Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2016
Publication Date: 9/7/2016
Citation: Holinka-Patterson, L.G., Largo, E., Gladue, D.P., O'Donnell, V.K., Risatti, G.R., Nieva, J.L., Borca, M.V. 2016. Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine. Journal of Virology. 90:10299-10308. doi: 10.1128/JVI.01530-16.

Interpretive Summary: Classical swine fever (CSF) is a highly contagious disease of swine caused by CSF virus (CSFV). The virus has a protein, called E2, that is involved in several critical virus functions including cell attachment and virulence in natural hosts. Computer based structural analysis of E2 allowed us to detect a small stretch of the protein that may help E2 to interact with the cell membranes during the infection. We named this protein stretch PFII. Using physicochemical we showed that PFII interacts with cell membranes in vitro. Using genetic engineering, we created CSF viruses containing E2 proteins with mutated PFII. These viruses showed a significantly decreased ability to replicate in cell cultures and was not capable of producing disease in swine. Furthermore, animals infected with this virus were protected against a virulent CSFV strain. The results are promising toward the rational design of new generation CSF vaccines.

Technical Abstract: E2, the major envelope glycoprotein of Classical Swine Fever Virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864-881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains a 872GG873 dipeptide, and two aromatic residues (871W and 875W) accessible from solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid residue substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated when inoculated in swine. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 28 days post-vaccination. Supporting a role for the E2 864-881 loop in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for inserting into model membranes and perturbing their integrity and this functionality is lost in synthetic peptides harboring substitutions at FPII.2 amino acid residue W871T, W875D, and V878T.