| Glycosylation and ubiquitination are two important post-translational modifications of host proteins,which play an important role in protein function,folding,intracellular localization and transport,and protein degradation.Since the virus is highly dependent on host cells,the virus protein will also undergo glycosylation and ubiquitination in the process of protein translation.How glycosylation and ubiquitination affect the function of virus protein and the biological characteristics of influenza virus,as well as the related molecular mechanism,are still unclear.This study focused on the biological significance of glycosylation and ubiquitination of influenza virus NA protein,as well as the effect of virus infection on the ubiquitination level of host proteins,in order to reveal the effect of glycosylation and ubiquitination on the biological characteristics of influenza virus and its molecular mechanism.N-type glycosylation(NLG)occurs in eukaryotic cells.Hemagglutinin(HA)and neuraminidase(NA)are highly glycosylated proteins of influenza virus.Studies have shown that N-glycosylation of HA protein can affect the antigenicity,immune escape,host adaptability and pathogenicity of influenza virus.Although NA protein plays a variety of functions in the life cycle of influenza virus,especially in the late stage of virus replication,the effect of N-glycosylation on the function of NA protein and the biological characteristics of influenza virus remains unclear.In this study,we identified three glycosylation sites(44,72219)in the NA protein of a / WSN / 1933 H1N1 virus.Tunicamycin treatment inhibited the occurrence of N-type glycosylation or the deletion of these three N-type glycosylation sites significantly inhibited the budding ability of NA protein,which indicated that N-type glycosylation was very important for the budding ability of NA protein.After the deletion of three N-type glycosylation sites,the mutant viruses were successfully rescued.Further studies showed that the deletion of glycosylation at 219 site significantly decreased the budding ability and replication level of the mutant virus.The results of mice study also showed that the pathogenicity of 219 site glycosylation deficient virus was siglucicantly attenuated,and it did not cause death in the infected mice,indicating that 219 site glycosylation modification played an important role in the budding ability of NA protein,in vitro replication ability,and pathogenicity of mice.In order to further explore its potential molecular mechanism,we detected UPR after overexpression of N-glycosylation knocking-out NA protein.The results showed that Nglycosylation knocking-out NA protein accumulated in endoplasmic reticulum(ER),resulting in UPR related proteins BIP and p-eif2 ? up-regulated,while XBP1 was down regulated,which indicated that Nglycosylation knocking-out NA protein accumulated in ER and induced UPR,which results in the failure of NA protein to transport and bud in cells,and affects the replication and pathogenicity of the virus.In addition to glycosylation,ubiquitination also exists in influenza virus proteins,which play an important role in viral protein function and influenza virus replication.Previous studies on ubiquitination of influenza virus proteins mainly focus on the internal proteins of the virus,including polymerase protein,NP protein,M1 protein and NS1 protein.However,there is no report on ubiquitination of surface protein NA.In this study,we found for the first time that the NA protein of influenza virus was also ubiquitinated.Further studies showed that K63 was the main type of ubiquitination of NA protein.In order to further identify the ubiquitination sites,we constructed truncated and point mutated NA protein,and found that lysine at position 6 of NA protein was one of its ubiquitination sites by immunoprecipitation,and that the ubiquitination type of K6 residue was K63 ubiquitination.The mutant virus congtaining K6 R mutation was successfully rescued.The results of in vitro experiments showed that the replication level of the mutant virus was 1000 times lower than that of the wild virus after the deletion of ubiquitination modification by K6 R mutation,and the mouse experiment also showed that the pathogenicity of NA-K6 R mutant virus was significantly attenuated and none of the infected mice died,which indicated that ubiquitination modification of this K6 site is very important for the replication and pathogenicity of the virus.In order to find the host E3 enzyme that ubiquitin modified NA protein,we identified a host E3 enzyme interacting with NA protein: HUWE1 by pull-down method,and verified that HUWE1 is ubiquitin E3 enzyme of ubiquitin modified NA protein by si RNA interference.In conclusion,K63 type ubiquitination exists in the K6 site of influenza virus NA protein,which plays an important role in the replication and virulence of influenza virus.Ubiquitination not only occurs directly in viral proteins to regulate their functions,but also regulates the host factors that are key to viral replication,affecting host immune response,protein quality control and antiviral signaling pathways,thus having an important impact on many aspects of influenza virus replication cycle.In order to further study the interaction between influenza virus infection and ubiquitination modification of host protein,the changes of ubiquitination level of host protein after influenza virus infection in U937 and A549 cells were studied by means of ubiquitination proteomics.After virus infection in U937,the ubiquitination levels of 18 proteins were significantly up-regulated,42 proteins were significantly down regulated,52 non ubiquitinated proteins were ubiquitinated,and ubiquitination of 57 proteins were completely disappeared.After influenza virus infection in A549 cells,ubiquitination levels of 43 protein were significantly up-regulated,19 protein ubiquitination levels were significantly down regulated,121 non ubiquitinated proteins were ubiquitinated,ubiquitination of 39 protein completely disappeared,all the changed host proteins were related to many important cellular pathways,such as influenza A,JAK-STAT signaling Pathway,NOD-like receptor signaling pathway,etc.Based on these results,we selected three proteins with significant changes in ubiquitination level for verification.The results showed that the ubiquitination levels of RFC2 and AMPKG1 were significantly up-regulated after influenza virus infection,while the ubiquitination level of RO60 protein was down regulated,which was consistent with the results of ubiquitinomics.RO60 is a kind of Y RNA binding protein.There is no report about the role of RO60 protein in influenza virus replication.Therefore,we studied the interaction between the ubiquitination of RO60 protein and influenza virus.The results showed that overexpression of RO60 protein could inhibit the replication of influenza virus in the early stage of infection,but the inhibition gradually disappeared with the increase of virus infection time.After that,we found that the anti influenza function of RO60 protein was related to its ubiquitination level.Ubiquitination mass spectrometry analysis showed that the ubiquitination site of RO60 was located at 342 K.After the point mutation,the mutant RO60 no longer inhibited the replication of influenza virus,which indicated that only the ubiquitination modified RO60 could effectively inhibit the replication of influenza virus,and the influenza virus down regulated the ubiquitination level of RO60 weakens its ability to inhibit influenza virus,which is beneficial to virus replication.In this study,we studied the effects of glycosylation and ubiquitination of influenza virus NA protein on protein function and virus biological characteristics,as well as the changes of ubiquitination level of host proteins after influenza virus infection.We further understood the key steps in the process of influenza virus replication and the interaction with the host,and provided reference,new ideas and theoretical basis for anti influenza virus drugs and vaccines development. |
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