Classical Swine Fever Virus Interferes with Cellular Antiviral Defense: Evidence for a Novel Function of N<sup>pro</sup>

Nicolas Ruggli

Jon Tratschin

Matthias Schweizer

Kenneth McCullough

Martin Hofmann

Artur Summerfield

Institute of Virology and Immunoprophylaxis, Mittelhäusern,^{Institute of Veterinary Virology, University of Berne, Switzerland}²

^{Corresponding author. Mailing address: Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland. Phone: 41 31 848 9211. Fax: 41 31 848 9222. E-mail:}hc.nimda.ivi@ilggur.salocin.

Received 2002 Oct 15; Accepted 2003 Apr 1.

Abstract

Classical swine fever virus (CSFV) replicates efficiently in cell lines and monocytic cells, including macrophages (MΦ), without causing a cytopathic effect or inducing interferon (IFN) secretion. In the present study, the capacity of CSFV to interfere with cellular antiviral activity was investigated. When the porcine kidney cell line SK-6 was infected with CSFV, there was a 100-fold increased capacity to resist to apoptosis induced by polyinosinic-polycytidylic acid [poly(IC)], a synthetic double-stranded RNA. In MΦ, the virus infection inhibited poly(IC)-induced alpha/beta IFN (type I IFN) synthesis. This interference with cellular antiviral defense correlated with the presence of the viral N^{gene. Mutants lacking the N}^{gene (ΔN}^{CSFV) did not protect SK-6 cells from poly(IC)-induced apoptosis, despite growth properties and protein expression levels similar to those of the wild-type virus. Furthermore, ΔN}^{CSFV did not prevent poly(IC)-induced type I IFN production in MΦ but rather induced type I IFN in the absence of poly(IC) in both MΦ and the porcine kidney cell line PK-15, but not in SK-6 cells. With MΦ and PK-15, an impaired replication of the ΔN}^{CSFV compared with wild-type virus was noted. In addition, ΔN}^{CSFV, but not wild-type CSFV, could interfere with vesicular stomatitis virus replication in PK-15 cells. Taken together, these results provide evidence for a novel function associated with CSFV N}^{with respect to the inhibition of the cellular innate immune system.}

Abstract

Classical swine fever (CSF) is a highly contagious disease of pigs caused by the classical swine fever virus (CSFV) and leads to important economic losses worldwide. CSFV, together with bovine viral diarrhea virus (BVDV) and border disease virus (BDV), form the genus Pestivirus within the family Flaviviridae. The two other genera of the family are the genus Flavivirus and the genus Hepacivirus (55). Pestiviruses carry an RNA genome which possesses a 5′ untranslated region (5′UTR), a single large open reading frame (ORF), and a 3′UTR. The 5′UTR functions as an internal ribosomal entry site for cap-independent translation initiation. The ORF encodes a polyprotein of approximately 3,900 amino acids which is processed into 12 mature proteins by virus-encoded and host cell proteases (for a review, see reference 35). The first protein encoded is the nonstructural protein N^{. It exhibits autoproteolytical activity and cleaves itself off the downstream nucleocapsid protein C (}43, 47, 56). Interestingly, it has no corresponding counterpart in flaviviruses and hepatitis C virus and was found to be dispensable for virus replication in cell culture (52). The N^{gene of the moderately virulent strain vA187-1 and of the highly virulent strain vEy-37 has been deleted or replaced with the murine ubiquitin gene, which substitutes for N}^{in the generation of the authentic nucleocapsid protein. Such mutants lacking the N}^{gene (ΔN}^{CSFV) displayed no major alteration of growth characteristics in the porcine kidney cell line SK-6 but were avirulent in pigs (}52; D. Mayer, M. A. Hofmann, and J. D. Tratschin, submitted for publication).

Monocytes and macrophages (MΦ) are among the main targets for CSFV infection (29, 53). They are potential factors in the spread of CSFV to different tissues, are major reservoirs for infectious virus, and are involved in CSFV-induced immunomodulation (29). Although CSFV replication in MΦ is sensitive to interferon (IFN), the virus does not induce IFN in these cells (28). Considering that MΦ possess the potential to produce alpha/beta IFN (type I IFN) in response to virus infection (39), it is plausible that CSFV may be capable of counteracting cellular antiviral activity. In this vein, Schweizer and Peterhans (44) recently demonstrated that noncytopathogenic (ncp) BVDV interfered with polyinosinic-polycytidylic acid [poly(IC)]-induced apoptosis in primary bovine turbinate cells and IFN induction in bovine MΦ. As CSFV is also ncp, the present work sought to determine the capacity of CSFV to interfere with apoptosis in the swine kidney cell line SK-6 and with IFN-α/β production in porcine MΦ. The results demonstrated that resistance to poly(IC)-induced cell death and control of IFN induction were dependent on the presence of the N^{gene, indicating a function of N}^{in innate immune evasion of CSFV.}

Acknowledgments

This work was in part funded by the Swiss National Science Foundation (grant # 31-56719-99).

We thank Markus Gerber, Luzia Liu, Viviane Neuhaus, René Schaffner, and Valérie TÂche for excellent technical assistance and Christian Griot for continuous support. We also thank Martin D. Fray for providing the Mx/CAT reporter gene assay and Bernard Charley for the recombinant porcine IFN-α.

Acknowledgments

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