The Molecular Mechanism Of CypB To Promote Orf Virus Replication

Contagious ecthyma is an acute,highly contagious disease which is caused by Orf virus(ORFV).It is characterized by the formation of erythema and pustules in the erythema and pustules of infected sheep.And the disease is also an important zoonosis.Our previous research confirmed that the host factor cyclophilin B(Cyp B)could contribute the proliferation of ORFV.However,the biological function of Cyp B in the ORFV infection process is still unknown.In addition,the mechanism of ORFV utilizing Cyp B to contribute its proliferation is still unclear.Therefore,the study investigated the potential biological functions of Cyp B in the infection process of ORFV,and screen viral proteins or protein molecules interacting with Cyp B by immunoprecipitation.In order to clarify the potential biological function of Cyp B in ORFV infection process,the localization distribution of Cyp B in infected cells and normal cells was detected by confocal laser scanning microscopy.Then,ultrathin sections of infected cells and normal cells were made and observed by transmission electron microscope.Changes in microstructure and distribution of Cyp B proteins showed that Cyp B mainly distributed on the rough endoplasmic reticulum of MDBK cells.Afterwards,the ER-Tracker Red was used for fluorescence labeling.The distribution of Cyp B at the subcellular level was observed by confocal laser scanning microscopy.The results showed that there was a clear co-localization between Cyp B and endoplasmic reticulum in ORFV-infected cells.This result further confirmed that Cyp B mainly located in the rough endoplasmic reticulum of MDBK cells.The specific inhibitor cyclosporin A(Cs A)of Cyp B was used to investigate the effect of the cis-reversion isomerase activity of Cyp B on ORFV proliferation.Western-blot was used to detect the effect of different concentrations of Cs A on the expression of Cyp B in early ORFV-infected cells.The results showed that the expression of Cyp B gradually decreased with the increase of Cs A concentration,which indicated that Cs A could inhibit the expression of Cyp B.Real-time PCR was applied to detect the effect of different concentrations of Cs A on virus replication.The results showed that Cs A not only could inhibit the expression of Cyp B,but also inhibit the proliferation of ORFV in host cells at low doses of drug concentration.The above results further confirmed that Cyp B was an important cofactor in the process of ORFV infection and could promote ORFV replication.However,the mechanism of ORFV utilizing Cyp B to contribute its proliferation is still unclear.Therefore,the viral proteins or protein molecules interacting with Cyp B was screened by immunoprecipitation.The results of SDS-PAGE and Western-blot analysis of co-immunoprecipitation products showed that there were distinct bands at the size of about 25 k D.The differential protein bands were separated for mass spectrometric identification.The original mass spectral data obtained were searched and identified using the MS/MS Ions Search tool(Mascot software).Six proteins related to ORFV were obtained,which might interact with Cyp B.The prospective result will not only help us to understand deeply the molecular pathogenesis of ORFV,but also provide valuable information for blocking infection process of ORFV by Cyp B pathways.