Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (10): 1995-2003.doi: 10.3864/j.issn.0578-1752.2018.10.018

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

The E2 Protein Mediates the Attachment and Internalization of Classical Swine Fever Virus in Cells

CaiXia YIN1,2(), ShaoXiong YU2, Kun SONG2, Su LI2, YuYing YANG1, HuaJi QIU2()   

  1. 1Collegeof Animal Science, Yangtze University, Jingzhou 434025, Hubei
    2State Key Laboratory of Veterinary Biotechnology / Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2017-12-13 Accepted:2018-04-16 Online:2018-05-16 Published:2018-05-16

RichHTML

8

PDF

166

Abstract:

【Objective】 It has been shown that the entry of classical swine fever virus (CSFV) into host cells is mediated by clathrin-mediated endocytosis. However, the viral protein involved in entry stage remains to be elucidated. This study aimed to clarify the role of the E2 protein in the entry of CSFV. 【Method】 Lentivirus carrying the E2 gene were packaged by transient transfection of HEK-293T cells. A cell line stably expressing the E2 protein was established through transducing the suspension 293 cell with the lentiviruses. The expression of the recombinant E2 (rE2) protein was optimized to achieve higher production, and the rE2 protein was purified by affinity chromatography. The expression level and reactivity of the rE2 protein was identified by SDS-PAGE and Western blotting. The effects of the E2 protein on CSFV infection, attachment and internalization were determined by respective assays. Polyclonal anti-E2 antibodies were prepared by immunizing BALB/c mice with the rE2 protein, and its blocking rate was determined by blocking ELISA. Attachment and internalization assays were performed using the prepared polyclonal antibodies to demonstrate the role of the E2 protein in virus attachment and internalization. 【Result】 The cell line 293 was successfully transduced with the lentivirus vector. The SDS-PAGE produced the anticipated band size of rE2 no matter the sample was treated with the reducing agent or not. Western blotting results showed that the rE2 protein could be recognized by the anti-E2 monoclonal antibody WH303. The results indicated that suspension 293 cell line could stably express the rE2 protein. Under the optimized expression condition, the concentration of the rE2 protein in the supernatants of the established suspension 293 cell line was up to 5.84 μg·mL-1. Soluble protein blocking assay results showed that the expressed rE2 protein exerted antiviral activity during the process of CSFV infection. CSFV infection were significantly inhibited by the rE2 protein treated in the entry step; Blocking ELISA and serum neutralization test showed that the anti-E2 polyclonal antibodies could neutralize CSFV infection; at the same time, attachment and internalization assays demonstrated that CSFV internalization could be inhibited by the rE2 protein and viral attachment was blocked by the polyclonal antibodies. 【Conclusion】 The E2 protein was involved in attachment and internalization of CSFV.

Key words: classic swine fever virus, E2 protein, suspension cell line, attachment, internalization

Fig. 1

Expression and identification of the recombinant E2 protein"

Fig. 2

The inhibitory activity of the recombinant E2 protein against CSFV"

Fig. 3

Effects of addition of the recombinant E2 protein during the entry or attachment on CSFV infection"

Fig. 4

The effects of treatment of CSFV with the anti-E2 pAb on its infection and attachment"

Fig. 5

The effects of the E2 protein on CSFV internalization"

[1] JI W, GUO Z, DING NZ, HE C Q.Studying classical swine fever virus: making the best of a bad virus.Virus Research, 2015, 197:35-47.
doi: 10.1016/j.virusres.2014.12.006 pmid: 25510481
[2] LUO Y, LI S, SUN Y, QIU H J.Classical swine fever in China: a minireview.Veterinary Microbiology, 2014, 172(1-2):1-6.
doi: 10.1016/j.vetmic.2014.04.004
[3] WANG F I, DENG M C, HUANG Y L, CHANG C Y.Structures and functions of pestivirus glycoproteins: not simply surface matters.Viruses, 2015, 7(7):3506-3529.
doi: 10.3390/v7072783 pmid: 4517112
[4] MEYERS G,RÜMENAPF T, THIEL H J. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology, 1989, 171(2):555-567.
doi: 10.1016/0042-6822(89)90625-9 pmid: 2763466
[5] BOYD B L, LEE TM, KRUGER E F, PINCHUK L M.Cytopathic and non-cytopathic bovine viral diarrhoea virus biotypes affect fluid phase uptake and mannose receptor-mediated endocytosis in bovine monocytes.Veterinary Immunology and Immunopathology, 2004, 102:53-65.
doi: 10.1016/j.vetimm.2004.06.009 pmid: 15451615
[6] FLORES E F, KREUTZ L C, DONIS R O.Swine and ruminant pestiviruses require the same cellular factor to enter bovine cells.Journal of General Virology, 1996, 77(Part6):1295-1303.
doi: 10.1099/0022-1317-77-6-1295 pmid: 8683219
[7] GRUMMER B, GROTHA S, GREISER-WILKE I.Bovine viral diarrhoea virus is internalized by clathrin-dependent receptor- mediated endocytosis.Journal of Veterinary Medicine Series B-infectious Diseases and Veterinary Public Health, 2004, 51: 427-432.
doi: 10.1111/j.1439-0450.2004.00798.x pmid: 15606865
[8] SHI B J, LIU C C, ZHOU J, WANG S Q, GAO Z, ZHANG X M, ZHOU B, CHEN P Y.Entry of classical swine fever virus into PK-15 cells via a pH-, dynamin-, and cholesterol-dependent, clathrin- mediated endocytic pathway that requires Rab5 and Rab7.Journal of Virology, 2016, 90(20):9194-9208.
[9] MOORMANN R J, WARMERDAM P A, VAN DER MEER B, SCHAAPER W M, WENSVOORT G, HULST M M. Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E1.Virology, 1990, 177(1):184-198.
doi: 10.1016/0042-6822(90)90472-4 pmid: 2162104
[10] HULST M M, VAN GENNIP H G P, MOORMANN R J M. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns.Journal of Virology, 2000, 74(20): 9553-9561.
doi: 10.1128/JVI.74.20.9553-9561.2000 pmid: 112386
[11] CHEN J, HE W R, SHEN L, DONG H, YU J, WANG X, YU S, LI Y, LI S, LUO Y, SUN Y, QIU H J.The laminin receptor is a cellular attachment receptor for classical swine fever virus.Journal of Virology, 2015, 89(9):4894-4906.
doi: 10.1128/JVI.00019-15 pmid: 25694590
[12] DRÄGER C, BEER M, BLOME S. Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachmentin vitro. Archives of Virology, 2015, 160(3):739-746.
doi: 10.1007/s00705-014-2313-y pmid: 25559665
[13] WEILAND F, WEILAND E, UNGER G, SAALMÜLLER A, THIEL H J. Localization of pestiviral envelope proteins Erns and E2 at the cell surface and on isolated particles.Journal of General Virology, 1999, 80(5):1157-1165.
doi: 10.1099/0022-1317-80-5-1157 pmid: 10355762
[14] WANG Z, NIE Y, WANG P, DING M, DENG H.Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry.Virology, 2004, 330(1): 332-341.
doi: 10.1016/j.virol.2004.09.023 pmid: 15527858
[15] EL OMARI K, IOURIN O, HARLOS K, GRIMES J M, STUART D I.Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry.Cell Report, 2013, 3(1):30-35.
doi: 10.1016/j.celrep.2012.12.001 pmid: 23273918
[16] EDWARDS S, SANDS J J.Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies.Deutsche Tierarztliche Wochenschrift, 1990, 97(2):79-81.
doi: 10.1111/j.1751-0813.1990.tb07708.x pmid: 2178905
[17] ZHAO J J, CHENG D, LI N, SUN Y, SHI Z, ZHU Q H, TU C, TONG G Z, QIU H J.Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of classical swine fever virus.Veterinary Microbiology, 2008, 126(1-3):1-10.
[18] WANG H B, ZHANG H, ZHANG J P, LI Y, ZHAO B, FENG G K, DU Y, XIONG D, ZHONG Q, LIU W L, DU H, LI M Z, HUANG W L, TSAO S W, HUTT-FLETCHER L, ZENG Y X, KIEFF E, ZENG M S.Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells.Nature Communications, 2015, 6:6240.
doi: 10.1038/ncomms7240 pmid: 25670642
[19] HOLLA P, AHMAD I, AHMED Z, JAMEEL S.Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.Traffic, 2015, 16:398-416.
doi: 10.1111/tra.12260 pmid: 25615268
[20] REED L J, MUENCH H.A simple method of estimating fifty percent endpoints.American Journal of Tropical Medicine and Hygiene, 1938, 27:493-497.
[21] HULST M M, MOORMANN R J.Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors.Journal of General Virology, 1997, 78(11):2779-2787.
doi: 10.1099/0022-1317-78-11-2779 pmid: 9367363
[22] HUANG H C, CHEN C C, CHANG W C, TAO M H, HUANG C.Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis.Journal of Virology, 2012, 86(17):9443-9453.
doi: 10.1128/JVI.00873-12 pmid: 3416113
[23] CEPPI M, DE BRUIN M G, SEUBERLICH T, BALMELLI C, PASCOLO S, RUGGLI N, WIENHOLD D, TRATSCHIN J D, MCCULLOUGH K C, SUMMERFIELD A. Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.Journal of General Virology, 2005, 86(9):2525-2534.
doi: 10.1099/vir.0.80907-0 pmid: 16099911
[24] GANGES L, BARRERA M,NÚÑEZ J I, BLANCO I, FRIAS M T, RODRÍGUEZ F, SOBRINO F. A DNA vaccine expressing the E2 protein of classical swine fever virus elicits T cell responses that can prime for rapid antibody production and confer total protection upon viral challenge.Vaccine, 2005, 23(28):3741-3752.
doi: 10.1016/j.vaccine.2005.01.153 pmid: 15882536
[25] RISATTI G R, BORCA M V, KUTISH G F, LU Z, HOLINKA L G, FRENCH R A, TULMAN E R, ROCK D L.The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.Journal of Virology, 2005a, 79(6):3787-3796.
doi: 10.1016/j.virol.2005.08.015 pmid: 15731272
[26] DOUAM F, LAVILLETTE D, COSSET F L.The mechanism of HCV entry into host cells.Progress in Molecular Biology and Translational Science, 2015, 129:63-107.
doi: 10.1016/bs.pmbts.2014.10.003 pmid: 25595801
[27] RISATTI G R, BORCA M V, KUTISH G F, LU Z, HOLINKA L G, FRENCH R A, TULMAN E R, ROCK D L.The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.Journal of Virology, 2005, 79(6):3787-3796.
doi: 10.1016/j.virol.2005.08.015 pmid: 15731272
[28] LI X, WANG L, ZHAO D, ZHANG G, LUO J, DENG R, YANG Y.Identification of host cell binding peptide from an overlapping peptide library for inhibition of classical swine fever virus infection.Virus GENES, 2011, 43(1):33-40.
doi: 10.1007/s11262-011-0595-7 pmid: 21400206
[29] MEI Y, YUE F, NING H M, ZHOU J J, WANG X N.Identification of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cells.Canadian Journal of Veterinary Research, 2017, 81(3):186-191.
pmid: 28725108
[1] YE Xing-Guo, WANG Xin-Min, WANG Ke, DU Li-Pu, LIN Zhi-Shan, XU Hui-Jun. A Review of Some Assisted Strategies for Improving the Efficiency of Agrobacterium-Mediated Plant Transformation [J]. Scientia Agricultura Sinica, 2012, 45(15): 3007-3019.
[2] ZOU Xing-qi,ZHAO Qi-zu,FAN Yun-feng,ZHU Yuan-yuan,WANG Qin,XU Lu,FAN Xue-zheng,NING Yi-bao
. Construction of the Full Length Infectious cDNA Clones of CSFV C Strain and Virus Rescue
 [J]. Scientia Agricultura Sinica, 2011, 44(2): 409-416 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd