Atomic structure of single-stranded DNA bacteriophage ΦX174 and its functional implications

Abstract

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage ΦX174. The F protein forms a T = 1 capsid whose major folding motif is the eightstranded antiparallel β barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight β barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure, accounting for about 12% of the total genome.Bacteriophage ΦX174 is a small icosahedral virus that contains a single-stranded, closed circular DNA molecule with 5,386 nucleotide bases (for a recent review, see ref. 1). Of the 11 gene products, four (J, F, G and H) participate in the structure of the virion. There are 60 copies each of the J, F and G proteins and 12 copies of the H protein per virion [2][3][4] (Table 1). Electron microscopy of shadowed or negatively stained particles [5][6][7][8] , as well as a threedimensional reconstruction of frozen-hydrated virions (Fig. 1a), clearly show large spikes at the vertices of the icosahedral particles (N.H.O., T.S.B., P.W. and N.L.I., manuscript in preparation). These spikes are about 32 Å long and have a mean diameter of 70 Å. Edgell et al. 3 have shown that these spikes, which can be removed from the capsid with 4 M urea treatment, are composed of G and H proteins. Assuming all 12 spikes are identical, then each spike will contain five G and one H protein 2 . The capsid itself has an external diameter of roughly 260 Å, with a protein shell that is about 30 Å thick, and contains the F and J proteins as well as the single-stranded (ss) DNA. The phage recognizes a lipopolysaccharide (LPS) receptor on the outer cell membrane of Escherichia coli [8][9][10][11] . Host range mutants, which determine which strains of E. coli are infected by ΦX174, map into the F, G and H genes [12][13][14][15] The infectious virion has a relative molecular mass of 6.2 × 10 6 (M r 6,200K) (26% is DNA) 32 . Lysates of infected E. coli contain two components which can be separated by sedimentation through a sucrose gradient 32,33 . The fast band contains the infectious 114S particles whereas the slow band contains noninfectious, 70S particles 32,34,35 . The latter contains about 20% of the normal DNA content with all of the genome represented in the population of particles. Both types of particles can be crystallized into isomorphous monoclinic crystals with space group P2 1 , and cell dimensions a = 305.6, b = 360.8, c = 299.5 Å, β = 92.89° that diffract at least to 2.6-Å resolution 33 . The asymmetric unit of the crystal cell contains one complete particle. We report here the three-dimensional structure...

Mutations in the N Terminus of the øX174 DNA Pilot Protein H Confer Defects in both Assembly and Host Cell Attachment

“…Again, these data indicate that mutant H proteins are misincorporated. Bacteriophages are generally synthesized in a high-energy state, the highly compacted DNA containing the potential energy to deliver genome to the subsequently infected cell (5,21,22). However, the requisite conformational change required for genome delivery most likely has a high activation energy, which confers stability to the packaged particle in the absence of the appropriate host.…”

Manipulation of the mechanical properties of a virus by protein engineering

“…However, in MVM (as in other viruses; see refs. [23][24][25][26][27][28], some segments of the viral nucleic acid molecule adopt very similar conformations at symmetrically equivalent positions inside the capsid and have been crystallographically visualized. Each of these DNA patches is noncovalently bound to several amino acid residues located at one of 60 equivalent sites on the inner capsid wall (refs.…”

Use of radial density plots to calibrate image magnification for frozen-hydrated specimens

“…For example, we calibrated the ΦX174 micrograph ( fig. 1B) on the basis of the known X-ray structure of ΦX174 [32]. The magnification value we obtained was 2% higher than that obtained using the polyoma X-ray standard.…”

“…The magnification value we obtained was 2% higher than that obtained using the polyoma X-ray standard. This discrepancy may be attributed in part to differences in the ΦX174 EM [33] and X-ray [32] structures. Though both structures were computed from "empty" capsids that contain up to 20% of the single-stranded DNA genome, only a small fraction of the DNA was included in the X-ray model and some of the protein density was not modeled in the X-ray map.…”