Mutation of Cysteine 171 of Pestivirus E^rnsRNase Prevents Homodimer Formation and Leads to Attenuation of Classical Swine Fever Virus

Abstract

Pestiviruses represent important pathogens of farm animals that have evolved unique strategies and functions to stay within their host populations. E rns , a structural glycoprotein of pestiviruses, exhibits RNase activity and represents a virulence factor of the viruses. E rns forms disulfide linked homodimers that are found in virions and virus-infected cells. Mutation or deletion of cysteine 171, the residue engaged in intermolecular disulfide bond formation, results in loss of dimerization as tested in coprecipitation and native protein gel electrophoresis analyses. Nevertheless, stable virus mutants with changes affecting cysteine codon 171 could be recovered in tissue culture. These mutants grew almost as well as the parental viruses and exhibited an RNase-positive phenotype. E rns dimerization-negative mutants of classical swine fever virus were found to be attenuated in pigs even though the virus clearly replicated and induced a significant neutralizing antibody response in the animals.

Substitution of Specific Cysteine Residues in the E1 Glycoprotein of Classical Swine Fever Virus Strain Brescia Affects Formation of E1-E2 Heterodimers and Alters Virulence in Swine

“…The effective protective immunity elicited by E1Cys24/94v suggests that modified E1 could be used for the development of live-attenuated vaccines. Similar studies have demonstrated that mutation or deletion of Cys171, one out of nine cysteine residues found in CSFV E rns protein that is involved in the formation of homodimers via disulfide bonds, yielded infectious viruses lacking the capability to form homodimers in infected SK6 cells (18). These CSFV E rns mutants were attenuated in pigs and induced a significant neutralizing antibody response in these animals.…”

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…This is reflected in numerous reports in which mutations of different functional domains in the structural proteins C, E rns , E1, and E2 and in the nonstructural proteins p7 and NS4B were shown to reduce the pathogenicity in pigs, indicating that these genes may be involved in determining CSFV virulence (7,8,9,10,11,12,13,32,36,37,38,39,40,46,54).…”

“…Comparison of the genomes of the GPE Ϫ vaccine virus and the parent ALD virus revealed 225 nucleotide substitutions and 46 amino acid differences (22), which did not provide any conclusive information on the molecular basis of the attenuation. Numerous reports suggest that the viral proteins N pro , C, E rns , E1, E2, p7, and NS4B may be involved in determining the virulence of CSFV in pigs (7,8,9,10,11,12,13,32,36,37,38,39,40,43,46,54). So far, however, all conclusions on CSFV virulence determinants rely on mutations that result in downregulation of pathogenicity.…”

Basic Science Paves the Way to Novel Safe and Effective Pestivirus Vaccines

“…The existence of such naturally occurring viruses and of several engineered virus mutants that lack C171 [90,91] proves that formation of E rns homodimers linked via C171 is not essential for pestivirus viability. Since E rns proteins lacking C171 do no establish dimers stable enough to allow purification and analysis under mild conditions, the formation of stable dimers does not appear necessary for pestivirus viability [91]. Nevertheless, E rns dimerization must have a distinct advantage, because C171 is conserved among the overwhelming majority of pestiviruses.…”

“…More important, two different CSFV mutants with exchange or deletion of C171 are very significantly attenuated in animals. Therefore, dimer formation seems to be connected with virulence [91].…”