Degradation Mechanism Of IAV HA Protein By Lysosome In Host Cells

Influenza A virus is an important zoonotic pathogen that poses a huge threat to public health.The cross-host transmission of influenza A virus poses great challenge for prevention and control.Currently,drugs targeting influenza virus have made drug-resistant strains constantly emerging,so it is imminent to develop new antiviral drugs.As the most abundant envelope protein on the surface of influenza virus,HA protein mainly mediates the binding of virions to target cell surface receptors.Therefore,the HA protein is correctly folded in host cells and transported to the membrane to assemble the virion,which is a prerequisite for the virus to acquire infectivity.It is worthy of further study by blocking the maturation of the HA protein as an effective antiviral means.The synthesis of glycoproteins is an error-prone process in which cells maintain their homeostasis by degrading misfolded proteins.HA proteins are highly glycosylated proteins whose intracellular synthesis and degradation are strictly regulated by host cells.Many studies have shown that glycosylation plays an important role in the function of HA protein,but how it regulates the maturation of HA protein and degradation in cells has rarely been reported.In this study,the N-linked glycosylation site of HA protein was first mutated,and it was found that the glycosylation mutation affected the maturation of HA protein.Three glycosylation sites,N26,N27 and N39,were mutated to significantly reduce protein expression,transport and virus assembly efficiency.Meanwhile,CO-IP showed that the interaction between the N-terminal glycosylated mutant HA and endoplasmic reticulum molecular chaperones and calcium reticulum protein was significantly enhanced.The above studies showed that the mutation of N-terminal glycosylation affects the maturity of HA protein,which plays a key role in the structure and function of HA protein.In order to maintain protein homeostasis,the misfolded protein is degraded by protein degradation pathway.The glycosylation mutant HA was rescued by Bafilomycin A1,and showed a strong colocalization with Lamp1,which indicated that the misfolded HA protein is degraded by lysosome.In order to verify the degradation of HA protein by lysosome,different inhibitors were used,and it was found that HA proteins of both H5 and H1 subtypes could be degraded by lysosome.Virus experiments proved that Baf-A1 could also save functional HA.In order to further study the specific mechanism of HA protein degradation by lysosome,different pathways of were analyzed by RNA interference,ELISA and inhibitors.It was found that molecular chaperon-mediated autophagy,endocytosis and macroautophagy were not involved in the degradation of HA protein.Finally,by separating the insoluble protein components,it was found that the HA protein aggregated after misfolding,and became insoluble.Misfolded proteins continue to aggregate,forming a dense and hydrophobic aggregate near the nucleus called aggresome.It was found that there was obvious colocalization between HA protein and vimentin,the marker protein of aggresome.In order to clarify that the aggresome of HA protein formation was degraded by lysosome,the aggresome was indicated by the marker protein GFP-250.It is found by fluorescence microscope that the HA proteins of H5 and H1 subtypes can form aggresomes,and more aggresome generated in the presence of Baf-A1.Taken together,we demonstrate glycosylation plays an important role in the maturation of proteins.Most of the HA proteins with deletion of the glycan are immature.HA proteins aggregated in host cells,and form aggresome which are degraded by the lysosome.Degradation inhibits the expression of HA protein,thereby inhibiting viral replication and infection.This study reveals the specific mechanism of host cell HA degradation protein which will identify new antiviral pathways and provide a basis for influenza drug development.