The Explortion Of The Interaction Between Classical Swine Fever Virus And Interferon-stimulated Gene 15

Interferon-stimulated gene 15(ISG15)is a kind of ubiquitin(Ub)-like protein,coded by isgl5 gene under interferon stimulation.It is similar to Ub in sequence,structure and function,covalently modifying target protein by enzyme cascade.ISG15 and its Ub-like modification system participate in the immune response,which is an important pathway for the interferon exerting antiviral effect.In this study,the recombinant swine IFN-a has been used as a standard stimulus to verify the feasibility for detecting ISG15 and ISGylation.Some methods have been applied including real-time quantitative reverse transcription PCR(RQ-RT-PCR)for detection of ISG 15 on the transcriptional level;indirect immunofluorescence analysis(IFA),flow cytometry(FCM)and western-blot(WB)for detection of ISG 15 on the translational level;moreover WB for detection of ISG 15 on the modificatory level.The experimental results showed those methods could be effectively used to detect ISG 15 and ISGylation.By combination of the techniques mentioned above,this study verified that the infection of classical swine fever virus(CSFV)could up-regulate the transcriptional,translational and modificatory level of ISG15.After the PK-15 cells infected with 1 moi of CSFV,ISG15 mRNA levels were identified to up-regulate 15.89,65.80,121.94 and 128.89 folds at 9,12,24 and 48 hours post-infection(hpi)by RQ-RT-PCR,respectively;the up-regulation of ISG 15 was significantly observed at 9 hpi by IFA;the rate of ISG 15-expressing positive cells were indicated the increases from 0.27%at 0 hpi up to 3.37%,6.81%and 19.42%at 6,12 and 24 hpi by FCM,respectively;Using WB,the specific protein bands of ISG15 and ISGylation were determined at 12 hpi,demonstrating that there was a significant correlation between the amounts of ISG15/ISGylation and the amount of CSFV E2 protein.The experimental results showed that ISG15 and its ubiquitin-like modification system play an important role in the process of CSFV infection.Compared with the normal PK-15 cells,ISG15 overexpression in PK-15 cells infected with 1 moi of CSFV declined the CSFV 5'-UTR amounts of 34.47%,58.25%,63.40%and 49.30%at 9,12,24 and 48 hpi,respectively.In the ISG 15 overexpressed cells,the CSFV E2 protein was significantly decreased;furthermore,the CSFV titers were reduced 58.33%,62.50%,46.67%and 58.33%at 9,12,24 and 48 hpi,respectively.The experimental results showed that ISG 15 overexpression could not only significantly decline the CSFV RNA abundance and viral protein amounts,but also reduce the virus titer,confirming that ISG 15 possesses the biological activity to inhibit CSFV replication.In this study,we found that ISG 15 and its Ub-like modificatory level in UV-inactivated CSFV inoculated cells did not change significantly,indicating that the survival of CSFV is necessary for activating ISG 15 expression and its Ub-like modification.To expound the pathway of CSFV up-regulating ISG 15,WB has been used to detect IRF3,NF-kB and the influence of HDAC6 inhibitor on ISG 15 expression.The experimental results indicated that the expression of IRF3 and NF-?B did not be influenced in the process of classical swine fever virus regulating ISG 15,moreover classical swine fever virus did not regulate ISG 15 via HDAC6.