N-Glycosylation And Viral Infectivity Analysis Of BmNPV Membrane Fusion Protein GP64

Bombyx mori nucleopolyhedrovirus（BmNPV）is one of the main pathogens causing economic losses in sericulture industry,and also one of the baculovirus expression systems for efficient expression of foreign proteins.Major envelope protein GP64 of BmNPV,the enveloped double-stranded circular DNA virus,is involved in critical process of infection by mediating the fusion of the host cell membrane with the BV envelope to realize the virions release.BmNPV GP64 shares high similarity with Autographa californica multicapsid nucleopolyhedrovirus（AcMNPV）GP64,and the main difference between them is whether the signal peptide（SP）is cleaved in mature virions or not.AcMNPV GP64 is cleaved while our studies have shown that BmNPV GP64 retained its SP.SP-cleavage GP64(SP^?nGP64)failed on PM localization,however,intracellular localized SP^?nGP64 was also incorporated into BV and contributed to infection.Proteins are led into Endoplasmic reticulum-Golgi apparatus guided by SPs,where they are N-glycosylation modified.It has been known that many viral envelope proteins are modified by asparagine（N）-linked glycosylation,which affects protein structure,physicochemical properties,and intracellular transport,and plays an important role in infection,replication,and the generation of infectious progeny,while the modification of glycosylation in BmNPV GP64 retained SP remains unknown.The functional relevance of N-glycosylation and BmNPV GP64 was analyzed in this research.First,deglycosylation of membrane proteins on the two recombinant viruses containing SP-retained(v BmBac^?gp64-gp64)or SP-cleaved GP64(v BmBac^?gp64-SP^?ngp64)showed the N-glycosylation in both GP64 and SP^?nGP64.It was predicted that GP64 contains six potential N-linked glycosylation sites at positions 5,175,213,370,400,and 442.To verify the prediction,the sites above were inactivated by site-directed mutagenesis（N→Q）singly or in combination to construct recombinant viruses with gp64 or sp^?ngp64 N-glycosylation site mutants.The expression and the influence on viral infectivity of the mutants were detected.Western Blot showed that N213,N370,and N400 could be identified as N-glycosylation sites.gp64^N175Q and gp64^N442Q were disabled to rescue the infectivity of the deletion virus BmBac^?gp64,and BmBac^?gp64-gp64^N5Q showed the lowest infectivity.None of sp^?ngp64mutants could rescue the infectivity of BmBac^?gp64,indicating the different N-glycosylation-dependence between gp64 and sp^?ngp64 in viral infection-related functioning.The transient expression vectors of gp64 and sp^?ngp64 were constructed for immunofluorescence and fusion activity assays.Results showed that the absence of SP n-region prevented GP64 from being transported to cell surface.The mutagenesis of N175Q and N370Q showed severe effects on GP64 cell surface expression ratio.All the site mutations affected GP64-mediated fusion activity,and mutagenesis of N175Q and N442Q led to the incapacity of membrane fusion activity.N-glycosylation is the key to correct folding and functioning of viral glycoproteins.The research on N-glycosylation is significant to further understanding the functions of viral membrane fusion proteins.These results indicated that N-glycosylation affected both infectivities of mutant recombinant viruses and fusion activity of GP64,providing further elucidation for the mechanism of BmNPV infection mediated by SP-retained GP64.