The Study On The Molecular Mechanism Of Ebola Virus Switching From RNA Synthesis To Assembly/budding

Ebola virus(EBOV)is an enveloped,non-segmented and negative-sense RNA virus,belonging to the genus Ebolavirus in the family Filoviridae of the order Mononegavirales.The viral genome is encapsidated by NP to form NP-RNA template,and then interacts with VP35,VP30 and L for the formation of RNP,to facilitate viral transcription and replication.The inclusion bodies of EBOV are the factories for RNA synthesis,and NP expression alone could lead to the formation of IBs.The nucleocapsid(NC)is formed at the periphery of the IBs,moreover,NP together with VP35 and VP24 are indispensable for the formation of nucleocapsid-like(NC)structures,which indicates NP can recruit other viral proteins for viral replication and NC assembly.These results suggest that NP plays a critical role in lifecycle of EBOV.Virus-like particle(VLP)system is useful to study the mechanism of viral assembly/budding.VP40 is the major matrix protein of EBOV,when expressed alone,it can release as a form of VLP,thereby regulating the assembly/budding of inner viral proteins.A previous study showed that VP40 also inhibits the synthesis of viral RNA.Given the essential role played by NP and VP40 in viral lifecycle,and some studies have been focused on NP and VP40.A previous work showed that both NP N-terminal 150 amino acids and C-terminal 139 amino acids are important for the interaction with VP40.Another report showed that NP C-terminal 50 amino acids are critical for NP incorporation into VP40–VLPs.However,several questions have not been resolved: 1)The precise site(s)of NP that are essential for NP incorporation into VP40–VLPs;How do N-and C-termini of NP function during the process of NP being incorporated into VP40–VLPs? 2)What the mechanism of viral internal proteins assembly? 3)What the mechanism of switching from viral RNA synthesis to NC assembly/budding?Our results demonstrate that the amino acids 26 to 150 from NP N-terminus are critical for the interaction with VP40,as well as the incorporation of NP into VP40–VLPs,via co-IP and VLP budding assays.We have confirmed that NP C-terminal 50 amino acids are indeed critical for NP incorporation into VP40–VLPs by VLP budding assay.We constructed a series of deleted and point mutants of NP C-terminus,and identified the precise sites L692,P697,P698 and W699 that are critical for NP incorporation into VP40–VLPs.Furthermore,we observed that side chains of these amino acids constitute a hydrophobic core,via structural analysis.These results show that both N-terminus and hydrophobic core within the C-terminus are indispensable for NP incorporation into VP40–VLPs.When these sites mentioned above are mutated to alanines,the hydrophobic core at the NP C-terminus is destoried,which results in enhancing the interaction of VP40 and NP,sequestration of VP40 into their IBs,thereby inhibiting the release of VP40–VLPs.These results suggest the interaction of NP N-terminus with VP40 is a prerequisite for the incorporation of NP into VP40–VLPs and only contributes to recruiting VP40 into IBs.If only the N-terminal interaction with VP40 is preserved,and the C-terminal interaction of NP with VP40 is deprived,VP40 will be trapped in the IBs,thereby inhibiting the assembly/release of VP40–VLPs.We also found that the interaction of NP with VP40 mediates viral proteins assembly,and VP40 incorporates nucleocapsid-like structures into VLPs by interacting with NP C-terminal hydrophobic core.Furthermore,we found that the hydrophobic core is critical for NP encapsidating viral RNA,and VP40 prevents NP from encapsidating RNA by association with this domain,thus inhibiting RNA synthesis.In conclusion,the N-terminus of NP recruits VP40 into IBs,which results in a conformational change and exposure of the hydrophobic core within the NP C-terminus.Subsequently,VP40 interacts with the hydrophobic core,on one hand,inhibiting RNA synthesis;on the other hand,incorporation NC into VLPs.These results suggest that the two-stage interaction of VP40 with NP plays a critical role in switching from viral RNA synthesis to virion assembly/budding.