POTATO LEAFROLL VIRUS BINDS TO THE EQUATORIAL DOMAIN OF THE APHID ENDOSYMBIOTIC GROEL HOMOLOG

A GroEL homolog with a molecular mass of 60 kDa, produced by the prima ry endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and rele ased into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteo viruses and pea enation mosaic virus, PLRV readily binds to extracellu lar Buchnera GroEL, and in vivo interference in this interaction coinc ides with reduced capsid integrity and loss of infectivity. To gain mo re knowledge of the nature of the association between PLRV and Buchner a GroEL, the groE operon of the primary endosymbiont of M. persicae (M pB groE) and its flanking sequences were characterized and the PLRV-bi nding domain of Buchnera GroEL was identified by deletion mutant analy sis. MpB GroEL has extensive sequence similarity (92%) with Escherichi a coli GroEL and other members of the chaperonin-60 family. The genomi c organization of the Buchnera groE operon is similar to that of the g roE operon of E. coli except that a constitutive promoter sequence cou ld not be identified; only the heat shock promoter was present. By a v irus overlay assay of protein blots, it was shown that purified PLRV b ound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encodi ng MpB GroEL revealed that the PLRV-binding site was located in the so -called equatorial domain and not in the apical domain which is genera lly involved in polypeptide binding and folding. Buchnera GroEL mutant s lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicate d in virus binding.