Mutations abrogating the RNase activity in glycoprotein E(rns) of the pestivirus classical swine fever virus lead to virus attenuation

Abstract

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV). Amino acid exchanges or deletions introduced by site-directed mutagenesis into the putative active site of the RNase residing in the glycoprotein E(rns) of CSFV abolished the enzymatic activity of this protein, as demonstrated with an RNase test suitable for detection of the enzymatic activity in crude cell extracts. Incorporation of the altered sequences into an infectious CSFV clone resulted in recovery of viable viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. These RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable for at least seven passages. In contrast to animals inoculated with an RNase-positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cells, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchanges of both codons 297 and 346 showed that all these RNase-negative mutants were attenuated. All viruses with mutations affecting codon 346 were completely apathogenic, whereas those containing only changes of codon 297 consistently induced clinical symptoms for several days, followed by sudden recovery. Analyses of reisolated viruses gave no indication for the presence of revertants in the infected animals.

FIG. 1

Sequences encoded by CSFV Alfort/Tübingen (CSFV) and the different E^rns mutants. Only those parts of the E^rns sequence containing the conserved regions with the putative active-site residues of the RNase are shown. Residues conserved with regard to known RNases of fungal origin are indicated in boldface. Residues 297 and 346 of the polyprotein are shaded and underlined.

FIG. 2

Expression of E^rns in the vaccinia virus vTF7-3 system. The different cDNA constructs were transfected into BHK21 cells infected with vTF7-3. The cells in one set of dishes were labeled with radioactive amino acids. The labeled proteins were precipitated with antisera specific for N^pro or E^rns, and equal volumes of the precipitates were separated by PAGE. The gels were analyzed by fluorography (upper part) or by measuring the amount of radioactivity contained in the specific bands by using a phosphorimager (middle). The protein extracts of a second set of dishes were used for the determination of RNase activity (lower part). Cells infected with vTF7-3 but not transfected with a plasmid were used as negative controls. For the RNase test, 100 ng of RNase A from bovine pancreas (0.0085 Kunitz units) served as a positive control. RNase activity was determined by measuring the OD₂₆₀ due to the release of acid-soluble RNA. OD values of ca. 0.4 to 08 as measured for the negative control are not due to enzymatic degradation of RNA, since no decrease was observed when less of the cell extracts was used for the test.

FIG. 3

Influence of antibodies on the RNase activity. Diluted extracts of cells transfected with p704 or p705 were first incubated with a polyclonal antiserum directed against E^rns (anti-E^rns) or a rabbit preimmune serum (NS) and then tested for RNase activity. An extract from cells infected with vTF7-3 (control) or 10 ng of RNase A from bovine pancreas (0.00085 Kunitz units) served as controls.

FIG. 4

Northern blot with RNA from cells infected with cell extracts prepared from cells transfected with in vitro-transcribed RNA containing the mutations resulting in the indicated changes in the E^rns sequence. Total RNA of the infected cells was separated in an agarose gel under denaturing conditions, transferred to a nylon membrane, and hybridized with a CSFV-specific probe. On the left side of the gel, the bands of an RNA ladder are indicated.

FIG. 5

Analysis of RNase activity in extracts of cells infected with CSFV Alfort/Tübingen (Alfort), the virus recovered from the infectious clone pA/CSFV [V(pA/CSFV)], or the indicated mutants thereof at 1 passage postinfection. The given OD values represent the averages of two independent experiments.

FIG. 6

Body temperature curves of animals infected with V(pA/CSFV), the virus recovered from the infectious cDNA clone pA/CSFV (solid lines), or C-346-d, the mutant thereof that contains a deletion of codon 346 (broken lines).

FIG. 7

Body temperature curves of animals infected with C-346-d/Rs, the virus recovered from the infectious cDNA clone in which the deletion present in C-346-d had been removed (solid lines) or the original mutant C-346-d (broken lines).

FIG. 8

Body temperature curves of animals infected with CSFV mutants C-346-L (A), C-297-L/346-L (B), C-297-L (C), or C-297-K (D).

FIG. 8

Body temperature curves of animals infected with CSFV mutants C-346-L (A), C-297-L/346-L (B), C-297-L (C), or C-297-K (D).

FIG. 8

Body temperature curves of animals infected with CSFV mutants C-346-L (A), C-297-L/346-L (B), C-297-L (C), or C-297-K (D).

FIG. 8

Body temperature curves of animals infected with CSFV mutants C-346-L (A), C-297-L/346-L (B), C-297-L (C), or C-297-K (D).

FIG. 9

Percentage of CD21-positive B lymphocytes in the PBMC of animals infected with virus mutants. Swine infected with the original mutant (C-346-d) are indicated by broken lines. The group of animals infected with the virus recovered from the infectious cDNA clone, in which the deletion had been removed (C-346-d/Rs), is indicated by solid lines. The percentage of CD21-positive B lymphocytes was determined as described in Materials and Methods.