The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and re®ned to an R value of 0.237 for data in the range 3.4-40.0 A Ê . The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg 2 , was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The ®rst 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical``jellyroll'' b-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a signi®cant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg 2 is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A Ê diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.

No abstract

Brome mosaic virus (BMV), a T = 3 icosahedral plant virus, can be dissociated into coat protein subunits and subunit oligomers at pH 7.5 in the presence of concentrated salts. We have found that during the course of this treatment the coat protein subunits are cleaved, presumably by plant cell proteases still present in the preparation, between amino acids 35 and 36. The truncated protein subunits will then reorganize into T = 1 icosahedral particles and can be crystallized from sodium malonate. Quasi elastic light scattering and atomic force microscopy results suggest that the transition from T = 3 to T = 1 particles can occur by separate pathways, dissociation into coat protein subunits and oligomers and reassembly into T = 1 particles, or direct condensation of the T = 3 virions to T = 1 particles with the shedding of hexameric capsomeres. The latter process has been directly visualized using atomic force microscopy. Native T = 3 virions have been crystallized in several different crystal forms, but neither a rhombohedral form nor either of two orthorhombic forms diffract beyond about 3.4 A. Tetragonal crystals of the T = 1 particles, however, diffract to at least 2.5 A resolution. Evidence suggests that the T = 1 particles are more structurally uniform and ordered than are native T = 3 virions. A variety of anomalous virus particles having diverse sizes have been visualized in preparations of BMV used for crystallization. In some cases these aberrant particles are incorporated into growing crystals where they are frequently responsible for defect formation.

TZ1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type TZ3 virus with 1 M CaCl 2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 Å resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 378, and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original TZ3 BMV particles. The TZ1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the TZ1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the TZ3 to TZ1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and TZ1 particles now permit us to propose a tentative model for that process. A comparison of the BMV TZ1 particles was made with the reassembled TZ1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 Å , is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 Å , has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.

In situ atomic force microscopy (AFM) was used to investigate surface evolution during the growth of single crystals of turnip yellow mosaic virus (TYMV). Growth of the (101) face of TYMV crystals proceeded by two-dimensional nucleation. The molecular structure of the step edges and adsorption of individual virus particles and their aggregates on the crystalline surface were recorded. The surfaces of individual virions within crystals were visualized and seen to be quite distinctive with the hexameric and pentameric capsomers of the T ‫؍‬ 3 capsids being clearly resolved. This, so far as we are aware, is the first direct visualization of the capsomere structure of a virus by AFM. In the course of recording the in situ development of the crystals, a profound restructuring of the surface arrangement was observed. This transformation was highly cooperative in nature, but the transitions were unambiguous and readily explicable in terms of an organized loss of classes of virus particles from specific lattice positions. In some cases areas of a single crystal surface were recorded in which were captured successive phases of the transition. We believe this provides the first visual record of a cooperative restructuring of the surface of a supramolecular crystal. 1999 Academic Press