COUNTERMEASURES TO CONTROL FOREIGN ANIMAL DISEASES OF SWINE
Location: Foreign Animal Disease Research
Title: Classical Swine Fever Virus p7 protein is a viroporin involved in virulence in swine
| Gladue, Douglas - |
| Largo, E - |
| Fernandez Sainz, Ignacio |
| O'Donnell, Vivian - |
| Baker-Branstetter, Ryan - |
| Lu, Zhiqiang - |
| Ambroggio, Xavier - |
| Risatti, Guillermo - |
| Nieva, Jose - |
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 3, 2012
Publication Date: April 11, 2012
Citation: Gladue, D.P., Holinka-Patterson, L.G., Largo, E., Fernandez Sainz, I.J., O'Donnell, V., Baker-Branstetter, R., Lu, Z., Ambroggio, X., Risatti, G.R., Nieva, J.L., Borca, M.V. 2012. Classical Swine Fever Virus p7 protein is a viroporin involved in virulence in swine. Journal of Virology. 86(12):6778-6791.
Interpretive Summary: Classical swine fever virus (CSFV) causes severe and often lethal disease in swine. The disease has been eradicated in the US but it is still present in many countries around the world including areas of Central America and the Caribbean representing an important risk to the US swine industry. As part of our continuing effort to develop better vaccines through a better understanding of the function of CSFV viral proteins, we studied the viral proteins p7. The role of p7 in virus replication and virulence is not well understood. Here we demonstrated that p7 is essential for virus replication in cell cultures and that small areas (5-7 amino acid residue stretches) of p7 that were critical for virus growth in cell cultures. Importantly, we also created recombinant CSFV having mutated small areas of p7 and showed that some of these mutations produced attenuation of virus virulence. This is the first report indicating that p7 is involved in the disease process in swine. Interestingly, some of these mutated viruses, although being significantly attenuated, were able to immunize animals and protect them against the challenge with a virulent strain of viruses, showing some promise as potential live attenuated vaccine candidates. Finally, we were able to clearly demonstrate that p7 belongs to a group of proteins called viroporines. Viroporines are viral proteins that have the role of producing pores in the internal membranes of the cell where the virus replicates. It is known that the formation of those pores is a critical step in the process of virus replication.
The non-structural protein p7 of Classical Swine Fever Virus (CSFV) is a hydrophobic polypeptide with an apparent molecular mass of 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop respectively, suggesting that this structure of CSFV p7 is involved in membrane insertion and consequently membrane permeabilization. Partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus replication in cell cultures. A panel of CSFV recombinant mutant viruses was created using alanine scanning mutagenesis of the p7 gene harboring sequential 3-6 amino acid residues long substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus replication in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain indicating a significant role of p7 in CSFV virulence. Structure-function analyses demonstrated that CSFV p7 is an ion channel forming protein. Circular dichroism measurements demonstrated the adoption of main helical conformations in membrane-mimetics by p7-N and p7-C, two partially overlapping peptides that spanned amino- and carboxy-terminal CSFV p7 hydrophobic domains, respectively. Moreover, subsequent use of those peptides in membrane permeabilization assays revealed robust pore-forming activity for the C-terminal transmembrane helix in vesicles emulating the ER lipid composition. Therefore, p7 is a viroporin which is involved in the process of CSFV virulence.