Inactivation of the RNase Activity of Glycoprotein E ^rns of Classical Swine Fever Virus Results in a Cytopathogenic Virus

Abstract

Envelope glycoprotein Erns 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 Erns 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 Erns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact Erns, the mutated proteins had lost their RNase activity. The mutated proteins reacted with Erns-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 Erns. 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 Erns (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 Erns gene and by neutralizing polyclonal antibodies directed against Erns, 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 Erns 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.

Crystal Structure of the Pestivirus Envelope Glycoprotein Erns and Mechanistic Analysis of Its Ribonuclease Activity

“…The active site of BVDV E rns has been determined to consist of His32, His76, Glu77, Lys80, and His81 ) ( Figure 2D), in agreement with single-site mutations rendering the enzyme catalytically inactive (Hulst et al, 1998;Meyers et al, 1999). Superposition of the catalytic residues of E rns and MC1 demonstrates their structural homology (Figure 4A) .…”

“…The RNase activity of E rns is not essential for virus viability in tissue culture (Hulst et al, 1998), but its abrogation also results in attenuation in the animal host (Meyer et al, 2002;Meyers et al, 1999). Although the mechanism for this attenuation is not understood at present, the ribonucleolytic activity of E rns was reported to block activation of the innate immune system (Iqbal et al, 2004;Meyers et al, 1999).…”

Laboratory diagnosis, epizootiology, and efficacy of marker vaccines in classical swine fever: A review

“…Virus isolation is currently the most sensitive in vitro method for the detection of infectious CSFV. Several methods have been described for isolation of CSFV from tissue, whole blood, or blood components (2,18,36,39,66). Often the leucocyte fraction has been considered the sample of choice (1).…”

“…The three structural envelope proteins are glycosated (67). Recently, infectious cDNA clones of CSFV have become available, which has enabled researchers tostudy the mechanisms of viral replication, attenuation, and pathogenesis (36,52,54,57).…”

Identification of Antigenic Regions of the Erns Protein for Pig Antibodies Elicited during Classical Swine Fever Virus Infection

“…The biological function of Eros ribonuclease activity is not yet clear. Mutations in Erns destroying the RNase activity give rise to viruses that are more cytopathic in culture and are attenuated in vivo (14,15). Antibodies that inhibit ribonuclease activity also tend to neutralize virus infectivity (7).…”