Modification of the internal ribosome entry site element impairs the growth of foot-and-mouth disease virus in porcine-derived cells

Abstract

The 59 untranslated region (59UTR) of foot-and-mouth disease virus (FMDV) contains an internal ribosome entry site (IRES) that facilitates translation initiation of the viral ORF in a 59 (m7GpppN) cap-independent manner. IRES elements are responsible for the virulence phenotypes of several enteroviruses. Here, we constructed a chimeric virus in which the IRES of FMDV was completely replaced with that of bovine rhinitis B virus (BRBV) in an infectious clone of serotype O FMDV. The resulting IRES-replaced virus, FMDV(BRBV), replicated as efficiently as WT FMDV in hamster-derived BHK-21 cells, but was restricted for growth in porcine-derived IBRS-2, PK-15 and SK-6 cells, which are susceptible to WT FMDV. To identify the genetic determinants of FMDV underlying this altered cell tropism, a series of IRES-chimeric viruses were constructed in which each domain of the FMDV IRES was replaced with its counterpart from the BRBV IRES. The replication kinetics of these chimeric viruses in different cell lines revealed that the growth restriction phenotype in porcine-derived cells was produced after the replacement of domain 3 or 4 in the FMDV IRES. Furthermore, the change in FMDV cell tropism due to IRES replacement in porcine-derived cells was mainly attributed to a decline in cellspecific IRES translation initiation efficiency. These findings demonstrate that IRES domains 3 and 4 of FMDV are novel cell-specific cis-elements for viral replication in vitro and suggest that IRES-mediated translation determines the species specificity of FMDV infection in vivo.

A Temperature-Dependent Translation Defect Caused by Internal Ribosome Entry Site Mutation Attenuates Foot-and-Mouth Disease Virus: Implications for Rational Vaccine Design

“…The O/M98/CHA/2010 strain was used as a challenge virus, which belongs to the Mya98 lineage of the O/SEA topotype and shares high levels of homology with subtype strain O/BY/CHA/2010 (JN998085). The chimeric-IRES FMDV mutants FMDV(R2), FMDV(R3), FMDV(R4), FMDV(R5), FMDV(RM,) and FMDV(BRBV) used in this study were constructed as described in a previous report (25). The monoclonal antibody (MAb) 4B2, which recognizes a conserved linear epitope on the VP2 protein of FMDV, was produced as described previously (38).…”

“…Western blotting. Western blotting was performed as described previously (25). Protein samples were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane.…”

“…Previously constructed IRES domain-chimeric FMDV mutants, including FMDV(R2), FMDV(R3), FMDV(R4), FMDV(R5), FMDV(RM), and FMDV(BRBV) (25), were used to investigate the effect of IRES domains on FMDV virulence (Fig. 1A).…”

A Novel Live Attenuated Vaccine Candidate Protects Against Heterologous Senecavirus A Challenge

“…The mechanism(s) of attenuation of rSVA mSacII was/were not investigated in our study, however, it is possible that the nucleotide changes introduced in the 5′UTR and/or in the P1 coding region may have affected the conformation of RNA secondary structures present in these regions of the virus genome [e.g., internal ribosomal entry site [IRES] or cis-active RNA elements [CRE], respectively] (26–28). Changes affecting the conformation of these RNA structures have been linked to impaired protein expression and/or picornavirus replication, thus resulting in decreased virus virulence and attenuated disease phenotype (29–31). Results here showing lower viral yields and reduced protein expression levels in rSVA mSacII infected cells support this hypothesis.…”

Chicken eEF1α is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus

“…The plasmids expressing different cheEF1α domains, including cheEF1α-DI (aa: 1-245), cheEF1α-DII (aa: 246-335), cheEF1α-DIII (aa: 336-462), cheEF1α-DI+II (aa: 1-335), and cheEF1α-DII+III (aa: 246-462) were also cloned into the pCAGGS plasmid with a FLAG tag. We constructed dicistronic reporter plasmids encoding Firefly luciferase gene (Fluc) and Renilla luciferase gene (Rluc) under the control of a T7 promoter, and the vvIBDV or aIBDV 5 -UTR was inserted between Fluc and Rluc according to a previous publication [36]. For luciferase assays, the expression plasmids were generated as part of a previously established RNA polymerase II reverse genetics system preserved in our laboratory [37].…”