Location: Location not imported yet.Title: Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses
|LARGO, ENEKO - University Of Basque Country
|AGUILELLA, VICENTE - University Of Jaume
|ALCARAZ, ANTONIO - University Of Jaume
|NIEVA, JOSE - University Of Basque Country
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2018
Publication Date: 1/6/2018
Citation: Largo, E., Gladue, D.P., Aguilella, V.M., Alcaraz, A., Borca, M.V., Nieva, J.L. 2018. Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses. Biochimica et Biophysica Acta. 1860:1015-1021. https://doi.org/10.1016/j.bbamem.2018.01.012.
Interpretive Summary: Viroporins are virus proteins that permeabilizes cell membranes during their replication cycle. Usually, viroporin function is critical for virus replication. Previously, we discover p7, a protein that function as viroporine of classical swine fever virus (CSFV). Our investigation demonstrated that interaction of p7 with the host cell is essential for CSFV replication and that interaction is critical for production of disease in swine. Therefore, blocking viroporin activity of p7 would provide a generic methodology for antiviral and vaccine development. Here, we describes the discovery of the four amino acids responsible of mediating the permeabilization of the cell membrane as well as determination of the size of the pores formed by p7. We believe this information may allow the use of novel strategies to abort/reduce CSFV infection by blocking viroporin p7 function.
Technical Abstract: Permeabilization of the endomembrane system by viroporins is instrumental in the progression of host-cell infection by many viral pathogens. Thus, blocking/attenuation of viroporin activity provides a generic methodology for antiviral and vaccine development. We have described that permeabilization of membranes emulating the Endoplasmic Reticulum (ER) by the Classical Swine Fever Virus (CSFV) viroporin p7 depends on two sequence determinants: the pore-forming C-terminal helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions hamper virus production in cells following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (> 1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the pore-domain actually altered the dimension of the p7 pores. Collectively, our data support the idea that p7 viroporin may assemble into finite pores with approximate widths of 1 and 5 nm. Moreover, the observation that specific mutations can interfere with formation of the larger pores suggests prospective strategies to block/attenuate pestiviruses by targeting viroporin p7.