Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation

Autoři: Kyousuke Kobayashi aff001;  Katsumi Mizuta aff002;  Satoshi Koike aff001
Působiště autorů: Neurovirology Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan aff001;  Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, Japan aff002
Vyšlo v časopise: Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation. PLoS Pathog 16(3): e1008428. doi:10.1371/journal.ppat.1008428
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008428


Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.

Klíčová slova:

Cell cultures – Mammalian genomics – Microbial mutation – Mutation detection – RNA viruses – Substitution mutation – Viral genomics – Viral replication


1. Racaniello V. Picornaviridae: The Viruses and Their Replication. In: Knipe D, Howley PM, editors. Fields Virology. 6th ed ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2013. p. 453–89.

2. Pallansch M, Oberste MS, Whitton JL. Enteroviruses: Polioviruses, Coxackieviruses, Echoviruses, and Newer Enteroviruses. In: Knipe D, Howley PM, editors. Fields Virology. 6th ed ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2013. p. 490–530.

3. Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin Infect Dis. 2000;31(3):678–83. doi: 10.1086/314032 11017815

4. Khanh TH, Sabanathan S, Thanh TT, Thoa le PK, Thuong TC, Hang V, et al. Enterovirus 71-associated hand, foot, and mouth disease, Southern Vietnam, 2011. Emerging infectious diseases. 2012;18(12):2002–5. doi: 10.3201/eid1812.120929 23194699

5. Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. The New England journal of medicine. 1999;341(13):929–35. doi: 10.1056/NEJM199909233411301 10498487

6. Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol J. 2010;7:94. doi: 10.1186/1743-422X-7-94 20459851

7. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998–2005. Pediatrics. 2007;120(2):e244–52. doi: 10.1542/peds.2006-3331 17671037

8. Chua BH, Phuektes P, Sanders SA, Nicholls PK, McMinn PC. The molecular basis of mouse adaptation by human enterovirus 71. The Journal of general virology. 2008;89(Pt 7):1622–32. doi: 10.1099/vir.0.83676–0

9. Wang YF, Chou CT, Lei HY, Liu CC, Wang SM, Yan JJ, et al. A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection. Journal of virology. 2004;78(15):7916–24. doi: 10.1128/JVI.78.15.7916-7924.2004 15254164

10. Zaini Z, McMinn P. A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype. The Journal of general virology. 2012;93(Pt 9):1935–40. doi: 10.1099/vir.0.043893–0

11. Arita M, Ami Y, Wakita T, Shimizu H. Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. Journal of virology. 2008;82(4):1787–97. doi: 10.1128/JVI.01798-07 18057246

12. Huang SW, Wang YF, Yu CK, Su IJ, Wang JR. Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement. Virology. 2012;422(1):132–43. doi: 10.1016/j.virol.2011.10.015 22078110

13. Kobayashi K, Sudaka Y, Takashino A, Imura A, Fujii K, Koike S. Amino Acid Variation at VP1-145 of Enterovirus 71 Determines Attachment Receptor Usage and Neurovirulence in Human Scavenger Receptor B2 Transgenic Mice. Journal of virology. 2018;92.

14. Fujii K, Sudaka Y, Takashino A, Kobayashi K, Kataoka C, Suzuki T, et al. VP1 Amino Acid Residue 145 of Enterovirus 71 Is a Key Residue for Its Receptor Attachment and Resistance to Neutralizing Antibody during Cynomolgus Monkey Infection. Journal of virology. 2018;92.

15. Kataoka C, Suzuki T, Kotani O, Iwata-Yoshikawa N, Nagata N, Ami Y, et al. The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model. PLoS Pathog. 2015;11(7):e1005033. doi: 10.1371/journal.ppat.1005033 26181772

16. Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, et al. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol. 2005;43(12):6171–5. doi: 10.1128/JCM.43.12.6171-6175.2005 16333123

17. Mizuta K, Aoki Y, Suto A, Ootani K, Katsushima N, Itagaki T, et al. Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine. 2009;27(24):3153–8. doi: 10.1016/j.vaccine.2009.03.060 19446185

18. McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa MJ. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. Journal of virology. 2001;75(16):7732–8. doi: 10.1128/JVI.75.16.7732-7738.2001 11462047

19. Cordey S, Petty TJ, Schibler M, Martinez Y, Gerlach D, van Belle S, et al. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog. 2012;8(7):e1002826. doi: 10.1371/journal.ppat.1002826 22910880

20. Arita M, Shimizu H, Nagata N, Ami Y, Suzaki Y, Sata T, et al. Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys. The Journal of general virology. 2005;86(Pt 5):1391–401. 86/5/1391 [pii] doi: 10.1099/vir.0.80784–0

21. Yeh MT, Wang SW, Yu CK, Lin KH, Lei HY, Su IJ, et al. A Single Nucleotide in Stem Loop II of 5'-Untranslated Region Contributes to Virulence of Enterovirus 71 in Mice. PLoS One. 2011;6(11):e27082. doi: 10.1371/journal.pone.0027082 22069490

22. Li R, Zou Q, Chen L, Zhang H, Wang Y. Molecular analysis of virulent determinants of enterovirus 71. PLoS One. 2011;6(10):e26237. doi: 10.1371/journal.pone.0026237 22039449

23. Chang CK, Wu SR, Chen YC, Lee KJ, Chung NH, Lu YJ, et al. Mutations in VP1 and 5'-UTR affect enterovirus 71 virulence. Scientific reports. 2018;8(1):6688. doi: 10.1038/s41598-018-25091-7 29703921

24. Tan CW, Sam IC, Lee VS, Wong HV, Chan YF. VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction. Virology. 2017;501:79–87. doi: 10.1016/j.virol.2016.11.009 27875780

25. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature. 2006;439(7074):344–8. doi: 10.1038/nature04388 16327776

26. Huang SW, Huang YH, Tsai HP, Kuo PH, Wang SM, Liu CC, et al. A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans. Journal of virology. 2017;91(23). doi: 10.1128/jvi.01062-17 28931688

27. Xiao Y, Dolan PT, Goldstein EF, Li M, Farkov M, Brodsky L, et al. Poliovirus intrahost evolution is required to overcome tissue-specific innate immune responses. Nature communications. 2017;8(1):375. doi: 10.1038/s41467-017-00354-5 28851882

28. Yamayoshi S, Yamashita Y, Li J, Hanagata N, Minowa T, Takemura T, et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nature medicine. 2009;15(7):798–801. doi: 10.1038/nm.1992 19543282

29. Yamayoshi S, Iizuka S, Yamashita T, Minagawa H, Mizuta K, Okamoto M, et al. Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71. Journal of virology. 2012;86(10):5686–96. doi: 10.1128/JVI.00020-12 22438546

30. Yamayoshi S, Ohka S, Fujii K, Koike S. Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71. Journal of virology. 2013;87(6):3335–47. doi: 10.1128/JVI.02070-12 23302872

31. Zhou D, Zhao Y, Kotecha A, Fry EE, Kelly JT, Wang X, et al. Unexpected mode of engagement between enterovirus 71 and its receptor SCARB2. Nat Microbiol. 2019;4(3):414–9. doi: 10.1038/s41564-018-0319-z 30531980

32. Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nature medicine. 2009;15(7):794–7. nm.1961 [pii] doi: 10.1038/nm.1961 19543284

33. Tan CW, Poh CL, Sam IC, Chan YF. Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor. Journal of virology. 2013;87(1):611–20. doi: 10.1128/JVI.02226-12 23097443

34. Yang SL, Chou YT, Wu CN, Ho MS. Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity. Journal of virology. 2011;85(22):11809–20. JVI.00297-11 [pii] doi: 10.1128/JVI.00297-11 21900167

35. Su PY, Liu YT, Chang HY, Huang SW, Wang YF, Yu CK, et al. Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells. BMC microbiology. 2012;12:162. doi: 10.1186/1471-2180-12-162 22853823

36. Su PY, Wang YF, Huang SW, Lo YC, Wang YH, Wu SR, et al. Cell surface nucleolin facilitates enterovirus 71 binding and infection. Journal of virology. 2015;89(8):4527–38. doi: 10.1128/JVI.03498-14 25673703

37. Du N, Cong H, Tian H, Zhang H, Zhang W, Song L, et al. Cell surface vimentin is an attachment receptor for enterovirus 71. Journal of virology. 2014;88(10):5816–33. doi: 10.1128/JVI.03826-13 24623428

38. He QQ, Ren S, Xia ZC, Cheng ZK, Peng NF, Zhu Y. Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry. J Virol. 2018;92(9). doi: 10.1128/JVI.02251-17 29467312

39. Nishimura Y, Lee H, Hafenstein S, Kataoka C, Wakita T, Bergelson JM, et al. Enterovirus 71 binding to PSGL-1 on leukocytes: VP1-145 acts as a molecular switch to control receptor interaction. PLoS Pathog. 2013;9(7):e1003511. doi: 10.1371/journal.ppat.1003511 23935488

40. Nelson CW, Moncla LH, Hughes AL. SNPGenie: estimating evolutionary parameters to detect natural selection using pooled next-generation sequencing data. Bioinformatics (Oxford, England). 2015;31(22):3709–11. doi: 10.1093/bioinformatics/btv449 26227143

41. Lee E, Hall RA, Lobigs M. Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses. Journal of virology. 2004;78(15):8271–80. doi: 10.1128/JVI.78.15.8271-8280.2004 15254199

42. Lee E, Lobigs M. Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus. Journal of virology. 2002;76(10):4901–11. doi: 10.1128/JVI.76.10.4901-4911.2002 11967307

43. Lee E, Wright PJ, Davidson A, Lobigs M. Virulence attenuation of Dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination. The Journal of general virology. 2006;87(Pt 10):2791–801. doi: 10.1099/vir.0.82164–0

44. Anez G, Men R, Eckels KH, Lai CJ. Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques. Journal of virology. 2009;83(20):10384–94. doi: 10.1128/JVI.01083-09 19656873

45. Klimstra WB, Ryman KD, Johnston RE. Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. Journal of virology. 1998;72(9):7357–66. 9696832

46. Bernard KA, Klimstra WB, Johnston RE. Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. Virology. 2000;276(1):93–103. doi: 10.1006/viro.2000.0546 11021998

47. Mandl CW, Kroschewski H, Allison SL, Kofler R, Holzmann H, Meixner T, et al. Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. Journal of virology. 2001;75(12):5627–37. doi: 10.1128/JVI.75.12.5627-5637.2001 11356970

48. Gardner CL, Hritz J, Sun C, Vanlandingham DL, Song TY, Ghedin E, et al. Deliberate attenuation of chikungunya virus by adaptation to heparan sulfate-dependent infectivity: a model for rational arboviral vaccine design. PLoS neglected tropical diseases. 2014;8(2):e2719. doi: 10.1371/journal.pntd.0002719 24587470

49. Bochkov YA, Watters K, Basnet S, Sijapati S, Hill M, Palmenberg AC, et al. Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells. Virology. 2016;499:350–60. doi: 10.1016/j.virol.2016.09.025 27743961

50. Vlasak M, Goesler I, Blaas D. Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment. Journal of virology. 2005;79(10):5963–70. doi: 10.1128/JVI.79.10.5963-5970.2005 15857982

51. Sa-Carvalho D, Rieder E, Baxt B, Rodarte R, Tanuri A, Mason PW. Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. Journal of virology. 1997;71(7):5115–23. 9188578

52. Minor PD. Live attenuated vaccines: Historical successes and current challenges. Virology. 2015;479–480:379–92. doi: 10.1016/j.virol.2015.03.032 25864107

53. Tsai YH, Huang SW, Hsieh WS, Cheng CK, Chang CF, Wang YF, et al. Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate. J Virol. 2019;93(13). doi: 10.1128/JVI.02308-18 30996087

54. Kohara M, Omata T, Kameda A, Semler BL, Itoh H, Wimmer E, et al. In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain. Journal of virology. 1985;53(3):786–92. 2983090

55. Kärber G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn-Schmiedebergs Archiv für Experimentelle Pathologie und Pharmakologie. 1931;162(4):480–3. doi: 10.1007/bf01863914

56. Jonsson N, Gullberg M, Lindberg AM. Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units. Microbiol Immunol. 2009;53(3):149–54. doi: 10.1111/j.1348-0421.2009.00107.x 19302525

57. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9. doi: 10.1038/nmeth.1923 22388286

58. Hunt M, Gall A, Ong SH, Brener J, Ferns B, Goulder P, et al. IVA: accurate de novo assembly of RNA virus genomes. Bioinformatics. 2015;31(14):2374–6. doi: 10.1093/bioinformatics/btv120 25725497

59. Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic acids research. 2012;40(22):11189–201. doi: 10.1093/nar/gks918 23066108

60. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. 2012;6(2):80–92. doi: 10.4161/fly.19695 22728672

61. Lorenzo-Redondo R, Fryer HR, Bedford T, Kim EY, Archer J, Pond SLK, et al. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature. 2016;530(7588):51–6. doi: 10.1038/nature16933 26814962

62. Zagordi O, Bhattacharya A, Eriksson N, Beerenwinkel N. ShoRAH: estimating the genetic diversity of a mixed sample from next-generation sequencing data. BMC Bioinformatics. 2011;12(1):119. doi: 10.1186/1471-2105-12-119 21521499

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