| Ubiquitination is an important protein post-translational modif ication which was widely found in eukaryotic cells,involved in many cellular processes including protein degradation,cell signal transduction and cell cycle regulation.Many viruses have evolved multiple strategies to perturb the cell ubiquitin system.Fowlpox virus(FPV)ORF150 encodes a homologue of E3 ubiquitin ligase,belonging to N1R/p28 gene family,which contains a DNA-binding domain(KilA-N domain)and a RING finger domain.RING ubiquitin ligases have a feature that they can catalyze the addition of ubiquitins to themselves,referred to as autoubiquitination.To investigate whether ORF150 encoded protein is regulated by autoubiquitination,the eukaryotic expression vectors of ORF150 and Ub were constructed.Co-immunoprecipitation indicated that the FP V150 protein can be modified by ubiquitination;confocal microscopy experiments in LMH cells and 293 T cells showed that the subcellular localizations of FPV150 protein and Ub all changed after co-expression,from scattered to clustered.FPV150 protein colocalized with Ub near the nucleus.To explore the function of FPV ORF150,a ORF150 gene deletion recombinant virus was constructed in this study on Chick Embryo Fibroblast(CEF).First,we constructed rFPV-?150-EGFP with the reporter gene EGFP replacing the FPV NX10 ORF150 gene using homologous recombination and plaque purif ication on CEF cells.Then,we used Cre/loxP recombination enzyme system to knock out EGFP gene in the recombinant virus r FPV-?150-EGFP,thus obtaining r FPV-?150.By titrating the two recombinant viruses(r FPV-?150-EGFP F3 and r FPV-?150 F3)as well as wild virus,we drew their one-step growth curves which showed similar growth kinetics characteristics.To identify the genetic stability of the recombinant viruses,they had been serially passaged on CEF as well as the parential virus.After r FPV-?150-EGFP F6,the lesions began to decrease,so do the fluorescent markers;r FPV-?150 also showed a reduction in lesions after F5,but not the wild virus.PCR and Western blot of the recombinant viruse rFPV-?150-EGFP showed neither wild virus blending nor EGFP missing happened during passage.That of r FPV-?150 showed no wild virus blending happened during passage.The replication kinetics of the two recombinant viruses F5 and the parental virus were basically the same.With r FPV-?150 F3,we can further investigate the interaction between FPV150 encoded protein and host Ub on CEF cells.On the one hand,the difference of endogenous Ub expression level after inoculation of wild virus FPV and recombinant virus r FPV-?150 was analyzed at mRNA level using fluorescence quantitative PCR technology.The relative expression level of Ub was measured every 2 hours after infection from 1 h to 23 h.The results showed that the relative expression level of Ub in the r FPV-?150-infected group was higher than that in the FPV-infected group,the differences were signif icant(p < 0.05)at 3 h,7 h,21 h and extremely significant(p < 0.01)at 17 h,19 h and 23 h;FPV and r FPV-?150 promoted Ub transcription greatly at 19 h and 15 h post infection respectively,indicating that FPV promoted Ub transcription after a replication cycle.On the other hand,the effects of Ub overexpression on the titer of parental and recombinant viruses w ere analyzed by titration,indicating that overexpression of Ub without affecting cell viability can promote the replication of FPV,but inhibit the replication of r FPV-?150,indicating that FPV150 protein promotes virus replication by interacting with Ub.The interaction between FPV150 protein and Ub was studied preliminar ily in this study,which helps to shed light on the functional analysis of the ubiquitin system related genes of FPV and their relationship with virus replication. |
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