Atomic force microscopy and electron microscopy analysis of retrovirus Gag proteins assembled in vitro on lipid bilayers.
We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1, 2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'N-(5-amino-1-carboxy pentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-A distances and distances between dissimilar cage holes of 45.5 A. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-A distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.
Zuber
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Vollum Institute, Department of Microbiology, Oregon Health Sciences University, Portland, Oregon 97201-3098 USA.
Barklis
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D002213:Capsid / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli; D015683:Gene Products, gag / Q000378:metabolism* / Q000648:ultrastructure*; D008051:Lipid Bilayers / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D008979:Moloney murine leukemia virus / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure
D015683:Gene Products, gag; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011994:Recombinant Proteins