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.

Abstract

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