Inactivation of the RNase activity of glycoprotein E(rns) of classical swine fever virus results in a cytopathogenic virus

Abstract

Envelope glycoprotein E(rns) of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E(rns) are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E(rns) was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E(rns), the mutated proteins had lost their RNase activity. The mutated proteins reacted with E(rns)-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E(rns). This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E(rns) (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E(rns) gene and by neutralizing polyclonal antibodies directed against E(rns), indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E(rns) plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

FIG. 1

(A) Mutagenesis of E^rns H>K(2) by PCR. The amino acid sequence of the first (1) and the second (2) RNase domains are given in the one-letter code. a.a., amino acids in the ORF of CSFV strain C (24). The histidine residues at positions 297 and 346 and the introduced lysine residue in the second domain (K) are underlined. PCR primers are indicated by arrows. B, BamHI. pAcAS3.gXCE2 was the template for the first PCR (baculovirus transfer vector containing the nonmutated E^rns gene [12]). pGem4z-blue. E^rns was the template for the second PCR (containing the nonmutated E^rns gene recloned from pAcAS3.gXCE2 into the BamHI site downstream of the bacteriophage T7 promoter of pGem4z-blue). (B) Scheme for the construction of pPRKflc13, the full-length cDNA clone of CSFV strain C containing the mutated E^rns gene H>K(2). Npro, autoprotease; C, core protein; E1 and E2, envelope proteins. The pPRKc5.H>K(2) vector, containing the structural genes of CSFV strain C (amino acids 5 to 1063 [24]), including the H>K(2) substitution at position 346 (see panel A), transiently expressed E^rns and E2 in SK6 cells. pPRKflc133 is the nonmutated full-length DNA copy of CSFV strain C in plasmid pOK12 (24). phCMV is the promoter-enhancer sequence of the immediate-early gene of human cytomegalovirus. T7p, bacteriophage T7 promoter. CIP, calf intestinal phosphatase.

FIG. 2

Cytopathogenic effect induced by FLc13. Groups of SK6 cells infected with FLc13 were observed in end-point dilution titrations 96 h after infection before (A) and after (B) immunostaining with a MAb directed against E2 of CSFV. (C) Monolayer of SK6 cells infected with FLc13R before immunostaining. Immunostaining of this monolayer demonstrated that 100% of the cells were infected (data not shown).

FIG. 3

Single-step growth kinetics of FLc2 and FLc13. Confluent monolayers of SK6 cells were infected with an MOI of 2 to 5 TCID₅₀ of FLc2 and FLc13 per cell for 1.5 h at 37°C. The virus was removed, and fresh medium was added. After 0, 24, 48, 72, and 96 h of growth, the virus titer of the medium plus cells was determined by end-point dilution.

FIG. 4

RNase specific activity (A₂₆₀ min⁻¹ mg⁻¹) of E^rns expressed by FLc2, FLc13, and FLc13R but not of mutated E^rns purified from insect cells. Activity was measured at 37°C and pH 4.5 in an antigen capture RNase assay.

FIG. 5

DNA gel analysis. Confluent monolayers of SK6 cells were infected with an MOI of 2 to 5 TCID₅₀ of FLc2 (2), FLc13 (13), and FLc13R (13R) per cell for 1.5 h at 37°C. The virus was removed, and fresh medium was added. After 0, 24, 48, and 72 h of growth, cells released from the monolayer were recovered by centrifugation and extracted together with monolayer-associated cells. Extracted DNA (3 μg) was analyzed on a 1.5% agarose gel. PstI-digested lambda DNA was run in parallel as a molecular weight calibration (λ PstI). M, mock-infected SK6 cells.