Structure and assembly of large lipid-containing dsDNA viruses

摘要: Two large, lipid-containing, double-stranded DNA (dsDNA) icosahedral viruses, each ∼1 GDa (1 × 109 daltons) and unlikely to be amenable crystallization, have been studied by cryo-electron microscopy (cryo-EM) three-dimensional image reconstruction. The first, the 1,850 A diameter Chilo iridescent virus (CIV: genus Iridovirus, family Iridoviridae) infects rice stem borer insect1. second, 1,900 Paramecium bursaria chlorella virus, type 1 (PBCV-1: Chlorovirus, Phycodnaviridae) certain unicellular, eukaryotic, chlorella-like green algae2. Despite differences in their pathogenicity genome size, both viruses exhibit common structural features, suggesting a evolutionary origin therefore serve as paradigm for other large such African swine fever frog virus-3 many others (for example, see ref. 3). Both layered structure consisting of dsDNA–protein core, surrounded turn lipid bilayer one or more capsid shells thousands protein subunits4–7. Furthermore, external is essentially consistent with quasi symmetry predictions Caspar Klug8. This unexpected view now well-documented departure from much smaller virusses polyoma9 inner core RNA 10). CIV has 209 kbp linear, circularly permuted (a linear molecule that maps circle) genome, whereas PBCV-1 330 non-permuted covalently closed, hairpin ends11,12. Some iridoviruses, like CIV, also contain fibers protrude 350 outer surface capsid5,13 this was confirmed study. In contrast little known about chlorovirus structure. capsids accommodate wide range sizes14,15 295 kbp16 380 kbp14. virion contains ∼50 proteins, 54 kDa glycoprotein, VP54, accounting ∼40% total mass17. its close relatives are freshwater collected worldwide2,12. Like tailed bacteriophages, host, Chlorella NC64A, attaching cell wall, possibly through unique vertex tail-like wall then degraded at point attachment viral released into cell2,18. Analysis 330,740 bp predicts it 377 protein-encoding genes 10 tRNA genes, including interesting genes12. To put size perspective, smallest free-living organism, mycoplasma, encodes ∼470 proteins19. Our cryo-EM studies CIV show profiles characteristic icosahedra viewed different orientations. Most virions asymmetric masses distributed non-uniformly within them (Fig. 1a,d). forms crystalline, closely packed hexagonal arrays which neighboring separated 400–600 due halo surround capsid. Image reconstructions two ∼26 resolution, structures composed pseudo-hexagonal ‘capsomers’ 1b,e). Close inspection demonstrates hexavalent capsomer trimeric 2). Fig. 1 Comparison virions. a, d, Micrographs vitrified samples (black bar = 1000 A). Representative particles oriented two-, three-, five-fold axes nearly parallel direction ... Fig. 2 Close-up views trimers PBCV-1. Shaded-surface representations down an three-fold axis (triangles). Fibers centered over central, ... The diameters 1,615 1,650 along two- PBCV-1, respectively 1c,f). capsomers triangular profile (∼80 ∼75 high) associated project radially center but not pentavalent capsomers. Capsomers doughnut-shaped (∼70 most axial channels ∼17 diameter. interconnect bases form contiguous, 20–25 thick shells. protruding portion extends above 2a,d). Density corresponding bilayer, ∼40 thick, follows contour visible inside although prominent appears tethered connections additional shell density lies between layer (white arrow; Fig. 1c). Such second apparent 1f). Occasional disruptions membrane might reflect presence transmembrane proteins. centers bilayers occur radii 637 703 two-fold 729 756 These values mass measurements indicate consists ∼9% lipid11,17,20. shell, these rotated ∼60° relative those (data shown). Assuming all identical chemically, 5,040 copies 1,680 trimers, earlier biochemical measurements17. similar suggests likely oligomers protein. encapsidates 26 proteins composed, respectively, 51.4 major P50 P′50 (refs 21,22). sequence; however, exists non-covalent trimer linked disulfide bonds trimer21. Hence, there 4,380 (= 1,460 3) P′50. Composition unknown but, clearly larger than 2c,f). Inspection surfaces especially shows pentagonal facets 2c,f). were first recognized described (named pentasymmetrons trisymmetrons) Wrigley23, who examined negatively stained preparations disrupted Sericesthis facet, ‘groups nine’, later adenovirus24. Each twelve vertices 31 capsomers, remaining 30 occupy positions capsids. twenty trisymmetrons; 55 66 PBCV 1, respectively. All trisymmetron arranged produces two-dimensional, ‘crystal’ hence gives facet characteristic, near planar morphology leads overall assembled differ rotation 2b,e). lack direct evidence assembly pathway any icosahedral, dsDNA-containing existence they may mature self-assembled, intermediate substructures facilitate efficient morphogenesis capsids24. Such package genomes surprisingly precise highly symmetric structures. Striking similarities facets, hint follow analogous pathways. Indeed, dsDNA related, ∼50kDa associate trimers25. addition, adenovirus bacteriophage PRD1, among largest date cryo-EM, T 25 trimers26,27. Phylogenetic analyses polymerases phycodnaviruses root eukaryotic polymerase delta proteins28, long history. information reveal patterns exist same manner tertiary can used map phylogenetic trees.