|Piccone, Maria - PIADC & UCONN|
|Pacheco Tobin, Juan|
|Kramer Jr, Edward - 1940-35-00 COLLABORATOR|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 10, 2009
Publication Date: January 1, 2010
Citation: Piccone, M.E., Pacheco Tobin, J., Pauszek, S.J., Borca, M.V., Rieder, A.E., Kramer Jr, E.V., Zhu, J.J., Rodriguez, L.L. 2010. The region between the two polyprotein initiation codons of foot-and-mouth disease virus is critical for virulence in cattle. Virology. 396:152-159. Interpretive Summary: Foot-and-mouth disease virus (FMDV) is a highly contagious livestock disease that causes important economic loss throughout the world. There is limited understanding of the genetic sequences in the viral genome that determine pathogenesis. In this research, we describe the role of a region of the viral genome where viral protein synthesis is initiated (Inter AUG region). We used a genetic approach where the genetic sequence of FMDV was mutagenized. Mutant viruses containing mutations in the inter-AUG region were characterized. Cattle inoculated with one of the mutant viruses (A24-L1123) did not show clinical signs of disease or presence of virus in the blood. In animals sacrificed 24 h after infection with A24-L1123 virus, distribution was more restricted than that of the wild type parental virus. In summary, changes in the inter-AUG region of FMDV result in decreased growth and attenuation in cattle. These findings can be applied to further the understanding of disease mechanisms and rational development of appropriate countermeasures against FMDV.
Technical Abstract: Translation of the viral polyprotein in picornaviruses initiates at an AUG located immediately after the internal ribosome entry site (IRES) in the 5’UTR. In the case of foot-and-mouth disease virus (FMDV), there are two functional AUG in this region separated by 75-84 nucleotides (inter-AUG) with AUG-2 being the preferred initiation site. To characterize the function of the inter-AUG region we generated a series of mutant viruses by transposon-mediated mutagenesis of a cDNA infectious clone of FMDV (pA24-WT). In vitro transcription of RNAs containing insertions in the inter-AUG region resulted in initiation of translation predominantly from AUG-1. Mutant viruses A24-L1115 and A24-L1123 each containing an in-frame 57-nt transposon insertion grew at a slower rate and had a smaller plaque size phenotype than the parental virus (A24-WT). Mutant A24-L1123 showed a marked delay in viral protein synthesis. A mutant (A24-L1094del) which contained a 51-nt deletion in inter-AUG had similar in vitro phenotype to that of A24-WT. When tested by aerosol inoculation in cattle, mutant A24-L1094del was fully virulent as was A24-WT. Mutant A24-L1115 caused delayed clinical disease in 2 of 3 inoculated steers from which virus was recovered that had deleted the transposon insert. In contrast all three steers inoculated with A24-L1123 and the remaining steer inoculated with A24-L1115 did not show clinical signs or viremia. In animals sacrificed 24 h after infection with A24-L1123, virus distribution was more restricted than that of A24-WT, A24-L1094del or A24-L1115. In summary, insertions in the inter-AUG region of FMDV change the AUG preference for translation initiation resulting in decreased growth in vitro and an attenuated phenotype in vivo.