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Title: Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

item FERNANDEZ-SAINZ, IGNACIO - University Of Connecticut
item LARGO, E - University Of Basque Country
item GLADUE, DOUGLAS - University Of Connecticut
item FLETCHER, PAIGE - Oak Ridge Institute For Science And Education (ORISE)
item O'DONNELL, VIVIAN - University Of Connecticut
item Holinka-Patterson, Lauren
item CAREY, B - Pompeu Fabra University
item LU, ZHIQIANG - Us Deparment Of Homeland Security
item NIEVA, J - University Of Basque Country
item Borca, Manuel

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2014
Publication Date: 5/1/2014
Citation: Fernandez-Sainz, I.J., Largo, E., Gladue, D.P., Fletcher, P., O'Donnell, V., Holinka-Patterson, L.G., Carey, B., Lu, Z., Nieva, J.L., Borca, M.V. 2014. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication. Virology. 456-457:121-130.

Interpretive Summary: Classical swine fever is a serious disease that can cause high mortality in swine. The causing virus, Classical Swine Fever Virus (CSFV) particle contains an external membrane (envelope) with three proteins: E2, E^rns and E1. E2 is involved in several virus functions including virus attachment to the target cell, and determining severity of disease in swine. In this manuscript we evaluate the role of a specific E2 region, the one located between amino acid residue positions 818 and 828 which have been predicted by computer analysis as a putative fusion peptide (FP). Fusion peptides are amino acid stretches critical that mediate the interaction between proteins and membranes. FPs have been shown to facilitate processes such as virus attachment and penetration to target cell, virus replication and formation of virus particles. In this manuscript we report the characterization of this putative FP in E2 by evaluating the effect of mutations introduced at specific amino acids of the FP. Results presented show that the E2 FP is critical for CSFV replication and for the interaction of E2 protein with membranes. This information is useful for understanding CSFV mechanisms of infection and in development of effective vaccines.

Technical Abstract: E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, mediated by disulfide bridges between cysteine residues. Here we evaluate the role of a specific E2 region, ^818CPIGWTGVIEC^828, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. Individual substitutions of C818S, C828S, P819S, I820T, W822S or double substitution V825T/I826T did not significantly alter virus replication in SK6 cell cultures. However, double C818S/C828S and triple P819S/I820S/W822S substitutions prevented viral replication. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a Beta-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring the double C818S/C828S or the triple P819S/I820S/W822S substitution adopted comparable conformations but exhibited different membrane activities. Specifically, the triple substitution peptide inserted more shallowly and demonstrated a decreased capacity for perturbing membranes. Therefore, a preliminary characterization of the putative FP ^818CPIGWTGVIEC^828 indicates a membrane fusion activity and a critical role in virus replication.