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Tolerance to mutations in the foot-and-mouth disease virus integrin-binding RGD region is different in cultured cells and in vivo and depends on the capsid sequence context

¹ Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain
² Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid, Spain
³ INRA, UMR 1225, F-31076 Toulouse, France
⁴ Université de Toulouse, ENVT, UMR 1225, F-31076 Toulouse, France

Correspondence
Margarita Sáiz
msaiz{at}cbm.uam.es

Engineered RNAs carrying substitutions in the integrin receptor-binding Arg-Gly-Asp (RGD) region of foot-and-mouth disease virus (FMDV) were constructed (aa 141–147 of VP1 capsid protein) and their infectivity was assayed in cultured cells and suckling mice. The effect of these changes was studied in the capsid proteins of two FMDVs, C-S8c1, which enters cells through integrins, and 213hs^–, a derivative highly adapted to cell culture whose ability to infect cells using the glycosaminoglycan heparan sulfate (HS) as receptor, acquired by multiple passage on BHK-21 cells, has been abolished. The capsid sequence context determined infectivity in cultured cells and directed the selection of additional replacements in structural proteins. Interestingly, a viral population derived from a C-S8c1/L144A mutant, carrying only three substitutions in the capsid, was able to expand tropism to wild-type (wt) and mutant (mt) glycosaminoglycan-deficient CHO cells. In contrast, the 213hs^– capsid tolerated all substitutions analysed with no additional mutations, and the viruses recovered maintained the ability of the 213hs^– parental virus to infect wt and mt CHO cells. Viruses derived from C-S8c1 with atypical RGD regions were virulent and transmissible for mice with no other changes in the capsid. Substitution of Asp143 for Ala in the C-S8c1 capsid eliminated infectivity in cultured cells and mice. Co-inoculation with a neutralizing monoclonal antibody directed against the type C FMDV RGD region abolished infectivity of C-S8c1 virus on suckling mice, suggesting that FMDV can infect mice using integrins. Sequence requirements imposed for viral entry in vitro and in vivo are discussed.

The sequences of the oligonucleotides used in this study are available with the online version of this paper.