Mutation of cysteine 171 of pestivirus E rns RNase 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.

FIG. 1.

(A) Coprecipitation experiments. Tagged (pB-E^rns-V5 and pB-E^rns-V5^C171R) and untagged (pB-E^rns and pB-E^rns-C171R) variants of E^rns from BVDV CP7 with or without C171 were (co)expressed in BHK-21 cells and metabolically labeled. Cells were lysed with a lysis buffer containing 1% Triton X-100 and proteins precipitated from the lysate (right panels) or the cell culture supernatant (left panels) with an E^rns-specific serum (α-E^rns, upper panels) or a V5 specific antibody (α-V5, lower panels) and deglycosylated before separation through SDS-PAGE under nonreducing conditions. The transfected plasmids or plasmid combinations are indicated at the top. The positions in the gel of the tagged and untagged monomers as well as the positions of the different dimers are indicated by arrows. The antisera used for precipitation are indicated on the left, together with the molecular masses of the size marker bands. (B) Determination of membrane binding/secretion of wt and mutant E^rns. The upper part shows an example of SDS-PAGE under reducing conditions of the proteins precipitated from different fractions of cells transfected with expression plasmids given above the respective lanes. 1, secreted proteins; 2, debris; 3, membrane fraction; 4, soluble proteins. The lower panel shows for each construct the calculated level of membrane association in percent. Both constructs were tested at least three times, and the meridian, upper, and lower percentiles are shown. The wt membrane association is indicated by the horizontal line (mean of wt membrane association). The mutated protein was judged to have no significantly different membrane association compared to the wt E^rns using the Welch test for unequal variances.

FIG. 2.

Immunoprecipitation of E^rns proteins from different pestiviruses. Proteins were transiently expressed in BHK-21 cells and metabolically labeled, and the cells were lysed with a lysis buffer containing 1% Triton X-100. Proteins were precipitated with an E^rns-specific serum and the precipitates separated on SDS-PAGE under nonreducing conditions. The transfected plasmids are indicated above the lanes: pBN^ATCC-E^rns-171C and pBN^ATCC-E^rns-171R, E^rns sequences from variants of BVDV NADL (ATCC VR534); pBN^MC-E^rns, E^rns sequence derived from biologically cloned BVDV NADL (5); pC-E^rns, E^rns from CSFV Alfort/Tübingen, wt or with the indicated mutations. For the other constructs, see the legend to Fig. 1 and the text. Broad bands or double bands (pB-E^rns-V5 and pB-E^rns-V5^C171R) are due to variations in the native glycosylation of the proteins.

FIG. 3.

Blue native PAGE of E^rns proteins from different pestiviruses. Proteins were expressed in BHK-21 cells and lysed with a lysis buffer containing 1% digitonin. Proteins were separated through blue native PAGE with a histidine-containing buffer system (40) and blotted onto nitrocellulose membranes. The transfected plasmids are indicated above the lanes (see the legend to Fig. 2 for designations). Detection was carried out with an E^rns-specific serum. On the right, the results of a control experiment are shown, with the left two lanes containing proteins expressed from pB-E^rns and pB-E^rns-C171R (equivalent to lanes 1 and 2 of the middle panel) and the right lane showing a Coomassie blue-stained BSA marker. The monomeric and dimeric bands of the marker protein are visible, corresponding to ca. 66 and 132 kDa, respectively.

FIG. 4.

(A) Growth curves of the recombinant CSFV mutants and the wt virus. SK6 cells were infected with CSFV Alfort/Tübingen (wt), V-H297K (RNase negative), or V-C171d (dimerization negative) at an MOI of 0.1 and harvested by freezing and thawing at the indicated time points. Titers were determined by immunofluorescence staining with monoclonal antibody a18 at 72 h p.i. The curves represent the mean values from four individual experiments. Standard deviations are indicated. (B) Inactivation kinetics of wt CSFV and mutant V-C171d. Virus stocks (titers adjusted with tissue culture medium) were incubated at 37°C. Samples were taken for titration at the indicated time points. The graph shows the mean values from two independent experiments.

FIG. 5.

Immunoprecipitation of E^rns proteins from SK6 cells infected with different CSFVs. Infected cells were metabolically labeled and the cells lysed with a lysis buffer containing 1% Triton X-100. Proteins were precipitated with an E^rns-specific serum and treated with PNGase F, and the deglycosylated precipitates were separated by SDS-PAGE under nonreducing (left) or reducing (right) conditions. The viruses used for infection are indicated above the lanes. The positions of protein size marker bands are given on the left.

FIG. 6.

Determination of RNase activity present in crude extracts of SK6 cells infected with wt CSFV Alfort/Tübingen or the recombinant virus V-C171F (dimerization negative), V-C171d (dimerization negative), or V-H297K (RNase negative). Noninfected SK6 cells served as a negative control (mock). The enzymatic degradation of poly(U) was determined by measuring the optical density at 260 nm (OD260) as a marker of the release of small acid-soluble RNA fragments (27, 47).

FIG. 7.

Results of an animal experiment in which groups of four pigs were infected with the RNase-negative CSFV V-H297K (group 1), the dimerization-negative mutant V-C171d (group 2), or wt Alfort/Tübingen recovered from the infectious cDNA clone (group 3). Clinical scores, according to the system published by Mittelholzer et al. (38), are given as mean values for each group (A). Also shown are the average body temperature (B), the mean values of leukocyte counts (C), the percentage of virus-positive cells in complete blood samples (as described in reference 57) (D), the amount of type I IFN in plasma samples compared to an IFN-α standard (1 ng per well) (E), and the titers of virus-neutralizing serum antibodies of the pigs, given as the maximal reciprocal dilution of the sera that is able to neutralize 100 50% tissue culture infective doses of CSFV Alfort/Tübingen (F). Standard deviations are indicated in panels A, D, and E.

FIG. 8.

Growth curve of the recombinant CSFV mutant V-C171S (dimerization negative) compared with those of the wt virus Alfort/Tübingen (wt) and mutant V-H297K (RNase negative). SK6 cells were infected with the CSFV variants at an MOI of 0.1 and harvested by freezing and thawing at the indicated time points. Titers were determined by immunofluorescence staining with monoclonal antibody a18 at 72 h p.i. The curves represent the mean values from two individual experiments.

FIG. 9.

Results of an animal experiment in which pigs were infected with the RNase-negative CSFV V-H297K (group 1, 3 pigs), the dimerization-negative point mutant V-C171S (group 2, 4 pigs) or wt Alfort/Tübingen recovered from the infectious cDNA clone (group 3, 3 pigs). (A to C) Mean values of clinical scores (A), body temperature (B), and leukocyte numbers (C) for each group of animals. (D) Titers of virus-neutralizing serum antibodies of the pigs are given as the maximal reciprocal dilution of the sera that are able to neutralize 65 TCID₅₀ of CSFV Alfort/Tübingen. Standard deviations are indicated in panel A.