Functional Study Of Ubiquitin Proteasome System In Infectious Bronchitis Virus Replication Cycle

Avian infectious bronchitis(IB)is an acute highly exposed respiratory disease in chicken caused by avian infectious bronchitis virus(IBV),which belongs to Coronaviridae and Coronavirus-?.The ubiquitin proteasome system(UPS)is responsible for degradation of misfolded or damaged proteins,to maintain protein homeostasis in cells,as well as involved in regulation of cell cycle,signal transduction,transcription regulation,cell apoptosis,and antigen processing.The involvement of UPS in virus proliferation has been reported,including MHV,which belongs to Coronavirus-?..However,whether UPS is involved in IBV proliferation,is unclear.In this study,the role of UPS on IBV proliferation was studied by using IBV-Beaudette permissive cells Vero and H1299,or chick embryo fibroblast cells DF-1.1.Pharmacological inhibition of UPS suppresses IBV proliferationVero,H1299,or DF-1 cells were infected with IBV,followed with treatment with proteasome inhibitor MG132,Epoxomicin,or Bortezomib at different time points post-infection.Cells were harvested at 12 hours post-infection(hpi)and subjected to detecting the levels of IBV N protein and genomic RNA,to monitor IBV proliferation.Results showed that both IBV N protein and genomic RNA were greatly reduced by all three UPS inhibitors treatment at 0-6 hpi.Further experiment showed that UPS inhibitors mainly inhibited IBV proliferation when they were added at 0-2hpi,suggesting that UPS might promote IBV entry.Meanwhile,we carried out above experiment using PEDV and got similar results with IBV.Thus,UPS may be involved in different group of coronavirus entry.2.UPS plays a role after IBV internalization and before initial translationNext,we investigated the involvement of UPS in IBV entry steps: attachment,internalization,intracellular trafficking,membrane fusion,uncoating,initial translation.Initial translation refers to Open Reading Frame 1a(ORF1a)and ORF1 ab are translated from virus genomic RNA,producing polyprotein 1a and 1ab,which are processed by protease cleavage and produce the replicase(nsp2-nsp16),responsible for virus genome replication.By checking the virus genome internalization using quantitative real time RT-PCR at 2 hpi,we found that UPS inhibitors did not prevent IBV internalization.By monitoring the initial translation of IBV ORF1 a and ORF1 ab with Western blot to detect nsp3 at 5 hpi,we found the initial translation was suppressed by UPS inhibitors.We examined the global translation efficiency by puromycin labeling experiment,found that the translation rate was not affected by UPS treatment fo 2h.Thus,the decrease of ORF1 a and ORF1 ab products is probably due to less available RNA template,in stead of translational inhibition.In consideration of the internalized virus genome was not reduced,but the translation available virus genome was decreased,we speculate that UPS inhibitors may target to virus intracellular trafficking,membrane fusion,or uncoating.3.Ubiquitination of cellular proteins is involved in IBV entryFurther study showed that UPS inhibitors treatment resulted in depletion of cellular mono-ubiquitin,which may affect cellular proteins ubiquination and their function,thereby suppressing IBV entry.To validate above result,we employed uniquitin activating enzyme E1 inhibitor PYR-41 to inhibit the protein ubiquination process.Result showedthat PYR-41 suppressed IBV entry when it was added at 0-2 hpi.This result further substiate that ubiquitination of cellular proteins upon IBV infection is necessary for virus entry process.