Estudio de los requerimientos para la entrada del virus de la fiebre aftosa en cultivos celulares y caracterización del ácido valproico como compuesto antiviral

Abstract

Foot-and-mouth disease virus (FMDV) is a small non-enveloped virus belonging to the Picornaviridae family (Aphthovirus genus). The FMDV genome, which consists of a positive RNA strand, is enclosed inside an icosahedral capsid built up with 60 copies of each of the four structural proteins (VP1 to VP4). FMDV initiates infection of cultured cells via binding of different ¿v integrins and then is internalized by clathrin-mediated endocytosis. The current model suggests that uncoating should mainly take place inside early endosomes, since FMDV particles disassembly at pHs around 6.5, which is consistent with pH values inside early endosomes (6.1 to 6.8). The expression of a dominant negative form of dynamin protein, involved in the vesicle scission during clathrin-mediated endocytosis, inhibited the internalization and infection of FMDV (C-S8c1 and MARLS isolates) and of the rhabdovirus vesicular stomatitis virus (VSV), a model virus for clathrin-mediated endocytosis. Alterations in the plasma membrane level of PI(4,5)P2 (a phospholipid with an important role in clathrin-mediated endocytosis) using ionomycin, 1-butanol or an inducible system, revealed that VSV as well as FMDV C-S8c1 displayed a high dependence on PI(4,5)P2 for internalization. Interestingly MARLS, a FMDV variant that uses receptors other than integrins for cell entry, was less sensitive to PI(4,5)P2 depletion. The role of cellular Rab GTPases that govern traffic between different endosome populations was also analyzed. Changes on viral receptor specificity between C-S8c1 and MARLS did not alter Rab5 requirement for infection. However, a correlation between uncoating pH and requirement of Rab5 for infection was observed. A FMDV mutant with higher uncoating pH threshold was less sensitive to inhibition of Rab5 function, whereas another mutant with higher acidic requirements was more sensitive to inhibition of Rab5 function. On the contrary, an opposite correlation was found between uncoating pH and dependence of Rab function upon expression of dominant negative forms of Rab7 or 11. In addition, modulation of uncoating pH also reduced FMDV virulence in suckling mice. These results are consistent with FMDV uncoating occurring inside early endosomes and indicate that displacements from optimum pH for uncoating reduce viral fitness in vivo. Two biological clones resistant to NH4Cl (drug that raise endosomal pH) and with an enhanced sensitivity to acidic pH were isolated from biological samples of natural host (pigs) infected with C-S8c1. A single amino acid substitution VP3 A116V was responsible of the phenotype exhibited by the mutant that exhibited higher resistance to NH4Cl. Replacement VP3 A116V was located close to substitution previously found in cell culture isolated NH4Cl resistant mutants, indicating that the structure of the ß-E sheet from VP3 is important for capsid stability. When the effect of valproic acid (VPA) on the infection of cultured cells by different viruses was analyzed, it was observed that this drug drastically inhibited multiplication of all the enveloped viruses tested, including VSV and the zoonotic lymphocytic choriomeningitis virus (LCMV) and West Nile virus (WNV). VPA reduced VSV and LCMV infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.