Preliminary Study On The Formation Of Newcastle Disease Virus Inclusion Bodies

Newcastle disease(ND)caused by the Newcastle disease virus(NDV)is an acute,virulent infectious disease,which can infect a wide range of birds and poultry,causing up to100% morbidity in chickens,and causing significant damage to the poultry industry worldwide.NDV is a negative-sense RNA virus and belongs to the genus Orthoavulavirus of the Paramyxoviridae family.Many negative-strand RNA viruses infect host cells and form membraneless intracellular compartments called inclusion bodies(IBs),viral factories(VFs)or viroplasms.These membrane-less viral compartments are generally considered to be critical sites of viral replication.Up to now,the formation,organization and regulation of NDV IBs remain largely unknown.In this study,we explored the formation of NDV IBs using techniques such as immunofluorescence,live cell imaging and fluorescence recovery after photobleaching etc.The main contents and results of this study included the following several aspects.1.We used NP protein as a marker to analyse the formation of inclusion body-like structures after NDV infection and its relationship with the time of virus infection.The results showed that spherical inclusion body-like structures began to appear at 12 h post infection.As the time of infection increased,the size of in inclusion body-like structures increased and their shapes become irregular.To investigate whether the inclusion body-like structure contains newly synthesized viral RNA,we used Act D to inhibit cellular RNA synthesis(without affecting viral RNA synthesis)and detected EU-labelled newly synthesized viral RNA in the inclusion body-like structures.The inclusion body-like structures were observed using transmission electron microscopy(TEM),the results showed that these structures were membrane-less granular structures with high electron density.The above results demonstrated that the spherical structures formed by NDV infection were similar to IBs of single-stranded negative-stranded RNA viruses in terms of morphology,composition and ultrastructure,and we considered that NDV infection triggers the formation of IBs.2.The high electron density of NDV IBs and the fact that small IBs appear to be able to fuse with each other to form large IBs suggested that NDV IBs might had properties consistent with liquid-liquid phase separation.Currently,the commonly accepted criteria for defining a phase-separated structure are that it is spherical,fuses and recovers from photobleaching.In this study,we constructed plasmids in which EGFP was tagged to the N-terminal(EGFP-NP)and C-terminal(NP-EGFP)of NP proteins.Upon NDV infection,we found that the NP-EGFP re-localizes into newly formed IBs by immunofluorescence and laser scanning confocal microscopy,thus making it a suitable marker for live-cell imaging of NDV IBs.DF-1 cells were transfected with NP-EGFP and infected with F48E9.At 12 h post-infection,we monitored and observed fusion events between these IBs.We performed FRAP analyses using cells infected with NDV,rapid and efficient recovery after photobleaching for the indicated IBs could be observed,suggesting that IBs exchange substances with their surroundings We further investigated the effect of 1,6-hexanediol,which could dissolve the liquid-like membrane-less organelles,on the NDV IBs.In the presence of 1,6-hexanediol,NDV IBs were rapidly dissolved.Taken together,these data demonstrate that NDV IBs have properties consistent with liquid-liquid phase separation.3.The RNP complex,comprised of NP,P,and L proteins,is essential for viral RNA replication.Here,NP,P and L proteins were transfected separately or in combination in DF-1cells.Coexpression of NP and P was able to generate IB-like puncta in DF-1 cells.To further investigate the regions of viral proteins required for the IB assembly,NP and P were truncated based on their functional domains.Using co-expression,immunofluorescence microscopy and Co-IP,we conclude that the N-terminal of NP and the C-terminal of P are pivotal regions for IB-like puncta formation triggered by these two viral proteins.Furthermore,NDV P is a highly phosphorylated protein and a previous report found that protein kinase C(PKC)is involved in the NDV life cycle,we explored whether PKC plays a role in IB formation.NDV-infected cells were treated with staurosporine(PKC inhibitor)or PMA(PKC agonist)respectively.As expected,treating NDV-infected cells with staurosporine significantly decreased the IB size,the viral protein and viral titer,while PMA had no effect.The inhibitory effect of staurosporine was hindered by the PMA treatment.These results suggest that the PKC affects the formation of IBs and NDV proliferation.4.Some RNA viruses are able to use cellular organelles such as the endoplasmic reticulum,Golgi apparatus and microtubules to promote virus proliferation,therefore the relationship between NDV IBs and the endoplasmic reticulum,Golgi apparatus and microtubules was explored in this study.NDV IBs localized nearby the endoplasmic reticulum(ER)and Golgi apparatus.When the endoplasmic reticulum,Golgi apparatus and microtubules were disrupted by inhibitors,the size of IBs was reduced,the virus titer and viral protein expression were significantly decreased and NDV proliferation was inhibited.Thus the integrity of the endoplasmic reticulum,Golgi apparatus and microtubules is necessary for NDV IBs formation and NDV proliferation.In conclusion,this study determined that NDV infection triggers the formation of IBs which exhibited properties consistent with liquid-liquid phase separation(LLPS).IBs were membrane-less structures with high electron density containing newly synthesized viral RNA.We found the nucleoprotein(NP)and P were sufficient to generate IBs,with the N terminus of NP and the C terminus of P playing important roles in this process.The protein kinase C(PKC)could affect IBs formation and NDV proliferation.In addition,the integrity of ER,Golgi and microtubules was important for IBs formation.