A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

Department of Infectious Diseases, Institute of Virology, University of Veterinary Medicine, D-30559 Hannover, Germany.
Journal of Virology (Impact Factor: 4.44). 09/2009; 83(23):12415-23. DOI: 10.1128/JVI.01487-09
Source: PubMed


For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33 degrees C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5 degrees C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5 degrees C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33 degrees C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.

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    • "The cp biotypes of BVDV induce cell death by apoptosis [44], [52], triggered by the accumulation of viral double-strand RNA [45], [53] and the continuous expression of NS3, the cleaved form of the nonstructural protein NS2-3 [14], [15]. Although viral RNA production in cp BVDV infections is higher than that in ncp ones, both show similar production of infectious viral particles [18], [53]–[56]. Accordingly, cell cultures infected with BVDV R2, R3 and R5 showed a decrease in the induction of apoptosis and in the accumulation of viral RNA. However, BVDV R1–5 produced higher infectious virus titers than wt BVDV p0, and due to host cell survival, BVDV R2, R3 and R5 produced viral antigens and infectious viral progeny for at least four additional days. "
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