Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine.
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 a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an872GG873dipeptide, as well as two aromatic residues (871W and875W) accessible to 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 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 in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2.


OBJECTIVE
This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.
ARS, USDA, Plum Island Animal Disease Center, Greenport, New York, USA.
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LG
Holinka
Biophysics Unit (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.
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E
Largo
ARS, USDA, Plum Island Animal Disease Center, Greenport, New York, USA.
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DP
Gladue
ARS, USDA, Plum Island Animal Disease Center, Greenport, New York, USA.|Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, Connecticut, USA.
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O'Donnell
Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, Connecticut, USA.
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GR
Risatti
Biophysics Unit (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.
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JL
Nieva
ARS, USDA, Plum Island Animal Disease Center, Greenport, New York, USA manuel.borca@ars.usda.gov.
M V
MV
Borca
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006691:Classical Swine Fever / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000235:genetics*; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics; D014774:Virulence / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014759:Viral Envelope Proteins; C033717:structural-GP protein, Bos taurus