Structure and assembly of large lipid-containing dsDNA viruses

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