A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo

• Published in: Journal of Virology 1993-07, Vol.67 (7), p.3748-3755
• Main Authors: Martinez Salas E, Saiz J.C, Davila M, Belsham G.J, Domingo E
• Format: Article
• Language: English
• Subjects: Animals; aphthovirus; Aphthovirus - genetics; arn; arn mensajero; arn messager; Base Sequence; Cell Line; Hydrogen Bonding; In Vitro Techniques; induced mutation; messenger rna; Molecular Sequence Data; mutacion inducida; mutant; mutantes; mutants; mutation provoquee; Nucleic Acid Conformation; nucleotide sequence; Oligodeoxyribonucleotides - chemistry; Protein Biosynthesis; Regulatory Sequences, Nucleic Acid; ribosomas; ribosome; ribosomes; Ribosomes - metabolism; rna; RNA Caps - metabolism; RNA, Viral - genetics; secuencia nucleica; sequence nucleique; virus fiebre aftosa; virus fievre aphteuse
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.67.7.3748-3755.1993

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martinez-Salas, J. Diez, A. Villaverde, F. Gebauer, E. Rocha, M. Davila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.