| Feline calicivirus(FCV)is a highly infectious pathogen with a widespread distribution in the feline population,which usually causes upper respiratory diseases,oral ulcers and occasionally causes fever,lameness or subclinical symptoms in cats.Mixed infection of FCV and Feline herpesvirus type I(FHV-1)in cats can occur frequently.The presence of high virulence strains in recent years can cause edema,ulcerative skin lesions,jaundice and high mortality,which seriously threaten the health of cats.FCV is RNA virus with high levels of genetic diversity,which plays a key role in vaccine immune failure and is the basis for the emergence of highly virulent strains.The interaction between virus and the host can lead to the shutoff of the host gene.Virus regulate the transcriptional abundance of the themselves by closing the host gene expression,thus evading host immune surveillance.FCV infection whether lead to host gene shutoff or not,FCV unstructured proteins how to block host gene expression.Exploring the mechanism of host gene shutoff can better understand the FCV pathogenic mechanism and provide a theoretical basis for the prevention and control of FCV infection.FCV 2280 is a a moderately virulent strain,this study first performed isotope experiments to determine whether FCV 2280 infection can cause host gene expression shutoff.The results showed that FCV infection reduced host protein production early in infection but increased viral protein synthesis,such as VP1.FCV infection can cause host gene shutoff and affect host protein synthesis.To detect whether down-regulating the expression mRNA host gene after FCV 2280 infection,a Northern Blot method was established to prepare biotin-labeled GFP and GAPDH DNA probes.This study,by Northern blot detection method,found that FCV infection can inhibit the mRNA expression of host endogenous gene GAPDH and exogenous gene GFP,and shut down the expression of host gene by degrading host gene.Secondly,using Northern blot method,PP was selected as the main viral factor of host gene shuoff caused by FCV,and the key active sites of host gene shutoff caused by PP protein was identified.According to the fluorescent quantitative PCR test,it was detected that PP could down regulate the mRNA expression of host external gene and internal gene by inducing RNA degradation.The RNA type of PP was identified as mRNA transcribed by RNA polymerase II,which was combined to the mRNA in a ribosome independent manner.Finally,the degradation of host mRNA was completed by exonuclease Xrn1.In order to detect whether PP protein can show nuclease activity as other viral factors that cause the host gene shuoff in vitro,GST-PP fusion protein and its mutant PP H39 A were purified.The same amount of substrate RNA was incubated with GST-PP fusion protein,mutant GST-PP H39 A and GST in 30? water bath pots respectively for 90 min.The results showed that the RNA substrate incubated separately with GST protein remained stable during the whole incubation period,while the RNA substrate incubated with GST-PP fusion protein was cut into small fragments.With the prolongation of incubation time,the small fragments were not further degraded.In order to further verify the results in vitro,GST-PP H39 A fusion protein was purified.After incubation of RNA substrate with GST-PP H39 A,no degradation fragment was detected.These results showed that PP had nuclease activity and caused gene shutoff of the host by cutting the host mRNA.In conclusion,this study applied Northern blotting to identified that FCV shut down the expression of host gene by degrading the host mRNA;PP was the main viral factor that caused the host gene shuoff by FCV,and identified the key active sites that PP protein caused the host gene shutoff;the type of RNA that PP targets was the mRNA transcribed by RNA polymerase II,and PP bind to mRNA in a ribosome-independent manner,and finally complete the degradation of the host mRNA through Xrn1 host exonuclease;PP has nuclease activity and cause host gene shutoff by cleavage of host mRNA. |
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