Location: Location not imported yet.Title: Molecular characterization of the viroporin function of foot-and-mouth disease virus non-structureal protein 2B
|LARGO, E - University Of Basque Country|
|DE LA ARADA, I - University Of Basque Country|
|AGUILELLA, V.M. - University Of Jaume|
|ALCARAZ, A - University Of Jaume|
|ARRONDO, J.L.R - University Of Basque Country|
|BROCCHI, E - University Of Jaume|
|RAMIREZ-MEDINA, E - Oak Ridge Institute For Science And Education (ORISE)|
|VUONO, E.A. - Oak Ridge Institute For Science And Education (ORISE)|
|BERGGREN, K - Oak Ridge Institute For Science And Education (ORISE)|
|CARRILLO, C - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2018
Publication Date: 9/19/2018
Citation: Gladue, D.P., Largo, E., De La Arada, I., Aguilella, V., Alcaraz, A., Arrondo, J., Holinka-Patterson, L.G., Brocchi, E., Ramirez-Medina, E., Vuono, E., Berggren, K., Carrillo, C., Borca, M.V. 2018. Molecular characterization of the viroporin function of foot-and-mouth disease virus non-structureal protein 2B. Journal of Virology. https://doi.org/10.1128/JVI.01360-18.
Interpretive Summary: FMDV non-structural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows for the passage of ions and small molecules through the membrane. In this study we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in non-viable virus. This suggests that both pore forming functions are independently required during FMDV infection.
Technical Abstract: The non-structural protein 2B of foot-and-mouth disease virus (FMDV) is comprised of a small hydrophobic, 154 amino acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28-147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60-78 and 119-147, and a third transmembrane region between residues 79-106, adopting a mainly extended structure. Using synthetic peptides, ion-channel activity measurements in planar lipid bilayers and imaging of single Giant Unilamellar Vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55-82, and the other spanning the C-terminal region of 2B residues 99-147. Fine mapping of the latter sequence identified residues 119-147 as responsible for the activity. Experiments to assess the degree of insertion on the synthetic peptides in bilayers, and the inclination angle adopted by each peptide regarding the membrane plane normal confirm the residues 55-82 and 119-147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of thirteen FMD recombinant mutant viruses was designed harboring non-conservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to either of these two structures were lethal for virus replication. FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains.