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Apple latent spherical virus structure with stablecapsid frame supports quasi-stable protrusions
expediting genome release
Hisashi Naitow 1,5, Tasuku Hamaguchi 1,5, Saori Maki-Yonekura
Nobuyuki Yoshikawa 3 & Koji Yonekura 1,4 ✉

1, Masamichi Isogai

2,

Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of thesurvival of these viruses depends on their capsid being stable enough to harbour the viralgenome and yet malleable enough to allow its release. However, molecular mechanisms
remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent
spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM)
with a cold-ﬁeld emission beam. The cryo-EM map reveals a unique structure composed ofthree capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM
images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits.
The structures and observations suggest that genome release occurs through occasional
opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame ofthe other subunits.

1 Biostructural Mechanism Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. 2 Plant Pathology Laboratory, Faculty ofAgriculture, Iwate University, Ueda 3-chome 18-8, Morioka, Iwate 020-8550, Japan. 3 Agri-Innovation Center, Iwate University, Ueda 3-chome 18-8,
Morioka, Iwate 020-8550, Japan. 4 Advanced Electron Microscope Development Unit, RIKEN-JEOL Collaboration Center, RIKEN Baton Zone Program,
1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. 5These authors contributed equally: Hisashi Naitow, Tasuku Hamaguchi. ✉email: yone@spring8.or.jp

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lant viruses invade host cells through a surface wound in
the cell wall, often expedited by transmitting insects, andpromptly release their genome for translation of their own
proteins. Newly proliferating viruses spread to neighboring cells
through so-called plasmodesmata, which are channels traversing
the cell walls normally for the transport of chemicals and evensmall proteins between the cells (e.g., refs. 1,2). This tubular
structure is modiﬁed by a viral protein called movement protein(MP2) to facilitate the entry of viral replication complexes (e.g.,
refs. 3,4) or virus particles themselves (e.g., refs. 5–9). Then, theviral genome is released again, bound for new destinations. In
most cases, no speciﬁc host receptors are thought to be involved
in infection and proliferation. Thus, capsid design for the survivalof these viruses should require some basic functions, such asretention of the viral genome inside the virion, genome release,and then its encapsidation with newly synthesized capsid proteins. In addition, the capsid protein of viruses that form a virion
during transport through the modiﬁed plasmodesma needs to beable to interact appropriately with MP7,8.
Picorna-like plant viruses are one such group of plant-infecting
viruses. They are classiﬁed into a large virus order, Picornavirales,
members of which are nonenveloped RNA spherical viruses of~30 nm, and which infect various hosts, such as vertebrates,
insects, algae, and plants10. Although extensive studies have been
carried out on these simple plant viruses, key molecularmechanisms still remain unclear. In particular, genome release isa basic and essential step for virus proliferation, and yet little is
known about how this occurs.
Apple-latent spherical virus (ALSV) is a picorna-like plantvirus found in apple leaves11. No symptoms appear upon infection of the virus12, a phenomenon referred to as “latent infection”. Apple trees may be the only naturally infected host as notransmitting insects have been found13. Yet, in vivo experiments
show that ALSV has a relatively broad spectrum of host plants(Caryophyllaceae, Chenopodiaceae, Cryptomeria, Fabaceae,
Cucurbitaceae, Gentianaceae, Pinus, Rosaceae, Rutaceae, Solanaceae, and Arabidopsis)14. Because of this, biotechnological applications have been explored, such as virus-induced genesilencing15,16, virus-induced ﬂowering17–19, epitope presentationon the surface of virus particles19,20, and development of plantvaccines21–23.
Such picorna-like plant viruses are classiﬁed in the family
Secoviridae. This family contains approximately eight genera, andonly a few virion structures in the family have been reported, i.e.,
cowpea mosaic virus (CPMV24), broad bean stain virus (BBSV25),
bean pod mottle virus (BPMV26), and red clover mottle virus(RCMV27) in the genus Comovirus and tobacco ringspot virus(TRSV28) and grapevine fanleaf virus (GFLV29) in the genus
Nepovirus. The virions of the genus Comovirus are composed of
two proteins, and those of Nepovirus only a single protein. The
virions of ALSV consist of three proteins, Vp25, Vp20, and Vp24,
and they show only a weak sequence homology to other virus
capsid proteins. In fact, ALSV was initially classiﬁed separately inthe family Sequiviridae30, but is now placed in the genus Cheravirus, family Secoviridae, after the latter was formed from anamalgamation of families Sequiviridae and Comoviridae.
We have determined the structure of ALSV at 2.87-Å resolution by single-particle cryo-EM with a cold-ﬁeld emission beam,
which allowed de novo modeling of atomic coordinates on the
cryo-EM map from a relatively smaller number of particle images
with an improved B factor for map sharpening. The structure
reveals that the three capsid proteins have characteristic jellyroll
folds that contribute to capsid stabilization. Uniquely among the
known structures in this virus family, a long N-terminal extension of Vp25 interacts with all other subunits, including aneighboring Vp25 through the inner surface of the capsid, as well2

as the genomic RNA. Image analysis resolves RNA leaking fromprotrusions of the virus particles. Our results suggest how capsid
stabilization is achieved, and we propose a mechanism for genome release.
Results
Structure analysis of ALSV. ALSV consists of three protein
subunits, Vp25, Vp20, and Vp24, which are coded in the viralgenome in this order and named for their approximate molecularweights. We applied single-particle cryo-EM to frozen–hydrated
ALSV particles containing genome RNA, isolated from infected
plant leaf tissue. A 3D map was reconstructed to 2.87-Å resolution from dose-fractionated movies using RELION-331 (Table 1,
Supplementary Fig. 1, and Supplementary Movie 1). The resolution is better, and the absolute value of the estimated B factor
for map sharpening is low when compared with other viruses in
the family (Table 2). This should reﬂect that higher-resolution
signals are well retained, thanks to the cold-ﬁeld emission beam32.
The cryo-EM map (Supplementary Fig. 1) revealed side-chain
densities, and allowed de novo modeling for the whole virus
capsid, except for three short segments (41–55, 100–116, and
146–151) in Vp24 and several residues in the N- and C-termini(Supplementary Fig. 2). After real-space reﬁnement, the ﬁnal
model has good geometry and a high correlation coefﬁcient withrespect to the cryo-EM map (Table 1).
Table 1 Cryo-EM data collection, reﬁnement, and validation
statistics.
ALSV (EMD-30375) (PDB 7CHK)
Data collection and processing
Microscope
Detector
Magniﬁcation (nominal)
Voltage (kV)Electron exposure (e–/Å2)
Dose rate (e–/Å2/s)
Defocus range (μm)
Physical pixel size (Å)
Symmetry imposed
Initial particle images (no.)
Final particle images (no.)
Map resolution (Å)
FSC threshold
Reﬁnement
Initial model used (PDB code)
Model resolution (Å)
FSC threshold
Map-sharpening B factor (Å2)
Model composition
Nonhydrogen atoms
Protein residues
B factors (Å2)
ProteinR.m.s. deviations
Bond lengths (Å)
Bond angles (°)
Validation
MolProbity score58
Clashscore
Poor rotamers (%)
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)

CRYO ARM 300
K2 summit(superresolution mode)
40,000300~51~8.5−0.5 ~ −2.0
1.24
Ih
9285
8018
2.87
0.143
N/A
2.98
0.5
−29.7
251,820
31,680
49.42
0.007
0.635
2.46
6.68
6.38
92.19
7.81
0.0

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Table 2 Cryo-EM structures of the spherical viruses in the family Secoviridae.
Virus

Resolution (Å) Number of virus Estimated B factor (Å2) for
particles
map sharpening

Electron source Accelerating
voltage (kV)Content

Reference

Recombinant empty
virus-like particle
Including RNA1 (6 kDa)

Heskethet al.59
Heskethet al.59
Heskethet al.33
Heskethet al.33
Lecorreet al.25
This work

CPMV 3.04

4998

−74.6

Schottky

300

CPMV 3.44

4331−107.6

Schottky

300

CPMV 3.94

10,850−144.1

Schottky

300

CPMV 4.25

4696−185.8

Schottky

300

BBSV

3.22

17,233−101.0

Schottky

300

Including RNA2
(3.5 kDa)
Naturally formed
empty capsid
Including RNA

ALSV

2.87

8018

−29.7

Cold-ﬁeld
emission

300

Including RNA

Structure and subunit interactions. One protomer is made of
Vp25, Vp20, and Vp24 subunits (Fig. 1a), and 60 copies of thisunit compose one virion (Fig. 1b). The Vp24 subunits form apentameric protrusion around a ﬁvefold axis, and the protrusionsits on a base of Vp25 and Vp20. All the three proteins containjellyroll domains composed of all β-strands, except that Vp25 has
extra β1 and β2 (Fig. 1a and Supplementary Fig. 2), and the threeproteins also comprise short α-helices and loops. The densities forthree short segments (residues 41–55, 100–116, and 146–151) in
Vp24 are missing (Supplementary Fig. 2) probably due to theirhigh ﬂexibility (Fig. 1a, c). Residues connecting to and from themissing segments are located at the interfaces to the other surrounding subunits (residues 41–55 to Vp25 and neighboring
Vp24, 100–116 to Vp20, and 146–151 to Vp25). Another unit of
the capsid can also be chosen as a triangle enclosed with yellow
lines in Fig. 1c. It may be convenient to use this triangular unit todescribe the structure.
One of the striking features is an ~120-Å-long N-terminal
extension of Vp25 (Fig. 1 and Supplementary Fig. S2). When thevertex of the Vp24 pentamer is placed “top” and the structure
viewed from outside the virion, the N-terminal extension of Vp25
runs under Vp20 in the same triangle and extends to Vp24
(2Vp24 in Fig. 2a) through the main body of Vp25 (2Vp25) of theneighboring triangle on the right side (Fig. 1a, c). The longextension has intimate hydrogen bonds with Vp20 (0Vp20) andthe main body of Vp25 (2Vp25), and forms β-sheets with them
(between β1 of 0Vp25 and β9 of 2Vp25, and between β2 of 0Vp25
and β8 of 0Vp20, Fig. 2a, b). In contrast, a smaller number ofhydrogen bonds are found with Vp24 (Table 3). The numbers ofintersubunit hydrogen bonds in each monomer are 17 for Vp25,
12 for Vp20, and 7 for Vp24. We summarize all interactions
among the capsid proteins in Table 3 and Fig. 2a.
We then calculated the buried solvent-accessible surface area
(SASA) of each Vp25, Vp20, and Vp24 monomer among thesurrounding subunits in the virion (Table 3). The buried SASAs
are 6029 Å2 for Vp25, 5092 Å2 for Vp20, and 3607 Å2 for Vp24.
Thus, Vp25 and Vp20 have rather extensive interactions with
their neighbors, and Vp24, which forms the pentameric
protrusion, is more loosely held. Vp25 and Vp20 likely form arigid capsid frame that supports the looser Vp24 protrusions.
Comparison with other virus structures. Here we compare thestructure of ALSV with those of other picorna-like plant viruses.
We take CPMV (PDB ID: 5MS1)33 as representative of the genus
Comovirus (Fig. 3b). Structures in the same genus have beenreported for BBSV25, BPMV26, and RCMV27, and they are very
similar to that of CPMV. The CPMV capsid is composed of two
protein subunits, called large (L) and small (S). The L subunit

contains two domains that correspond to Vp25 and Vp20 of
ALSV, and the S subunit corresponds to Vp24—the overall
structures and folding are similar (Fig. 3). However, sequencealignment by a widely used program Clustal Omega34 shows
relatively lower homology and poorer match of secondary
structure elements (Supplementary Fig. 3), indicating a genetically distant relationship. The Dali server with 3D structure ﬁtting35 yielded a sequence identity of 9.7% between ALSV and
CPMV (all amino acid residues of ALSV were included in the
denominator for the calculation).
TRSV28 and grapevine fanleaf virus (GFLV29) from the genus
Nepovirus in the family Secoviridae show the same protein fold(Fig. 3c), but here one large capsid protein comprises threedomains, analogous to the three separate proteins of ALSV, andsequence homology is also low between TRSV and ALSV(Supplementary Fig. 3). The structure of ALSV is the ﬁrst solvedin the genus Cheravirus to our knowledge, and is the only one
with three separate capsid proteins. Interactions of the L and S
subunits in the genus Comovirus and the corresponding domains
in the genus Nepovirus are also loose as observed in ALSV.
The long N-terminal extension in Vp25 of ALSV is uniqueamong picorna-like viruses, except that some insect36 andmammalian viruses37 exhibit different extensions with a twostranded β-sheet, which do not interact with the pentameric
protrusion (Fig. 3d, e). The capsids of the insect and mammalianviruses are composed of four separate protein subunits, three coreproteins (Fig. 3d, e), and one short segment, but the sequencehomology is again low (~12.9% between ALSV and insect
Triatoma virus (TrV, PDB ID: 3NAP)36 and ~7.7% between
ALSV and mammalian Hepatitis A virus (HAV, PDB ID: 4QPI)37,
obtained in the same way as above using the Dali server foralignment of 3D structures35.
Retention and release of genome. One ALSV virion contains one
longer or two shorter single-strand RNA chains. The former(RNA1) encodes proteins for replication, such as helicase, protease, and RNA polymerase, and the latter two (RNA2s) encode a
MP and the capsid proteins30. There are concentric shells ofdensity inside the capsid and humps of density beneath the Vp24
pentameric protrusions (Fig. 2c). The density corresponds to asymmetrized average of the RNA genome, and this feature iscommon in single- particle reconstructions of other picorna-likeplant viruses (e.g., ref. 24). The original map showed no clear
interaction of capsid protein density with the genome density.
However, after applying the autosharpening algorithm implemented in the Phenix suite38 to the map (Fig. 2c, d), we foundthat His 113 of Vp20 and Trp 9 of the Vp25 N-terminal extension
are involved in a clear interaction with the RNA inside the capsid

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Fig. 1 Structure of apple-latent spherical virus (ALSV) in the ribbon diagram, determined by single-particle cryo-EM. a The protomer structure, which iscomposed of three protein subunits, Vp25 in red, Vp20 in blue, and Vp24 in green, disposed as in other picorna-like plant viruses. Secondary structureelements refer to Supplementary Fig. 2. b Overall structure, which comprises 60 copies of the protomer. c Five protomers around the ﬁvefold axis. Subunits
in one protomer are colored as in (a) and others in gray, except for the N-terminal extensions of Vp25 subunits in orange and one Vp20 inside a yellow
triangle in light blue. The N-terminal extensions appear to encircle the ﬁvefold axis. The icosahedral two-, three-, and ﬁvefold axes are marked with anellipse, triangle, and pentamer, respectively. Dashed lines represent three missing segments (41–55, 100–116, and 146–151) in Vp24 and numbered one forresidues 41–55, two for 100–116, and three for 146–151 in (a) and (c).

4

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COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01217-4(Fig. 2d). These residues are at different positions from thosefound in the L subunit in the genus Comovirus25,33. It is not clear
whether this difference represents unique sites for RNA binding
in the genus Cheravirus, or rather arises just from the enforced
symmetry of the RNA density. Nonetheless, the N-terminal
extension of Vp25 extensively covers the inner surface of thecapsid shell, and likely interacts with the internal RNA genome.

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Cryo-EM images of ALSV sometimes contained string-likedensities around virus particles as in Fig. 4a and Supplementary
Fig. 4a, and they increased after the sample solution wasfrozen–thawn several times and/or incubated for several days.
These strings clearly leaked from the virus particles as someparticles appeared partly empty in approximate proportion to thevolume of the surrounding strings extruded (Fig. 4b). The strings

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Fig. 2 Interaction of apple-latent spherical virus (ALSV) subunits. a Arrangements of Vp25, Vp20, and Vp24 subunits in the ALSV virion. Subunits in the
protomer are shown in the same color scheme as in Fig. 1, but in deeper colors. Subunits that have contacts with the three in the center triangle (position 0)
are also colored and those with no contact are in white. See also Table 3 for buried solvent-accessible surface area (SASA) and numbers of hydrogen bonds
between the center subunits and their partners. b Interface between Vp25 and Vp20 subunits viewed from inside the capsid. Ribbon diagram showing thesecondary structures. Hydrogen bonds in cyan lines are formed between O and NH in peptide bonds, except for Arg 15 of 0Vp25 and Glu 160 of 2Vp25
displayed in stick representation. Dashed lines represent the missing segments in Vp24 and numbered 1, 2, and 3 in black and gray as in Fig. 1a and c.
c, d Interaction between the capsid and internal RNA genome. Cross-sectional views of a sharpened ALSV map by phenix.auto_sharpen38. c Whole virus
showing concentric shells of symmetrized averaged density of RNA genome (shown in ocher) inside the capsid and protrusions beneath the Vp24
pentamer. d Zoom-up view of the area enclosed by a black rectangle in (c). His 113 of Vp20 and Trp 9 of Vp25 appear to be in contact with the RNA
genome. The viewing direction is slightly adjusted from that in (c) for clarity. The map is contoured at 3.3 δ.

Table 3 Interactions of ALSV capsid proteins.
Interaction partnera

Buried SASAb

Number ofhydrogen bonds

Interaction partnera

Buried SASAb

Number ofhydrogen bonds

0Vp25

1786
591
1032
0
367
829
889
535
0
0
0
6029
443
582
871
667
355
684
0
0
5
3607

7
1
3
0
0
6
0
0
0
0
0
17
0
1
2
2
0
2
0
0
0
7

0Vp20

1771
443
872
1032
0
890
39
6
39

7
0
2
3
0
0
0
0
0

5092

12

Sum
0Vp24

Sum

0Vp20
0Vp24
1Vp20
1Vp24
2Vp24
2Vp25
3Vp20
3Vp25
9Vp25
10Vp20
11Vp25
0Vp20
0Vp25
1Vp20
1Vp24
1Vp25
2Vp24
2Vp25
4Vp24
5Vp24

Sum

0Vp25
0Vp24
2Vp24
2Vp25
3Vp20
3Vp25
6Vp20
7Vp25
8Vp20

aRefers to Fig. 2a for subunit numbers and positions.
bBuried solvent-accessible surface area (SASA).

must correspond to viral RNA genome. Leaking genomes havebeen reported previously39,40. We cut out images that included
RNA and part of the virion, and applied 2D classiﬁcation.
Thereby, the 2D-class averages resolved that RNA chains extrude
from one of the vertices of the virion, which likely corresponds tothe tip of a pentameric protrusion (Fig. 4c). Also, there is a humpor protuberance of genome density beneath each pentameric
protrusion of the capsid (Fig. 2c) and a pore with a diameter of~4 Å there (Fig. 4d). No other pores or clefts with around this sizeor larger than this are found on the capsid surface. Theseobservations suggest that the genome escapes from the virion at
one of the pentameric protrusion sites. Of course, the pore is too
small for the genome to pass through and needs to be larger, but
the pore would be the only candidate where such opening could
occur.
Previous studies by cryo-electron tomography of a poliovirus,
which is a human picorna-like virus, suggest that genome releaseoccurs at a deformed part of the capsid around the twofold axis40.
However, this is unlikely in ALSV, as there are tight interactionsof the capsid base composed of Vp25 and Vp20 around thetwofold axis (Table 3), and the 2D-class averages in Fig. 4c point
to release being at the tip of the pentameric protrusion. Of course,
the genome-release mechanism may be different between plant6and human-infecting viruses, as the mechanisms of infection andspread are different. Nonetheless, there are indications in otherviruses that the pentameric protrusion is related to genome
release: the structure of an insect picorna-like virus, honeybeedeformed wing virus, reveals that a RNA segment is bound inside
the pentameric protrusion41; cryo-EM analysis showed that atailless bacteriophage ΦΧ174, which is a spherical virus inanother family, is fused to the membrane with a pentameric
protrusion of the virion, and the genome is thought to be injected
into the host cell through this contact point42.
Discussion
The capsid structure requires two apparently contradictory characteristics: it needs to be rigid enough to hold the internal genome,
but ﬂexible enough to release the genome to the host cell. The
ALSV structure shows a solid base composed of ﬁve pairs of Vp25
and Vp20 proteins, and a turret of ﬁve Vp24 proteins on top(Fig. 1c). The surface of the virion is tightly patched with thispentagonal unit, except for holes of ~4 Å at the center of ﬁve
Vp24 subunits (Fig. 4d). The three subunits are structurally supported by the long N-terminal extension of Vp25. This extension
makes many intimate hydrogen bonds with Vp20 and neighboring
Vp25, but only a few with Vp24 (Fig. 2b; Table 3). The buried

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Fig. 3 Structure comparison of picorna-like plant viruses, an insect and a mammalian picorna viruses. a Apple-latent spherical virus (ALSV) in the genus
Cheravirus. Two protomers are shown with the viral lattice and colored as in Fig. 1c, except for the left Vp25 subunit in light red and the right Vp20 in gray.
b Cowpea mosaic virus (CPMV) (PDB ID: 5MS1)33 in the genus Comovirus. Colored in red for the L and in green for the S subunits. c Tobacco ringspot virus(TRSV) (PDB ID: 1A6C)28 in the genus Nepovirus composed of the single-capsid protein colored in green. The sequence identity is 9.7% between ALSV and
CPMV, and 8.5% between ALSV and TRSV. The Dali server35 was used for the alignment (see text). d Insect Triatoma virus (TrV, PDB ID: 3NAP)36.
e Mammalian Hepatitis A virus (HAV, PDB ID: 4QPI)37. The capsids of TrV and HAV have four separate protein subunits. Two protomers of three coreproteins are shown with viral lattices as in (a), and one short segment was not identiﬁed in the crystal structures. The structures of CPMV, TRSV, TrV, and
HAV are superimposed onto that of the ALSV protomer (green, blue, and red in a). The sequence identity is 12.5% between ALSV and TrV, and 7.7%
between ALSV and HAV calculated in the same way above.

SASAs show the same feature of a looser interaction of Vp24 withthe base (Table 3), and the positioning of ﬂexible segments in Vp24
at the interfaces to Vp25 and Vp20 supports this (Fig. 1). Cryo-EM
images and the 2D-class averages suggest that RNA could leak fromthe pentameric protrusion (Fig. 4c).
Recent reports proposed that geometric defects found in
spherical viruses by single-particle analysis without symmetry
enforcement might be a site for infection and genome release.
However, 2D-class averages of ALSV images (Supplementary
Fig. 4b) and a 3D reconstruction assuming no icosahedral symmetry do not indicate such defects (Supplementary Fig. 4c). Herewe picked up more particles from different datasets and processed
together for this test (see Image analysis without icosahedral
symmetry in “Methods”). Relatively large defects (e.g., in Fig. 1 ofWang et al.43) are unlikely as genome release would be too facile,and the virion would not be suitable for carrying the genome to
nearby cells. Then, the defect should only appear at a single
deﬁned site rather than at random sites or solid parts of thecapsid. The pentameric protrusion in the capsid of ALSV appears
eminently suitable to be a release site.
Many ALSV viruses are produced in a plant cell and spread toneighboring cells through a plasmodesma modiﬁed with MP5.
The cryo-EM images (Fig. 4a) suggest that there is a certain low
probability that particles lose the RNA genome from the pentameric protrusions (Figs. 2c and 4c; Table 3). The protrusions may
make a hinge motion to open a channel to release the genome due
to less intimate interactions among themselves and ﬂexible segments of Vp24 at the subunit interface (Figs. 1, 2a, b, and 4d;Table 3). The turret of ﬁve Vp24 subunits seems an unstablestructure compared with the solid circular base of Vp25 and
Vp20 subunits strongly linked through the interfaces and the Nterminal extension. The strongest link of 0Vp24 with a neighbor,judging from the number of hydrogen bonds and buried surface
area, is with 1Vp20 of the juxtaposed trimer (Figs. 1, 2a, b;
Table 3). This link together with an interaction with the Nterminal extension of 1Vp25 may serve as an anchor for a hinge-

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bending motion of Vp24 along the relatively weak 0Vp25 and
0Vp20 interfaces. A mechanism whereby all ﬁve Vp24s of a
protrusion open like the petals of a ﬂower seems feasible. Making
an escape route by dissociation of some or all ﬁve Vp24 subunits
may also be possible for ALSV and viruses of the Comovirus
genus since they have separate proteins. The rate of opening of
the Vp24 subunits and genome release may depend on RNA
8interactions with the Vp25 extension, or may occur spontaneously at a certain low level. Replication of the virus seems toproceed with the leaked RNA from a limited number of viruses.
Our cryo-EM analysis of ALSV reveals the ﬁrst structure of apicorna-like plant virus in the genus Cheravirus to our knowledge. The structure consists of three capsid proteins Vp25, Vp24,
and Vp20, arranged so that a rigid frame of Vp25 and

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Fig. 4 Cryo-EM micrographs of apple-latent spherical virus (ALSV) embedded in vitreous ice, and volume rendering of a pore at the center of ﬁve
Vp24 subunits. a A typical image used for single-particle reconstruction at 2.87-Å resolution. b Images of RNA leakage from many virus particles. Vitriﬁed
sample solution was frozen–thawn several times and incubated for several days. Arrows indicate string-like density of leaked RNA. Densities inside viruses
appear to be lower in (b) than those in (a) and lowest in the rightmost one in (b), as most RNA had leaked out. c Top three of 2D-class averages. RNAleakage is resolved to come from the vertex of the virion. Numbers of particle images that contributed to the corresponding class averages are shown at thebottom. Individual images are shown in Supplementary Fig. 4a. d Side view of the pentameric protrusion overlaid with volume rendering inside the central
pore in gray. Colored in the same scheme as in Fig. 1 and prepared with PyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC). e Plot of thepore radii corresponding to (d).

Vp20 subunits is topped by a pentameric protrusion of
Vp24 subunits with a center hole of ~4 Å (Figs. 1 and 4d). Image
analysis shows the RNA genome leaking from the tip of a protrusion (Fig. 4). The structure of ALSV and observations reported
here suggest how the virion is stabilized and points to a possible
mechanism for genome release through opening of weaklyinteracting Vp24 subunits. The N-terminal extension of Vp25
may help in packing of RNA as it is located on the inner surfaceof the capsid, and does in fact contact the genome (Fig. 2c, d).
The structure presented here could, in the future, provide a
structural basis to study MP-assisted movement of the picornalike plant virus through the tubule between cells.
Methods
Sample preparation. ALSV was isolated and puriﬁed from infected Chenopodium
quinoa leaf tissue as described in Li et al.44. Puriﬁed viruses were suspended in
0.1 M Tris-HCl (pH 7.8), 0.1 M NaCl, and 5 mM MgCl, concentrated to about
10 mg/ml, and kept at −80 °C before use. The sample was negatively stained andchecked with a JEM-2100 electron microscope (JEOL, Tokyo). It revealed virus
particles of round shape with ~300-Å diameter.
To prepare frozen–hydrated samples, gold was ﬁrst sputtered on holey carbon
ﬁlm-coated copper grids (Quantifoil R1.2/1.3 or R2/1, Quantifoil Micro Tools
GmbH). This reduces beam-induced movement45, when the samples are exposedto the electron beam. Three microliters of sample solution containing virus
particles at a concentration of 1–2 mg/ml were applied onto the grid, blottedmanually with ﬁlter paper from the reverse side of the grid, and rapidly frozen inliquid ethane with a homemade plunger in a cold chamber with a humidiﬁer.
Data collection. The samples were examined at a specimen temperature of ~96 K
with a JEOL CRYO ARM 300 electron microscope. A parallel electron beam with adiameter of ~2.3 μm illuminated the sample with a condenser aperture of 150 μm, aspot size of 4, and a convergence angle of 2. Inelastically scattered electrons were
removed through an in-column energy ﬁlter with an energy slit width of 20 eV. Beamtilt was adjusted to the coma-free axis, and twofold astigmatism was removed bySerialEM46. The distribution of ALSV particles in ice appeared to be quite uneven,and most images collected by automated data collection47 showed no virus particles
or very few. Hence, we manually collected images of ALSV particles at a nominal
magniﬁcation of ×40,000. Dose-fractionated frames were recorded on a Gatan
K2 summit in superresolution mode using Gatan DigitalMicrograph. The setting of
critical lenses and deﬂector coils was monitored with ParallEM32,48. The physical pixel
size of the K2 detector corresponded to 1.24 Å, and one image view included ~100
virus particles, each with a diameter of ~300 Å. Thus, a total of 279 movies werecollected. Imaging parameters are shown in Table 1. Magniﬁcation was calibrated
from gold particles sputtered on carbon, which yields 2.3469- or 2.0325-Å rings.
Image analysis. Image processing was performed with RELION-3.031. Image
stacks were ﬁrst drift-corrected, dose-weighted, and summed with the MotionCor2
algorithm49 implemented in RELION-3. Contrast- transfer function (CTF) parameters were estimated with CTFFIND50, and particles were manually picked up
through the image display of RELION for initial templates. Then, automatic particle picking and reference-free 2D classiﬁcation were carried out by following thestandard protocol of RELION-331. Here, picked particles were edited by visualinspection before the 2D classiﬁcation. Creation of a de novo initial 3D map, 3Dclassiﬁcation based on the initial map, and 3D auto-reﬁnement were all done with
icosahedral symmetry enforcement. After applying reﬁnement of defocus andBayesian polishing for motion correction of individual particles31,51, the ﬁnal 3D
structure was reconstructed from particle images (8,018) recorded on 110 movie
stacks. Beam tilts were also corrected for each micrograph (Supplementary Fig. 1f).
Image analysis statistics are shown in Table 1.
A soft mask was estimated and applied to the two half-maps in the postprocessing process of RELION. B-factor estimation and map sharpening were also
carried out in this step. The resolution was measured to be 2.87 Å based on the gold
standard FSC 0.143 criteria (Supplementary Fig. 1e)52. The density map clearly
reveals the side-chain densities of amino acids (Supplementary Figs. 1b–d),
supporting the estimated resolution. Local resolution was calculated using RELION

from unﬁltered half-maps (Supplementary Fig. 1a). Details related to cryo-EM
single-particle analysis are summarized in Table 1.
De novo model building. As virus structures in this genus, and even parts thereof,
have not been solved before, we carried out de novo model building onto the 3D
reconstruction. As the cryo-EM map clearly revealed side-chain densities, anautomated procedure implemented in ARP/wARP53 produced a reasonable initialatomic model. The protomer model was manually adjusted with COOT54, andthen the model of the whole virus was reﬁned by using phenix.real_space_reﬁne55
with icosahedral symmetrization. This gave good geometries and high crosscorrelation (CC) values against the cryo-EM map (Table 1). The structures in
Figs. 1, 2b–d, 3, Supplementary Figs. 1a–d and 4c were prepared with UCSF
Chimera56. Intersubunit-buried SASAs and hydrogen bonds were calculated withUCSF Chimera56. The surface pore of the capsid was analyzed by HOLE57.
The 3D-density map resolved amino acids 2–216 (Vp25), 3–168 (Vp20), and6–190 (Vp24), and residues 41–55, 100–116, and 146–151 in Vp24 are missing on
the map.
Image analysis of the genome. The reconstructed volume of ALSV was sharpened by phenix.auto_sharpen38 for visualization of ﬁner structures. This revealed
residues contributing to interactions with the internal densities of the symmetrized
average RNA genome (Fig. 2c, d).
Images, including leaked RNA and part of the virion, were cut out(Supplementary Fig. 4a) from motion-corrected micrographs of frozen–hydrated
virus after the sample solution was frozen–thawn several times and/or incubatedfor several days. Then, a total of 1142 cut-out images were subjected to 2Dclassiﬁcation with an option of “Ignore CTFs until ﬁrst peak”. The top three classaverages revealing leaked RNA (Fig. 4c) include 243, 190, and 90 particle images.
Image analysis without icosahedral symmetry. We picked up more ALSV
particles from images taken with automated data collection software JADAS47,
although the yield rate was not good due to the uneven distribution of viruses in ice
as above. Particle images were processed together with manually collected ones,
and 2D classiﬁcation was applied to a total of 37,328 particles from 1,296 movie
stacks with an option of “Ignore CTFs until ﬁrst peak,” the setting of which is
expected to be suitable for detection of ﬁner features. A 3D map was reconstructedwithout icosahedral symmetry from a total of 28,633 particles. Icosahedral symmetrization of this combined dataset did not improve the resolution of thestructure probably due to an increased heterogeneity among the datasets.
Statistics and reproducibility. The parameters and statistics of data collection,
image analysis, and de novo atomic model building are shown in Table 1.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.

Data availability
A cryo-EM-density map of ALSV and its atomic coordinates have been deposited in
EMDataResource under the accession number EMD-30375 and in the Protein Data Bank
under the accession number 7CHK, respectively. Sequence data of ALSV RNA1 andRNA2 are accessible in the DNA Data Bank of Japan under accession numbers
AB030940 and AB030941, respectively. Source data for the graph in Fig. 4e are available
as Supplementary Data.

Received: 14 March 2020; Accepted: 11 August 2020;

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Acknowledgements
We thank D.B. McIntosh for help in improving the paper. This work was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for ScientiﬁcResearch Grant 16H04757 (to K.Y.), Japan Society for the Promotion of Science Grantin-Aid for Challenging Exploratory Research Grant 24657111 (to K.Y.), the RIKEN
Pioneering Project, Dynamic Structural Biology (to K.Y.), and the Cyclic Innovation for
Clinical Empowerment (CiCLE) from the Japan Agency for Medical Research andDevelopment, AMED (to K.Y.).

Author contributions
H.N., T.H., S.M.-Y., M.I., N.Y., and K.Y. designed the experiments; M.I. prepared thesample; H.N., T.H., S.M.-Y., and K.Y. collected image data; H.N., T.H., S.M.-Y., and K.Y.
analyzed the data; T.H. reconstructed the cryo-EM map; H.N. built the atomic model;
N.H., H.T., N.Y., and K.Y. wrote the paper.

Competing interests
The authors declare no competing interests.

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Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s42003020-01217-4.

© The Author(s) 2020

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