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2000
Volume 32, Issue 5
  • ISSN: 0929-8665
  • E-ISSN: 1875-5305

Abstract

Background

Human papillomavirus type 16 (HPV16) is implicated in various malignancies. The virus enters host cells through endocytosis, during which the minor capsid protein L2 interacts with the S100A10 subunit of the annexin A2 heterotetramer (A2t) on the host cell membrane. This interaction is critical for facilitating HPV entry and subsequent infection of human cells. Therefore, examining the interaction between the L2 protein and S100A10 is crucial for advancing our understanding of the mechanisms by which HPV16 infiltrates cells.

Objective

The cell-free expression (CFE) system was investigated for L2 purification. The structure of L2 was characterized and its interaction with S100A10 was explored.

Methods

The L2 protein was expressed using a CFE expression system, and its expression was verified Western blotting. L2 was further purified through size-exclusion chromatography (SEC), and its structural features were preliminarily assessed using transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Additionally, surface plasmon resonance (SPR) was employed to analyze the interaction between L2 and S100A10.

Results

Western blotting confirmed the successful expression of L2. TEM and CD provided preliminary structural observations of L2, and SPR measurements yielded precise kinetic parameters for the interaction between L2 and S100A10.

Conclusion

In this study, we successfully expressed the HPV16 L2 protein using a cell-free protein expression system. Preliminary structural analysis using TEM and CD revealed key structural features of L2. Furthermore, SPR analysis provided detailed kinetic parameters for its interaction with S100A10. These findings provide more details on understanding L2’s structural features, with broader implications for antipathogen studies.

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References

  1. de MartelC. GeorgesD. BrayF. FerlayJ. CliffordG.M. Global burden of cancer attributable to infections in 2018: A worldwide incidence analysis.Lancet Glob. Health202082e180e19010.1016/S2214‑109X(19)30488‑731862245
     [Google Scholar]
  2. Mlynarczyk-BonikowskaB. RudnickaL. HPV infections-classification, pathogenesis, and potential new therapies.Int. J. Mol. Sci.20242514761610.3390/ijms2514761639062859
     [Google Scholar]
  3. BuckC.B. DayP.M. TrusB.L. The papillomavirus major capsid protein L1.Virology20134451-216917410.1016/j.virol.2013.05.03823800545
     [Google Scholar]
  4. OzbunM. CamposS.K. The long and winding road: Human papillomavirus entry and subcellular trafficking.Curr. Opin. Virol.202150768610.1016/j.coviro.2021.07.01034416595
     [Google Scholar]
  5. MikuličićS. StrunkJ. FlorinL. HPV16 entry into epithelial cells: Running a gauntlet.Viruses20211312246010.3390/v1312246034960729
     [Google Scholar]
  6. BuckC.B. ChengN. ThompsonC.D. LowyD.R. StevenA.C. SchillerJ.T. TrusB.L. Arrangement of L2 within the papillomavirus capsid.J. Virol.200882115190519710.1128/JVI.02726‑0718367526
     [Google Scholar]
  7. GoetschiusD.J. HartmannS.R. SubramanianS. BatorC.M. ChristensenN.D. HafensteinS.L. High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility.Sci. Rep.2021111349810.1038/s41598‑021‑83076‑533568731
     [Google Scholar]
  8. ChenJ. WangD. WangZ. WuK. WeiS. ChiX. QianC. XuY. ZhouL. LiY. ZhangS. LiT. KongZ. WangY. ZhengQ. YuH. ZhaoQ. ZhangJ. XiaN. LiS. GuY. Critical residues involved in the coassembly of L1 and L2 capsid proteins of human papillomavirus 16.J. Virol.2023973e01819-2210.1128/jvi.01819‑2236815785
     [Google Scholar]
  9. KirnbauerR. BooyF. ChengN. LowyD.R. SchillerJ.T. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic.Proc. Natl. Acad. Sci. USA19928924121801218410.1073/pnas.89.24.121801334560
     [Google Scholar]
  10. ConartyJ.P. WielandA. The tumor-specific immune landscape in HPV+ head and neck cancer.Viruses2023156129610.3390/v1506129637376596
     [Google Scholar]
  11. AmiriS. RasekhS. MoezziS.M.I. SeifiN. FatemiS.A. FathiS. BagheriA. NegahdaripourM. Prophylactic vaccines against HPV-caused cervical cancer: Novel vaccines are still demanded.Infect. Agent. Cancer20252011610.1186/s13027‑025‑00643‑540059217
     [Google Scholar]
  12. DiamosA.G. LariosD. BrownL. KilbourneJ. KimH.S. SaxenaD. PalmerK.E. MasonH.S. Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen.Vaccine201937113714410.1016/j.vaccine.2018.11.02130459071
     [Google Scholar]
  13. YadavR. ZhaiL. TumbanE. Virus-like particle-based L2 vaccines against HPVs: Where are we today?Viruses20191211810.3390/v1201001831877975
     [Google Scholar]
  14. MarkowitzL.E. UngerE.R. Human papillomavirus vaccination.N. Engl. J. Med.2023388191790179810.1056/NEJMcp210850237163625
     [Google Scholar]
  15. TsukamotoK. YamashitaA. MaekiM. TokeshiM. ImaiH. FukaoA. FujiwaraT. OkuderaK. MizukiN. OkudaK. ShimadaM. Enhanced broad-spectrum efficacy of an L2-based mRNA vaccine targeting HPV types 6, 11, 16, 18, with cross-protection against multiple additional high-risk types.Vaccines20241211123910.3390/vaccines1211123939591142
     [Google Scholar]
  16. ȘandruF. RaduA.M. PetcaA. DumitrașcuM.C. PetcaR.C. RomanA.M. Unveiling the therapeutic horizon: HPV vaccines and their impact on cutaneous diseases—A comprehensive review.Vaccines202412322810.3390/vaccines1203022838543862
     [Google Scholar]
  17. OzbunM.A. Extracellular events impacting human papillomavirus infections: Epithelial wounding to cell signaling involved in virus entry.Papillomavirus Res.2019718819210.1016/j.pvr.2019.04.00930981651
     [Google Scholar]
  18. Kombe KombeA.J. LiB. ZahidA. MengistH.M. BoundaG.A. ZhouY. JinT. Epidemiology and burden of human papillomavirus and related diseases, molecular pathogenesis, and vaccine evaluation.Front. Public Health2021855202810.3389/fpubh.2020.55202833553082
     [Google Scholar]
  19. KnappeM. BodevinS. SelinkaH.C. SpillmannD. StreeckR.E. ChenX.S. LindahlU. SappM. Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate.J. Biol. Chem.200728238279132792210.1074/jbc.M70512720017640876
     [Google Scholar]
  20. HanR. HuaC. SunS. ZhangB. SongY. van der VeenS. ChengH. Autophagy is induced in human keratinocytes during human papillomavirus 11 pseudovirion entry.Aging20201222230172302810.18632/aging.10404633197887
     [Google Scholar]
  21. CerqueiraC. Samperio VentayolP. VogeleyC. SchelhaasM. Kallikrein-8 proteolytically processes human papillomaviruses in the extracellular space to facilitate entry into host cells.J. Virol.201589147038705210.1128/JVI.00234‑1525926655
     [Google Scholar]
  22. RichardsR.M. LowyD.R. SchillerJ.T. DayP.M. Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.Proc. Natl. Acad. Sci. USA200610351522152710.1073/pnas.050881510316432208
     [Google Scholar]
  23. SmithJ.L. LidkeD.S. OzbunM.A. Virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix.Virology20083811162110.1016/j.virol.2008.08.04018834609
     [Google Scholar]
  24. DziduszkoA. OzbunM.A. Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes.J. Virol.201387137502751510.1128/JVI.00519‑1323637395
     [Google Scholar]
  25. GaoL. NieX. GouR. HuY. DongH. LiX. LinB. Exosomal ANXA2 derived from ovarian cancer cells regulates epithelial-mesenchymal plasticity of human peritoneal mesothelial cells.J. Cell. Mol. Med.20212523109161092910.1111/jcmm.1698334725902
     [Google Scholar]
  26. GerkeV. MossS.E. Annexins: From structure to function.Physiol. Rev.200282233137110.1152/physrev.00030.200111917092
     [Google Scholar]
  27. ZhangH. LuD. ZhangY. ZhaoG. RaheemA. ChenY. ChenX. HuC. ChenH. YangL. GuoA. Annexin A2 regulates Mycoplasma bovis adhesion and invasion to embryo bovine lung cells affecting molecular expression essential to inflammatory response.Front. Immunol.20221397400610.3389/fimmu.2022.97400636159852
     [Google Scholar]
  28. Rintala-DempseyA.C. RezvanpourA. ShawG.S. S100–annexin complexes – Structural insights.FEBS J.2008275204956496610.1111/j.1742‑4658.2008.06654.x18795951
     [Google Scholar]
  29. YanagiH. WatanabeT. NishimuraT. HayashiT. KonoS. TsuchidaH. HirataM. KijimaY. TakaoS. OkadaS. SuzukiM. ImaizumiK. KawadaK. MinamiH. GotohN. ShimonoY. Upregulation of S100A10 in metastasized breast cancer stem cells.Cancer Sci.2020111124359437010.1111/cas.1465932976661
     [Google Scholar]
  30. OkuraG.C. BharadwajA.G. WaismanD.M. Recent advances in molecular and cellular functions of S100A10.Biomolecules20231310145010.3390/biom1310145037892132
     [Google Scholar]
  31. BharadwajA.G. KempsterE. WaismanD.M. The ANXA2/S100A10 complex—regulation of the oncogenic plasminogen receptor.Biomolecules20211112177210.3390/biom1112177234944416
     [Google Scholar]
  32. TantyoN. KaryadiA. RasmanS. SalimM. DevinaA. SumarpoA. The prognostic value of S100A10 expression in cancer (Review).Oncol. Lett.20181721417142410.3892/ol.2018.975130675195
     [Google Scholar]
  33. Umbrecht-JenckE. DemaisV. CalcoV. BaillyY. BaderM.F. Chasserot-GolazS. S100A10-mediated translocation of annexin-A2 to SNARE proteins in adrenergic chromaffin cells undergoing exocytosis.Traffic201011795897110.1111/j.1600‑0854.2010.01065.x20374557
     [Google Scholar]
  34. WoodhamA.W. Da SilvaD.M. SkeateJ.G. RaffA.B. AmbrosoM.R. BrandH.E. IsasJ.M. LangenR. KastW.M. The S100A10 subunit of the annexin A2 heterotetramer facilitates L2-mediated human papillomavirus infection.PLoS One201278e4351910.1371/journal.pone.004351922927980
     [Google Scholar]
  35. TaylorJ.R. FernandezD.J. ThorntonS.M. SkeateJ.G. LühenK.P. Da SilvaD.M. LangenR. KastW.M. Heterotetrameric annexin A2/S100A10 (A2t) is essential for oncogenic human papillomavirus trafficking and capsid disassembly, and protects virions from lysosomal degradation.Sci. Rep.2018811164210.1038/s41598‑018‑30051‑230076379
     [Google Scholar]
  36. WoodhamA.W. RaffA.B. RaffL.M. Da SilvaD.M. YanL. SkeateJ.G. WongM.K. LinY.G. KastW.M. Inhibition of Langerhans cell maturation by human papillomavirus type 16: A novel role for the annexin A2 heterotetramer in immune suppression.J. Immunol.2014192104748475710.4049/jimmunol.130319024719459
     [Google Scholar]
  37. HernandezB.Y. TonT. ShvetsovY.B. GoodmanM.T. ZhuX. Human papillomavirus (HPV) L1 and L1-L2 virus-like particle-based multiplex assays for measurement of HPV virion antibodies.Clin. Vaccine Immunol.20121991348135210.1128/CVI.00191‑1222761294
     [Google Scholar]
  38. HartmannS.R. GoetschiusD.J. HuJ. GraffJ.J. BatorC.M. ChristensenN.D. HafensteinS.L. Cryo EM analysis reveals inherent flexibility of authentic murine papillomavirus capsids.Viruses20211310202310.3390/v1310202334696452
     [Google Scholar]
  39. ChabedaA. van ZylA.R. RybickiE.P. HitzerothI.I. Substitution of human papillomavirus type 16 L2 neutralizing epitopes into L1 surface loops: The effect on virus-like particle assembly and immunogenicity.Front. Plant Sci.20191077910.3389/fpls.2019.0077931281327
     [Google Scholar]
  40. BurkertO. KreßnerS. SinnL. GieseS. SimonC. LilieH. Biophysical characterization of polyomavirus minor capsid proteins.Biol. Chem.20143957-887188010.1515/hsz‑2014‑011424713574
     [Google Scholar]
  41. BreinerB. PreussL. RoosN. ConradyM. LilieH. IftnerT. SimonC. Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2.Biol. Chem.2019400451352210.1515/hsz‑2018‑031130375341
     [Google Scholar]
  42. MiguezA.M. ZhangY. PiorinoF. StyczynskiM.P. Metabolic dynamics in Escherichia coli -Based cell-free systems.ACS Synth. Biol.20211092252226510.1021/acssynbio.1c0016734478281
     [Google Scholar]
  43. WangT. LuY. Advances, challenges and future trends of cell-free transcription-translation biosensors.Biosensors202212531810.3390/bios1205031835624619
     [Google Scholar]
  44. HuntA.C. RasorB.J. SekiK. EkasH.M. WarfelK.F. KarimA.S. JewettM.C. Cell-free gene expression: Methods and applications.Chem. Rev.202512519114910.1021/acs.chemrev.4c0011639700225
     [Google Scholar]
  45. BoydM.A. ThavarajahW. LucksJ.B. KamatN.P. Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles.Sci. Adv.202391eadd660510.1126/sciadv.add660536598992
     [Google Scholar]
  46. MaharjanA. ParkJ.H. Cell-free protein synthesis system: A new frontier for sustainable biotechnology-based products.Biotechnol. Appl. Biochem.20237062136214910.1002/bab.251437735977
     [Google Scholar]
  47. SilvermanA.D. KarimA.S. JewettM.C. Cell-free gene expression: An expanded repertoire of applications.Nat. Rev. Genet.202021315117010.1038/s41576‑019‑0186‑331780816
     [Google Scholar]
  48. GaoW. ChoE. LiuY. LuY. Advances and challenges in cell-free incorporation of unnatural amino acids into proteins.Front. Pharmacol.20191061110.3389/fphar.2019.0061131191324
     [Google Scholar]
  49. LoC.H. ZengJ. Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives.Bioeng. Transl. Med.202381e1035010.1002/btm2.1035036684106
     [Google Scholar]
  50. YanX. Lebel-BeaucageM.F. TremblayS. CantinL. ShawG.S. BoisselierE. Optimized transformation, overexpression and purification of S100A10.Biotechniques201967524624810.2144/btn‑2019‑008131475584
     [Google Scholar]
  51. BöhmG. MuhrR. JaenickeR. Quantitative analysis of protein far UV circular dichroism spectra by neural networks.Protein Eng. Des. Sel.19925319119510.1093/protein/5.3.1911409538
     [Google Scholar]
  52. PletanM.L. TsaiB. Non-enveloped virus membrane penetration: New advances leading to new insights.PLoS Pathog.20221812e101094810.1371/journal.ppat.101094836480535
     [Google Scholar]
  53. GilsonT.D. GibsonR.T. AndrophyE.J. Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer.Sci. Rep.20201011551710.1038/s41598‑020‑72027‑132968082
     [Google Scholar]
  54. LiuY. WuZ. WuD. GaoN. LinJ. Reconstitution of multi-protein complexes through ribozyme-assisted polycistronic co-expression.ACS Synth. Biol.202312113614310.1021/acssynbio.2c0041636512506
     [Google Scholar]
  55. EisenhutM. The identification of native epitopes eliciting a protective high-affinity immunoglobulin subclass response to blood stages of plasmodium falciparum: Protocol for observational studies.JMIR Res. Protoc.202097e1569010.2196/1569032706743
     [Google Scholar]
  56. WallisJ. ShentonD.P. CarlisleR.C. Novel approaches for the design, delivery and administration of vaccine technologies.Clin. Exp. Immunol.2019196218920410.1111/cei.1328730963549
     [Google Scholar]
  57. WangJ.W. RodenR.B.S. L2, the minor capsid protein of papillomavirus.Virology20134451-217518610.1016/j.virol.2013.04.01723689062
     [Google Scholar]
  58. JinX.W. CowsertL.M. PilacinskiW.P. JensonA.B. Identification of L2 open reading frame gene products of bovine papillomavirus type 1 using monoclonal antibodies.J. Gen. Virol.19897051133114010.1099/0022‑1317‑70‑5‑11332471804
     [Google Scholar]
  59. KomlyC.A. BreitburdF. CroissantO. StreeckR.E. The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.J. Virol.198660281381610.1128/jvi.60.2.813‑816.19862430112
     [Google Scholar]
  60. ChanginO. Sequence independent activity of a predicted long disordered segment of the human papillomavirus L2 capsid protein during virus entry.Proc. Natl. Acad. Sci. USA202312042e230772112010.1073/pnas.230772112037819982
     [Google Scholar]
  61. HanoulleX. VerdegemD. BadilloA. WieruszeskiJ.M. PeninF. LippensG. Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded.Biochem. Biophys. Res. Commun.2009381463463810.1016/j.bbrc.2009.02.10819249289
     [Google Scholar]
  62. MilesA.J. RamalliS.G. WallaceB.A. DichroWeb, a website for calculating protein secondary structure from circular dichroism spectroscopic data.Protein Sci.2022311374610.1002/pro.415334216059
     [Google Scholar]
  63. ManyilovV.D. IlyinskyN.S. NesterovS.V. SaqrB.M.G.A. DayhoffG.W.II ZinovevE.V. MatrenokS.S. FoninA.V. KuznetsovaI.M. TuroverovK.K. IvanovichV. UverskyV.N. Chaotic aging: Intrinsically disordered proteins in aging-related processes.Cell. Mol. Life Sci.202380926910.1007/s00018‑023‑04897‑337634152
     [Google Scholar]
  64. KalitaP. TripathiT. PadhiA.K. Computational protein design for COVID-19 research and emerging therapeutics.ACS Cent. Sci.20239460261310.1021/acscentsci.2c0151337122454
     [Google Scholar]
  65. CriteM. DiMaioD. Human papillomavirus L2 capsid protein stabilizes γ-secretase during viral infection.Viruses202214480410.3390/v1404080435458534
     [Google Scholar]
  66. CuiZ. LiC. ChenP. YangH. An update of label-free protein target identification methods for natural active products.Theranostics20221241829185410.7150/thno.6880435198076
     [Google Scholar]
  67. Higuera-RodriguezR.A. De PascaliM.C. AzizM. SattlerM. RantU. KaiserW. Kinetic FRET assay to measure binding-induced conformational changes of nucleic acids.ACS Sens.20238124597460610.1021/acssensors.3c0152738060303
     [Google Scholar]
  68. NguyenH. ParkJ. KangS. KimM. Surface plasmon resonance: A versatile technique for biosensor applications.Sensors2015155104811051010.3390/s15051048125951336
     [Google Scholar]
  69. ButtM.A. Surface plasmon resonance-based biodetection systems: Principles, progress and applications—A comprehensive review.Biosensors20251513510.3390/bios1501003539852086
     [Google Scholar]
  70. MignonC. Ortiz MorenoA.R. ShirzadH. PadamatiS.K. DamleV.G. OngY. SchirhaglR. ChipauxM. Fast, broad-band magnetic resonance spectroscopy with diamond widefield relaxometry.ACS Sens.2023841667167510.1021/acssensors.2c0280937043367
     [Google Scholar]
  71. NiuZ. DuH. MaL. ZhouJ. YuanZ. SunR. LiuG. ZhangF. ZengY. Wavelength division multiplexing-based high-sensitivity surface plasmon resonance imaging biosensor for high-throughput real-time molecular interaction analysis.Molecules20242912281110.3390/molecules2912281138930876
     [Google Scholar]
  72. SchmidtD. MaierJ. BernauerH. Nesterov-MuellerA. Label-free imaging of solid-phase peptide synthesis products and their modifications tethered in microspots using time-of-flight secondary ion mass spectrometry.Int. J. Mol. Sci.202324211594510.3390/ijms24211594537958928
     [Google Scholar]
  73. MaL. LiuH. LiuX. YuanX. XuC. WangF. LinJ. XuR. ZhangD. Screening S protein – ACE2 blockers from natural products: Strategies and advances in the discovery of potential inhibitors of COVID-19.Eur. J. Med. Chem.202122611385710.1016/j.ejmech.2021.11385734628234
     [Google Scholar]
  74. ShewellL.K. DayC.J. JenF.E.C. HaselhorstT. AtackJ.M. ReijneveldJ.F. Everest-DassA. JamesD.B.A. BoguslawskiK.M. BrouwerS. GillenC.M. LuoZ. KobeB. NizetV. von ItzsteinM. WalkerM.J. PatonA.W. PatonJ.C. TorresV.J. JenningsM.P. All major cholesterol-dependent cytolysins use glycans as cellular receptors.Sci. Adv.2020621eaaz492610.1126/sciadv.aaz492632494740
     [Google Scholar]
  75. DasS. DevireddyR. GartiaM.R. Surface plasmon resonance (SPR) sensor for cancer biomarker detection.Biosensors202313339610.3390/bios1303039636979608
     [Google Scholar]
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Cell-Free Expression of HPV16 Minor Capsid Protein L2 and Its Interaction with S100A10
PPL 32, 376 (2025); https://doi.org/10.2174/0109298665390494250513110604
/content/journals/ppl/10.2174/0109298665390494250513110604
/content/journals/ppl/10.2174/0109298665390494250513110604
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  • Article Type:     Research Article
Keyword(s): cell-free protein synthesis; HPV16; minor capsid protein L2; S100A10; size-exclusion chromatography; SPR measurements

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