Structure of AYVV

To address these questions, we determined the structure of AYVV using single particle cryo-EM (Fig. 1). AYVV (and, indeed, the majority of the Begomoviruses) are phloem-limited and consequently are not mechanically transmissible, so we used Agrobacterium transformed with a plasmid containing a partial tandem copy of AYVV DNA-A13 (see Methods), to agroinfect14 N. benthamiana plants (Supplementary Fig. 1). Geminate particles were purified from symptomatic leaves (~2 mg/Kg of fresh leaf tissue) and a cryo-EM dataset collected (see Methods). Structure refinement with D5 symmetry, yielded a density map at 3.3 Å resolution (Fig. 1a and Supplementary Fig. 2), and an atomic model for the CP layer was built and refined (see Supplementary Table 1), starting from a homology model of the STNV CP (see methods). The capsid is composed of 110 copies of the CP, and we built and refined a complete D5 asymmetric unit of 11 unique chains (chains A-K) (Fig. 1b–e). As described previously7,8,12, each hemicapsid is formed from a T = 1 particle from which a penton of CP subunits is missing. The resulting facets on each hemicapsid associate to form the geminate particle (Fig. 1a). The body of the CP is similar to that of STNV and many other viruses, consisting of a jelly-roll fold with two twisted, 4-stranded β-sheets. This core domain structure is identical in all 11 chains in the asymmetric unit (Fig. 1e).

Fig. 1 The structure of Ageratum yellow vein virus. a EM density for AYVV at 3.3 Å resolution. The density is segmented and colored to highlight the 11 unique copies of the AYVV CP in the D5 asymmetric unit (labeled A-K). b Complete atomic model for all 110 subunits in the capsid, with a polyhedral cage showing the symmetry of the particle. The fivefold symmetry axis is vertical and through the center of the particle in this view. c Representative regions of EM density with the corresponding section of atomic model. d A “zoomed in” view of the N-terminal segment of all 11 unique CPs aligned. The N-terminal of chain I, chain H and the remaining 9 CPs is highlighted. The position of a polar patch (residues 214–215) is also shown. e Alignment of residues 64–256 from all 11 unique copies of the CP. The RMSD between structures is ~0.2–0.3 Å. The position of the N terminal and the polar patch of residues is indicated Full size image

The structure of the CP N-terminal conformations

The different conformations needed to build a geminivirus arise through differences in the N-terminal domain of the CP, rather than in its core fold. Many ssRNA plant viruses, including STNV, have positively-charged, N-terminal domains that play roles in binding RNA (e.g. refs. 15,16), and which are disordered in all structures (both X-ray and EM) that have been determined so far. A similar phenomenon is observed here for this ssDNA virus. The N-termini of the CP of AYVV are considerably longer than those seen in STNV, presumably at least in part because the first ~20–22 residues constitute the nuclear localization signal which is required for a DNA virus. However, they share the common theme seen in all N- and C-terminal extensions to viral CPs that project into the encapsidated space, in that they are strongly positively charged (Supplementary Fig. 3). In the subunits that comprise the majority of the hemicapsids, all subunits have a common first resolved residue (residue 63). However, additional amino acids are resolved in the subunits that make the equatorial interface (chains H (blue) and I (maroon)) (Fig. 2a). The biggest change is in subunit H, where an extra 23 residues are resolved as a helix-loop-helix motif (first ordered residue: 40) (Fig. 2b). By contrast in chain I, an extra 8 residues (first ordered residue: 55) become ordered, but in an extended conformation that is different to that seen for the same residues in chain H (Fig. 2b). The N-terminal extensions in subunits H and I are positively charged (Supplementary Fig. 3). These two new segments play very different roles in stabilizing the capsid. The helix-loop-helix motif from chain H is a major component of the equatorial interface (Fig. 2c), making a series of H-bonding interactions to a patch of predominantly polar residues in the body of the CP subunit across the interface (residues 214 & 215 in chain I; see Figs. 1d, e and 2c, and Supplementary Fig. 4), which is surrounded by van der Waals interactions. This polar patch is normally solvent-exposed on the surface of the virus in the subunits that form the hemicapsid (see Supplementary Fig. 4). The ordered segment from chain I extends across to stabilize the base of the helix-loop-helix motif in an adjacent H subunit, presumably stabilizing the ring of equatorial subunits (Fig. 2d). This interaction is mediated by backbone hydrogen bonding; essentially, a short (~3 residue) domain-swapped, antiparallel β-strand interaction is formed. The geminate particle therefore critically relies on a previously disordered segment of the N-terminus of the CP acquiring two different conformations to build the equatorial interface, which local resolution analysis suggests is marginally the most ordered part of the structure (Supplementary Fig. 5).

Fig. 2 Building a geminate capsid. a Rear half of the EM density for AYVV at 3.3 Å resolution. The cut surface of the EM density is colored bright magenta for clarity. Three CP subunits from each hemisphere are colored as in Fig. 1. In the top hemisphere, these are H-I-H (blue-maroon-blue) and in the bottom hemisphere these are I-H-I (colored maroon-blue-maroon). b Zoomed-in view of the interactions that form the interface. A helix-loop-helix motif comprising residues 40–63, becomes ordered only in subunit H (green halo), whilst an extra segment (residues 55–63) in subunit I also becomes ordered (orange halo) but in a different conformation to that seen by the same residues in chain H. c Details of the interactions across the equatorial interface. The loop in the helix-loop-helix motif makes H-bonding and van der Waals interactions with a patch of polar residues (214–215) on the opposing subunit. This patch is normally exposed on the surface of the subunits that form the isometric parts of the capsid (see Supplementary Fig. 4). d Details of the interaction between the two different N-terminal conformations. Effectively a beta-strand interaction is formed from backbone H-bonding of residues 59–61 in H (blue) and residues 56–59 in I, which helps to stabilize the ring of H & I subunits within a hemisphere Full size image

The role of the N-terminal extension in chains H and I

Our structure makes a simple functional prediction: that interactions between chain H, and chain I are critical to particle assembly, and that disrupting these interactions might favor single rather than geminate particle formation. However, the geminate capsid is required to encapsidate AYVV’s genomic DNA, the 2.7 kb DNA-A; destabilization of the geminate interface could therefore lead to degradation of the genomic DNA in vivo and abolish particle formation of all types.

We therefore exploited our knowledge of geminivirus genetics to establish an in vivo assay for encapsidation and assembly. AYVV infection of Ageratum conyzoides requires its 2.7 kb genomic DNA-A, together with its 1.3 kb β-satellite DNA, to give the classic yellow vein symptoms on this host3. However, in experimental hosts such as N. benthamiana, leaf curl symptoms are evident with inoculations of DNA-A alone. In the natural host, this “A + β” infection complex is always associated with DNA-α (again, ~1.3 kb in length). DNA-α encodes a Rep gene that facilitates its own replication, but is encapsidated by the CP encoded by DNA-A, and it is this encapsidation that is essential for vector transmission and ultimately for maintenance of the DNA-α itself. We therefore took advantage of this by over expressing geminiviral CP, in the absence of a geminivirus infection, and at the same time introduced the self-replicating DNA-α into the infiltration system. The result was the formation of a mixture of “geminate” and “single” virus-like particles (VLPs). Importantly no VLPs are generated by the expression of geminivirus CP alone, strongly indicating that the provision of circular ssDNA is essential for capsid formation irrespective of whether “geminate” or “single” VLPs are generated. In the presence of wildtype CP and DNA-α, the ratio of geminate to single VLPs is ~40:60, compared to essentially 100% geminate particles for the wildtype virus (Fig. 3a–c). We then made two mutations within the CP to probe the effect of interface destabilization. Firstly, we made R48A, which we predicted would disrupt the H-bonding between to the ‘top’ of the helix-loop-helix motif and chain I in the opposite hemicapsid (shown in Fig. 2c). We also made M59D, which we predicted would disrupt the main chain H-bonding interaction between chain I and the base of the helix-loop-helix motif in chain H (shown in Fig. 2d). This region is stabilized by backbone hydrogen bonds between residue M59 in the two chains, as part of the short β-strand interaction described above, and we rationalized that the introduction of a negative charge in this region would disrupt this specific interaction. For both mutations, an almost complete switch from geminate to single particles was observed with DNA-α (Fig. 3c–e), confirming the importance of these regions for geminate capsid formation. Strikingly for both the R48A and M59D variants, some of the resulting ‘single’ VLPs appear to have a missing pentameric capsomer in negative stain EM image averages (Fig. 3d, e).

Fig. 3 In vivo assembly assay for genome encapsidation. Representative negative stain micrographs of particles produced by infiltration with AYVV DNA-A (a) or co-infiltration with AYVV CP and DNA−α (b). Relative composition of “geminate” particles (pink in bar chart) compared with “single” particles (green in bar chart) from AYVV (c), and co-infiltrations of AYVV CP, R48A, and M59D, each with DNA−α (c). Representative negative stain micrographs and 2D class averages are shown for R48A variant (d) and M59D variant (e). The yellow asterisk highlights 2D class averages which appear to be “single” VLPs with a missing pentameric capsomer. Error bar is 100 nm Full size image

Interactions between the genomic DNA and CP subunits

A geminate capsid thus requires three distinct conformations, which although not classically ‘quasi-equivalent’ as they do not map to positions within an icosahedral lattice, fulfill a similar role in allowing a bigger capsid to be built. However, a fundamental question remains: how does a CP subunit ‘know’ what position it occupies within the overall architecture of the capsid? One solution would be for the conformation to be specified through interaction with the genome, as has been shown for single-stranded RNA viruses17,18,19. We therefore looked at the extensive density in our EM map that was unoccupied by the atomic model for the CP (Fig. 4; Supplementary Movies 1 and 2). Density is present beneath every CP in the capsid, consistent with an interaction with a ssDNA stem-loop. The map was of sufficient quality to build 7 nucleotides into the density beneath each subunit, except for subunit H at the interface where there are 6. This density is as strong as that for the CP, suggesting that occupancy is high, but owing to the D5 symmetry averaging applied, we cannot definitively say that all sites in the capsid are occupied. However, assuming that they are, our structure resolves ~28% of the genomic DNA molecule at high resolution (Figs. 4 and 5). There is some suggestion from the density, which is slightly different for each of the 11 DNA chains in the asymmetric unit, as to whether each position is a purine or pyrimidine, and thus we have built a very tentative consensus sequence of YRRYYRY into the density (where Y represents a generic pyrimidine, and R a generic purine; using adenine for R, and cytosine for Y).

Fig. 4 Single-stranded DNA binding. a Rear half of the EM density for AYVV at 3.3 Å resolution. The cut surface of the EM density is colored bright magenta for clarity. Two D5 asymmetric units are shown at the back of the capsid (colored as in Fig. 1a). High-resolution density corresponding to ssDNA is in red. b The EM density for 7 nucleotides of DNA is shown as a red mesh and the modeled atomic coordinates for CPs C and D and DNA stem loop N (CD-type DNA) is shown. These 7 nucleotides and the mode of binding to the neighboring subunits is identical for the majority of the CPs (i.e. A-G & I-K). At the interface (i.e. CPs H and I) these interactions are different, c shows 6 nucleotides, stem loop S, and the neighboring CPs H and I (HI-type DNA), depicted as described in b Full size image

Fig. 5 Details of interactions between DNA and CP. The details of the interactions between CP subunits and bound DNA. a The atomic model of “CD-type” DNA (i.e. DNA:CP interactions in the majority of the capsid (CPs A-G & I-K)) and the neighboring CPs (C and D). b as in a rotated 55 degrees as indicated. a, b show the interactions between polar sidechains and both the DNA bases and backbone at positions 2, 3, and 4, along with a base stacking interaction between F203 and the base of nucleotide 4. c, d The atomic model of “HI-type” DNA (i.e. DNA:CP interactions at the interface) and the neighboring CPs (H and I). At the interface, we see an extra interaction formed from the extreme N-terminus of the ordered density (residue 41) and the fifth nucleotide in the stem loop, which appears to stabilize the ordered N-terminal helix-loop-helix domain (c, d). e The overall conformation of the DNA stem-loop in each position is essentially identical. In e all chains are colored gray, except for H (blue) Full size image

For each DNA chain in the body of the capsid (i.e. chains A-G and J-K), and for chain I, interactions are made between one CP by four residues (S114, Y116, R248, and Y250) to the top of the bound stem loop (nucleotides 3 and 4) (between the DNA and the blue subunit in Fig. 5a, b; Supplementary Movie 3). Additional contacts are made between arginine residues in a second CP subunit (R142, R144 & R174) (the yellow in Fig. 5a, b; Supplementary Movie 3) and the DNA backbone at the 3′ end of the bound stem loop (nucleotides 5 & 7). In chain H, we again see a difference compared to the other chains. At the top of the stem loop, the interactions remain unchanged (between the DNA and the blue subunit in Fig. 5c, d; Supplementary Movie 4). More significantly, a new interaction occurs between the backbone of nucleotide 5 and R41, at the extreme N-terminus of the helix-loop-helix motif in chain H. This motif forms the equatorial interface, and presumably the interaction with DNA stabilizes this structural element. Despite this, overall the conformation of the bound DNA is very similar at each position, including at the equatorial interface (Fig. 5e).

To test the importance of DNA binding, we also made mutant R41A, to disrupt the interaction between R41 and the DNA backbone. When this was expressed alongside DNA-α, no particles were recovered from leaves, suggesting that DNA binding and/or assembly is severely compromised, as expected from our predicted role for R41.