Relationship Of Structure And Function Of Autographa Californica Multiple Nucleopolyhedrovirus GP64 Protein

Baculoviruses are a group of large double-stranded DNA viruses,specifically infect insects.In a life cycle,baculoviruses usually produce two kinds of virions with distinct morphologies: budded virions(BV)and occlusion-derived virions(ODV).GP64 is the major envelope glycoprotein of BV and has been classified as a member of class III viral fusion protein family,which includes other viral fusion proteins,such as vesicular stomatitis virus glycoprotein(G)and herpes simplex virus type 1(HSV-1)gB.The post-fusion structure(under a low-pH condition)of Autographa californica multiple nucleopolyhedrovirus(AcMNPV)GP64 has been solved and it contains five domains(domain I-V,abbreviated as DI-V).However,the pre-fusion structure(under a neutral-pH condition)and the mechanism of low-pH triggered conformational change of GP64 are not clear.In this study,we focused on the potential interaction between DI and DV,and the structure and function relationship of DIV of AcMNPV GP64.Based on alanine-scanning mutagenesis,we characterized the conserved amino acids and the interatomic contacts within and/or between DI and DV or within DIV in GP64 expression,oligomer formation,cell surface localization,syncytium formation,AcV1-binding and infectious virus production.The main results are listed as following:1.Roles of the interactions between DI and DVAmino acid sequence analysis revealed that most of the amino acids in domain I and domain V were relatively conserved,and it was predicted that domain V interacted with two segments that flank fusion loop2 in domain I.To evaluate the significance of the amino acids conserved or involved in these interactions,we replaced the 24 amino acids in these two domains with alanine substitution to examine the importance of the amino acids or the interaction of amino acid pairs on the structure or function of GP64.In several cases,substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity,but no single amino acid pair appears to be absolutely required.We identified 4 critical residues in domain V(G438,W439,T452,and T456)that are important for membrane fusion.Among them,the mutation of amino acids G438 and W439 affects the formation of the fusion pore,while the mutation of T452 and T456 does not affect the formation of the fusion pore,but leads to a decrease in the formation efficiency of the fusion pore.Further studies found that two amino acid residues(G438 and W439)that are critical for membrane fusion appear to be important for the formation or stability of the GP64 pre-fusion conformation.2.Roles of DIV in GP64-mediated membrane fusionIn determined post-fusion structure of GP64,DIV is composed of residues from 374 to 407 and few amino acids in this domain(V394 ~ D398)have not been solved.In a homology-modelling structure of GP64,DIV contains two parallel loops(loop1 and loop2).Interatomic contacts analysis revealed that there are 12 amino acid-pair interactions in DIV.These interaction contacts mainly distributed at top,middle,and bottom regions of DIV.Among these contacts,the interaction of N384-Y388 locks the top region of loop 1.Nine interaction pairs locate in the middle region,including N381-N385,N381-K389,N385-K389(in loop 1),D398-S400,D398-Q401(in loop2),and N381-Q401,N381-I403,N385-W393,K389-W393(link loop1 and loop2).At the bottom region,two contact pairs separately locate in loop2(D404-S406)and between loop 1 and loop2(T379-F405).Amino acid sequences alignment showed that the contact and neighboring residues are highly conserved in the GP64 proteins family.To determine the roles of DIV in GP64 function,single or double alanines were used to substitute the conserved or contact residues in DIV.Our results indicated that all of the substitutions did not affect the expression and oligomerization of GP64.However,the distinct negative effects of different mutations on GP64 were observed.1)In plasmids-transfected Sf9 cells,the constructs D404 A and N407 A has no effect on cell surface localization of GP64,but both dramatically restricted the fusion pore expansion.In contrast,the substitution of other 5 conserved residues,including Y388,E390,G391,R392,and W393,significantly decreased the cell surface level of the mofidied GP64 and completely inhibited the initiation of membrane fusion.Interestingly,in viral bacmid-transfected cells,E390 A and G391 A induced the formation of small syncytia and significantly decreased the efficiency of entry of budded virions.2)For double analnies substituted constructs,T379/F405 A,N385/K389 A,D398/S400 A,and D398/Q401 A have no significant negative effect on the modified GP64.However,the mutations N381/Q401 A,N381/I403 A,and D404/S406 A restricted the fusion pore expansion,whereas N381/N385 A,N381/K389 A,N384/Y388 A,N385/W393 A,K389/W393 A inhibit the initiation of membrane fusion.In addition,in bacmid transfected cells,N381/K389 A induced the formation of small syncytia and significantly decreased the efficiency of entry of budded virions.3)For the same group amino acid-substitution constructs,including Y388 F,Y388W,E390 D,R392H,R392 K,W393F,and W393 Y,only Y388 F,E390D,and W393 Y induced the syncytia formation as that of wild-type GP64,however,the other substitutions resulted in the dramatic reduction of cell surface level of mutated GP64 proteins.Structural analysis revealed that alanine,phenylalanine,or histidine substitution of Y388 disrupted the interaction of N384-Y388.Although Y388 W also disrupt the interaction,however,the newly formed interactions with other residues might maintain the structure stability of DIV.Another aromatic residue,W393,controls the conformation of DIV.Alanine substitution of this residue resulted in a dramatic conformational change of DIV.The major change of the confirmation is about a 2.5 ? shift of the top region of loop 2 toward the center of trimer core that resulted from the formation of the interaction of S386-N396 and I387-N396.Additionally,the mutation N381/K389 A disrupted the interaction pairs N381-K389,N385-K389,N381-Q401,and N381-I403 in predicted structures of GP64,whereas N381/I403 A disrupted the interaction pairs N381-Q401 and N381-I403,the negative effect of N381/K389 A and N381/I403 A on fusion pore expansion suggest that the interactions bridge loop1 and loop2 are critical in maintaining the fusion activity of GP64.Besides of the above mentioned interactions,N381-N385 and N385-K389 might be redundant in maintaining the conformation of DIV.Disruption of the interactions,the close contact of the other residues resulted from the local conformational change formed additional interaction pairs to maintain the entire structural conformation of DIV.In conclusion,our results suggested that the conserved residues G438 and W439 in DV,and N381,Y388,E390,G391,R392,and W393 in DIV and the contacts formed by these and other neighboring residues are critical for maintaining the neutral pH structure of GP64 and also necessary for low pH-triggered membrane fusion initiation and fusion pore expansion.Based on our results and the pre-and post-fusion structures of few class III viral fusion proteins,we proposed a model in which DI,DIV,and DV coordinate to regulate the conformational change of GP64.