16 does not require cathepsin L or B for infection. J Gen Virol 94, 1865–1869 doi: 10.1099/vir.0.053694-0 23677785

62. Campos S. K., Chapman J. A., Deymier M. J., Bronnimann M. P., and Ozbun M. A. (2012) Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration. J Virol 86, 4169–4181 doi: 10.1128/JVI.05493-11 22345461

63. Sapp M., Kraus U., Volpers C., Snijders P. J., Walboomers J. M., and Streeck R. E. (1994) Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type 33 and in infected tissues using monoclonal antibodies to the major capsid protein. J Gen Virol 75 (Pt 12), 3375–3383

64. Spoden G., Freitag K., Husmann M., Boller K., Sapp M., Lambert C., and Florin L. (2008) Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs). PLoS ONE 3, e3313 doi: 10.1371/journal.pone.0003313 18836553

65. DiGiuseppe S., Luszczek W., Keiffer T. R., Bienkowska-Haba M., Guion L. G., and Sapp M. J. (2016) Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis. Proc Natl Acad Sci U S A 113, 6289–6294 doi: 10.1073/pnas.1600638113 27190090

66. Schatz P. J. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Bio/technology 11, 1138–1143 doi: 10.1038/nbt1093-1138 7764094

67. Fernandes A. P., and Holmgren A. (2004) Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxidants & redox signaling 6, 63–74

68. Deponte M. (2013) Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochimica et biophysica acta 1830, 3217–3266 doi: 10.1016/j.bbagen.2012.09.018 23036594

69. Lash L. H., Jones D. P., and Orrenius S. (1984) The renal thiol (glutathione) oxidase. Subcellular localization and properties. Biochimica et biophysica acta 779, 191–200 doi: 10.1016/0304-4157(84)90008-x 6375723

70. Lushchak V. I. (2012) Glutathione homeostasis and functions: potential targets for medical interventions. Journal of amino acids 2012, 736837 doi: 10.1155/2012/736837 22500213

71. Lowenstein C. J., and Tsuda H. (2006) N-ethylmaleimide-sensitive factor: a redox sensor in exocytosis. Biol Chem 387, 1377–1383 doi: 10.1515/BC.2006.173 17081110

72. Washbourne P., Cansino V., Mathews J. R., Graham M., Burgoyne R. D., and Wilson M. C. (2001) Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting. The Biochemical journal 357, 625–634 doi: 10.1042/0264-6021:3570625 11463334

73. Kinsella B. T., and Maltese W. A. (1991) rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif. The Journal of biological chemistry 266, 8540–8544 1850747

74. Seabra M. C. (1998) Membrane association and targeting of prenylated Ras-like GTPases. Cellular signalling 10, 167–172 doi: 10.1016/s0898-6568(97)00120-4 9607139

75. Yamaguchi T., Nakayama K., Hatsuzawa K., Tani K., Himeno M., and Tagaya M. (1998) ADP-ribosylation factor-1 is sensitive to N-ethylmaleimide. Journal of biochemistry 124, 1229–1236 doi: 10.1093/oxfordjournals.jbchem.a022242 9832629

76. Grek C. L., Zhang J., Manevich Y., Townsend D. M., and Tew K. D. (2013) Causes and consequences of cysteine S-glutathionylation. The Journal of biological chemistry 288, 26497–26504 doi: 10.1074/jbc.R113.461368 23861399

77. Hess D. T., Matsumoto A., Kim S. O., Marshall H. E., and Stamler J. S. (2005) Protein S-nitrosylation: purview and parameters. Nature reviews. Molecular cell biology 6, 150–166 doi: 10.1038/nrm1569 15688001

78. DiGiuseppe S., Bienkowska-Haba M., Hilbig L., and Sapp M. (2014) The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network. Virology 458–459, 93–105 doi: 10.1016/j.virol.2014.04.024 24928042

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