J Vet Sci.  2015 Sep;16(3):265-272. 10.4142/jvs.2015.16.3.265.

Establishment and evaluation of a murine alphavbeta3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus

• Zhang W
• Lian K
• Yang F
• Yang Y
• Zhu Z
• Zhu Z
• Cao W
• Mao R
• Jin Y
• He J
• Guo J
• Liu X
• Zheng H

• KMID: 2344298
• DOI: http://doi.org/10.4142/jvs.2015.16.3.265

Abstract

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.

Keyword

alphavbeta3; cell line; Foot-and-mouth disease virus; integrin; receptor

MeSH Terms

Animals
Animals, Suckling
CHO Cells
Cloning, Molecular
Cricetulus
DNA, Complementary/genetics/metabolism
Disease Susceptibility/virology
Foot-and-Mouth Disease/*genetics/virology
Foot-and-Mouth Disease Virus/*physiology
Integrin alphaVbeta3/*genetics/metabolism
Mice
DNA, Complementary
Integrin alphaVbeta3

Figure

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  Fig. 1 PCR products of integrin subunits αv (A) and β3 (B) genes extracted from suckling mouse tongue or lung tissues. 1, αv gene fragment (3135 bp); 2, β3 gene fragment (2364 bp); M, 5000 bp ladder.

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  Fig. 2 (A) Phylogenetic relationships of suckling mouse integrin αv at the nucleotide level among suckling mice and other species. (B) Phylogenetic analysis of suckling mouse integrin β3 at the nucleotide level among suckling mice and other species.

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  Fig. 3 Enzyme identification of the lentiviral recombinant plasmid pLVX-αv-IRES-β3. Lane M, molecular weight marker λ-EcoT14 I fragment (Takara Bio); Lane 1, double enzyme identification of αv fragment; Lane 2, double enzyme identification of the β3 fragment.

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  Fig. 4 Identification of CHO-677-mαvβ3 cell line. (A) Detection of murine integrin subunits αv and β3 genes from the CHO-677-mαvβ3 cell line and CHO-677 cells by PCR. PCR products of αv and β3 genes in the CHO-677-mαvβ3 cell line were detected in line 1 and line 3. As a negative control, PCR products of αv and β3 genes in CHO-677 cells were not detected in line 2 and line 4, respectively. (B) The protein expression of integrin subunits αv and β3 was detected by IFA using rabbit polyclonal antiserum against integrin subunit αv or β3 and FITC-anti-rabbit IgG secondary antibody.

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  Fig. 5 The results of infectivity assays. (A) Plaque morphology of CHO-677-mαvβ3 and CHO-677 cells after inoculation with FMDV/O/ZK/93. (B) One-step growth kinetic curve of FMDV/O/ZK/93 in CHO-677-mαvβ3 and CHO-677 cells. (C) Immunofluorescence analysis of FMDV/O/ZK/93 in CHO-677-mαvβ3 and CHO-677 cells. The virus proteins were probed with guinea pig anti-FMDV serum, then reacted with FITC-conjugated goat anti-guinea pig IgG antibody. (D) The levels of O/ZK/93 FMDV RNA copy numbers were determined in infected CHO-677-mαvβ3 and CHO-677 cells at different time points by RT-PCR.

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  Fig. 6 Results of the antibody blockade assay. The virus titers (mean ± SEM of three independent experiments) were detected by TCID50 on BHK cells.

Reference

1. Akers WJ, Zhang Z, Berezin M, Ye Y, Agee A, Guo K, Fuhrhop RW, Wickline SA, Lanza GM, Achilefu S. Targeting of α_vβ₃-integrins expressed on tumor tissue and neovasculature using fluorescent small molecules and nanoparticles. Nanomedicine (Lond). 2010; 5:715–726.

2. Argraves WS, Pytela R, Suzuki S, Millán JL, Pierschbacher MD, Ruoslahti E. cDNA sequences from the α subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J Biol Chem. 1986; 261:12922–12924.

3. Banasik MB, McCray PB Jr. Integrase-defective lentiviral vectors: progress and applications. Gene Ther. 2010; 17:150–157.

4. Berman AE, Kozlova NI. Integrins: structure and functions. Membr Cell Biol. 2000; 13:207–244.

5. Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin α_vβ₃ antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 1994; 79:1157–1164.

6. Clark RA, Tonnesen MG, Gailit J, Cheresh DA. Transient functional expression of αvβ3 on vascular cells during wound repair. Am J Pathol. 1996; 148:1407–1421.

7. Coffin JM, Hughes SH, Varmus HE. The Interactions of Retroviruses and their Hosts. In : Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. New York: Cold Spring Harbor Laboratory Press;1997.

8. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010; 10:9–22.

9. Domingo E, Baranowski E, Escarmís C, Sobrino F. Foot-and-mouth disease virus. Comp Immunol Microbiol Infect Dis. 2002; 25:297–308.

10. Gu YX, Gao ZL, Zhou JH, Zhang J, Liu YS. Establishment and evaluation of stable cell lines inhibiting foot-and-mouth disease virus by RNA interference. Biomed Res Int. 2014; 2014:109428.

11. Hegde S, Raghavan S. A skin-depth analysis of integrins: role of the integrin network in health and disease. Cell Commun Adhes. 2013; 20:155–169.

12. Humphries MJ. Integrin structure. Biochem Soc Trans. 2000; 28:311–339.

13. Hussain AF, Tur MK, Barth S. An aptamer-siRNA chimera silences the eukaryotic elongation factor 2 gene and induces apoptosis in cancers expressing αvβ3 integrin. Nucleic Acid Ther. 2013; 23:203–212.

14. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002; 110:673–687.

15. Isberg RR, Tran Van Nhieu G. Binding and internalization of microorganisms by integrin receptors. Trends Microbiol. 1994; 2:10–14.

16. Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J Cell Sci. 2000; 113:3563–3571.

17. Ma X, Li P, Bai X, Sun P, Bao H, Lu Z, Cao Y, Li D, Chen Y, Qiao Z, Liu Z. Sequences outside that of residues 93-102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells. Virus Res. 2014; 191:161–171.

18. Miller LC, Blakemore W, Sheppard D, Atakilit A, King AM, Jackson T. Role of the cytoplasmic domain of the β-subunit of integrin αvβ6 in infection by foot-and-mouth disease virus. J Virol. 2001; 75:4158–4164.

19. Neff S, Baxt B. The ability of integrin α_vβ₃ to function as a receptor for foot-and-mouth disease virus is not dependent on the presence of complete subunit cytoplasmic domains. J Virol. 2001; 75:527–532.

20. Neff S, Sá-Carvalho D, Rieder E, Mason PW, Blystone SD, Brown EJ, Baxt B. Foot-and-mouth disease virus virulent for cattle utilizes the integrin α_vβ₃ as its receptor. J Virol. 1998; 72:3587–3594.

21. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol Chem. 2000; 275:21785–21788.

22. Pytela R, Pierschbacher MD, Ruoslahti E. A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc Natl Acad Sci U S A. 1985; 82:5766–5770.

23. Pytela R, Pierschbacher MD, Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985; 40:191–198.

24. Ruoslahti E, Pierschbacher MD. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986; 44:517–518.

25. Stupack DG, Cheresh DA. ECM remodeling regulates angiogenesis: endothelial integrins look for new ligands. Sci STKE. 2002; 2002:pe7.

26. Stupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol. 2001; 155:459–470.

27. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28:2731–2739.

28. van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell Tissue Res. 2001; 305:285–298.

29. Zhang Y, Zheng HX, Zhang ZD, Jin Y, Yang F, He JJ, Cao WJ, Sun DH, Lv L. Establishment of a murine αvβ1 transgenic CHO-K1 cell line and its susceptibility to foot-and-mouth disease virus type Asia I/HN/2006 in China. J Anim Vet Adv. 2013; 12:108–117.

30. Zhang Y, Sun Y, Yang F, Guo J, He J, Wu Q, Cao W, Lv L, Zheng H, Zhang Z. Induction of partial protection against foot and mouth disease virus in guinea pigs by neutralization with the integrin β6-1 subunit. Viruses. 2013; 5:1114–1130.

31. Zhao H, Ross FP, Teitelbaum SL. Unoccupied α_vβ₃integrin regulates osteoclast apoptosis by transmitting a positive death signal. Mol Endocrinol. 2005; 19:771–780.