Nucleolar Trafficking Of Classical Swine Fever Virus N^pro Protein And Its Effects On Viral Replication

Classical swine fever virus （CSFV） is the causative agent of Classical swine fever, a devastatingdisease of pigs worldwide. The N^proprotein of CSFV is involved in several functions, including anautoproteolytic activity, preventing the type I interferon （IFN） production and an association with thevirulence of CSFV. Interestingly, a recent study revealed that impairment of interferon regulatory factor3（IRF3）-dependent IFN-α/β inhibition is not a prerequisite for CSFV virulence. Therefore, N^promightplay more roles than expected in CSFV replication and/or virulence. Nucleolar-cytoplasmic shuttling ofviral proteins has been shown to be critical for the replication of some viruses. Furthermore, it has beenreported that N^prolocalizes in the cytoplasm and nucleus during CSFV infection. However, the relevanceof nucleus localization of N^proto viral replication remains unclear.In the present study, the kinetics of N^prosubcellular transport was described in details using CSFVmutants bearing the TC tag and biarsenical labeling technology. Two viable CSFV mutants with thetetracysteine （TC） tag （CCPGCC） inserted between amino acids4and5（vSMTC385） or13and14（vSMTC412） of the N^proprotein were recovered and showed similar growth characteristics to the parentvirus Shimen strain （wtShiman） or vShimen-II, the virus rescued from the cloned cDNA of Shimenstrain. Using the TC-biarsenical system to fluorescently label the N^proprotein, we found the protein waslocalized in the cytoplasm at27h post-infection （hpi） and present in the nucleolus at48hpi, withnucleolar import and export obviously observed from36.5to37hpi in living CSFV-infected cells. TheN^proprotein was also localized to the nucleolus of transiently transfected cells. EGFP-TC-N^proandEGFP-N^prohad similar subcellular localization, suggesting that the TC tag did not alter the subcellularlocalization of the N^proprotein. Western blot analysis of the subcellular fractions showed that EGFP-N^prowas present in the cytoplasmic and nuclear fractions of transfected cells.N^proprotein has a molecular weight of17kDa and there is no predicted nucleus localization signal（NLS） or nucleolus localization signal （NoLS） in the N^proprotein; all truncated N^proproteins werepresent in nucleus but not nucleoli. IRF3is known to interact with the N^proprotein and distributes in thecytoplasm of CSFV-infected cells. IRF3was shown to prevent N^proand its mutants from targeting thenucleus/nucleolus in infected or transfected cells coexpressing N^proand IRF3. Furthermore,EGFP-IRF3-N^profusion protein was only localized in the cytoplasm of transfected cells and retained theautoprotease activity of N^pro. In addition, the mutant N^pro-D136N not inhibiting the induction ofinterferon was able to present in the nucleoli. The results indicated that nucleolar targeting of the N^prowas independent of its known functions.To evaluate whether nucleolar trafficking of N^proaffects viral replication, the mutant vSM-IRF3expressing the IRF3-N^profusion protein that prevented N^profrom nucleolar trafficking was generated. The mutant vSM-D136N carrying the D136N mutation in N^proincapable of inhibiting IFN-β productionwas used to compare with vSM-IRF3. Compared to TC-N^proof vSMTC412present in the nucleolus,IRF3-N^proof vSM-IRF3was only localized to the cytoplasm at48hpi. The mean titer of vSM-IRF3inthe culture supernatant was1.7104TCID50/mL, approximately200-fold lower than that of wtShimen（3.2106TCID50/mL）, in contrast with the titer （105TCID50/mL） of vSM-D136N in the supernatant,which was30-fold lower than that of wtShimen. The multiple growth curve demonstrated that thegrowth of vSM-IRF3was greatly impaired at the late stage of replication cycle （36-72hpi）. IFN-β wasnot induced by the mutant vSM-IRF3as well as wtShimen in PK-15cells. Our results showed thatnucleolar-cytoplasmic shuttling of CSFV N^prois critical for viral replication.Taken together, the present study shows that nucleolar trafficking of N^prois a novel functionindependent of its known functions, and it is modulated by IRF3and plays a key role in the late stage ofCSFV replication cycle.