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Atomic force microscopy and electron microscopy analysis of retrovirus Gag proteins assembled in vitro on lipid
1Vollum Institute, Department of Microbiology, Oregon Health Sciences University, Portland, Oregon 97201-3098 USA.
Abstract:
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.
Insights
Moloney murine leukemia virus (M-MuLV) Gag proteins form ordered arrays on membranes. These viral proteins assemble into hexamers, suggesting dimers are key building blocks for M-MuLV particle assembly.
Area of Science:
- Virology
- Structural Biology
- Biophysics
Background:
- Understanding viral structural protein (Gag) oligomerization is crucial for viral assembly.
- Moloney murine leukemia virus (M-MuLV) provides a model system for studying Gag protein assembly dynamics.
- Membrane interfaces play a significant role in initiating viral particle formation.
Purpose of the Study:
- To investigate the oligomerization of M-MuLV Gag proteins at membrane interfaces.
- To elucidate the structural organization of viral proteins during particle assembly.
- To determine the role of membrane interactions in Gag protein assembly.
Main Methods:
- Utilized an in vitro system mimicking M-MuLV assembly.
- Employed membrane bilayers with phosphatidylcholine (PC) and a nickel-chelating lipid (DHGN).
- Analyzed membrane-bound protein arrays using electron microscopy (EM) and atomic force microscopy (AFM).
Main Results:
- Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) formed on lipid bilayers.
- EM revealed cage-like structures with specific spacing between protein-free holes.
- AFM showed a network of symmetrical hexamers with precise spacing, indicating specific protein arrangements.
Conclusions:
- M-MuLV Gag proteins form ordered, symmetrical hexameric structures at membrane interfaces.
- These findings support a model where protein dimers act as fundamental building blocks.
- Interactions between membrane-proximal and distal protein domains influence array formation.