Effects of the nuclear localization of the Npro protein of classical swine fever virus on its virulence in pigs

•A mutant CSFV was generated stably expressing IRF3-Npro fusion protein.•Npro of the virus failed to localize in the nucleus in contrast with that of its parent virus or EGFP-CSFV.•The mutant virus was markedly attenuated both in vitro and in vivo.•Pigs inoculated with the virus were protected from the lethal CSFV challenge.•The virus did not induce IFN response yet retained the autoproteolytic activity of Npro.

The Npro protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of Npro correlates with the virulence of CSFV in the host. Previously, we showed that the Npro protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the Npro gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-Npro fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-Npro fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of Npro plays a significant role in the CSFV replication and virulence.