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Articleshttps://doi.org/10.1038/s41564-020-0758-1
Structures of capsid and capsid-associated tegument complex inside the Epstein–Barr virusWei Liu1,2,3,8, Yanxiang Cui 1,8, Caiyan Wang1,2,4, Zihang Li 1,2, Danyang Gong5,6, Xinghong Dai1,2,7, Guo-Qiang Bi 3, Ren Sun1,5 and Z. Hong Zhou 1,2 ✉
1California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA. 2Department of Microbiology Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA. 3Center for Integrative Imaging, Hefei National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, China. 4International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China. 5Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA. 6Present address: Department of Therapeutics Discovery, Amgen Research, Amgen Inc., Thousand Oaks, CA, USA. 7Present address: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. 8These authors contributed equally: Wei Liu, Yanxiang Cui. ✉e-mail: [email protected]
SUPPLEMENTARY INFORMATION
In the format provided by the authors and unedited.
NatuRE MiCRoBioLoGY | www.nature.com/naturemicrobiology
http://orcid.org/0000-0002-8909-6576http://orcid.org/0000-0002-4625-9283http://orcid.org/0000-0001-8735-3818http://orcid.org/0000-0002-8373-4717mailto:[email protected]://www.nature.com/naturemicrobiology
Supporting Information
Structures of Capsid and Capsid-Associated Tegument Complex inside the Epstein-Barr Virus
Wei Liu, Yanxiang Cui et al.
List of Content Supplementary Discussion
Supplementary Tables 1-3
Supplementary Figures 1-5
1
Supplementary Discussion Inter-MCP interactions and structural plasticity of the 20 MCP conformers
First recognized in HCMV1 and subsequently confirmed in HSV2-4, HHV-6B5, and
KSHV6, the herpesvirus capsid floor is characterized by either type I, II, or III MCP-MCP
interactions (Extended Data Fig. 3e-l). Type I interaction is intra-capsomeric while type
II and type III are inter-capsomeric. Types I, II, and III interactions are formed between
two or three adjacent MCP subunits by either β-strands in the N-lasso, E-loop in the
Johnson-fold and dimerization domains (Extended Data Fig. 3g,i-j), or α-helices in the
dimerization domains (Extended Data Fig. 3h) of the neighboring MCPs. The lasso
action (type III) occurs only between hexon-hexon MCPs (Extended Data Fig. 3i-j) but
not penton-hexon MCPs (Extended Data Fig. 3k,l); this is due to the flexibility of the
penton MCP N-terminus, which causes P6 MCP to refold into a conformation that
effectively eliminates its lassoing ability (Extended Data Fig. 3k). Additionally, the inter-
capsomeric interactions between α-helices in the dimerization domains (i.e., type II) also
only occur between hexon-hexon MCPs because the dimerization domain of penton
MCP adopts a configuration which makes it unable to form type II interactions with the
dimerization domain of P1 hexon MCP, rendering the P1 dimerization domain flexible
(Extended Data Fig. 3k,l).
In contrast to these conserved MCP domain organization and MCP-MCP
interaction schemes, the structures among individual MCP subunits of the capsid exhibit
an unprecedented level of variability which we refer to as structural plasticity
(Supplementary Video 8). We identified 20 unique conformations among the MCP
structures resolved in each EBV capsid (Fig. 3; Extended Data Fig. 4): 16 within an
icosahedral asymmetric triangle (Fig. 3e), 2 portal proximal (Extended Data Fig. 4a,
right panel), and 2 related to CATC binding (Extended Data Fig. 4a, middle panel).
These MCPs show structural plasticity at different degrees depending on their
surroundings. 16 MCPs within the asymmetric unit vary in their local structures
throughout multiple domains, particularly at their floor regions (Fig. 3e-p). Compared
with P1, P6 and Pen MCPs, the structures of C1-C6, E1-E3, and P2-P5 MCPs do not
have dramatic changes. The alignment of these 13 MCP models highlights not only the
2
conformational changes at their N-lasso domains (Fig. 3j) and two fragments (a.a. 124-
172 and a.a. 1072-1091) in their Johnson-fold domains (Fig. 3k), but also the orientation
switch (i.e., being “up” and “down”) of two MCP fragments (a.a. 1149-1169 and a.a.
1256-1267) in their buttress domains (Fig. 3h,i). Each triplex adopts a unique direction,
but still can be stabilized by interacting with three quasi-equivalent neighboring MCPs.
Different interacting sites on the heterotrimeric triplexes (Supplementary Figs. 4-5)
might be responsible for the multiple binding states found in the buttress sub-domains
(Fig. 3h,i).
Variations among MCP subunits can be observed in the floor regions of P1, P6
and Pen MCPs, particularly in N-lasso (Fig. 3o) and dimerization (Fig. 3n) domains. N-
lasso of P1 MCP folds similarly with that of C1 MCP, but is completely different from
those of P6 and Pen MCPs (Fig. 3o). Likewise, the fragment of a.a. 295-374 of P6 MCP
folds in conformity with that of C1 MCP while differing vastly compared with those of P1
and Pen MCPs (Fig. 3n). It is worth noting that the fragment a.a. 1277-1323 in the
buttress domain of Pen MCP folds into a long helix instead of two short helices in those
of other 15 MCPs (Fig. 3m). As shown in Fig. 3p, the spine helix in the Johnson-fold
domain of Pen MCP tilts downward ~40º compared with the 15 hexon MCPs.
There are also variations in the P1 (and P6) MCP structures in the CATC-absent
vertex (Extended Data Fig. 4a, left panel), CATC-binding vertex (Extended Data Fig. 4a,
middle panel), and portal vertex (Extended Data Fig. 4a, right panel). Among these
three, the portal vertex displays the greatest level of structural plasticity (P1 in Extended
Data Fig. 4b-e; P6 in Extended Data Fig. 4f,g). Such structural variations may result
from differences in capsid-bindings by CATC and portal proteins. For instance, fragment
a.a. 80-96 of P1 MCP in the portal vertex flips up rather than down as those in the P1
MCPs of CATC-absent and CATC-binding vertices (Extended Data Fig. 4d). Fragments
of a.a. 305-340 and a.a. 1146-1168 are missing in P1 MCP (Extended Data Fig. 4c,e)
while a.a. 26-63 is invisible in P6 MCP (Extended Data Fig. 4g) in the portal vertex
reconstruction. These variations in hexons MCPs, particularly P1 and P6 MCPs, and
penton MCP reveal the intrinsic plasticity of MCP structure in EBV; the folding of
individual domains is preserved with gradual structural changes spreading out across
3
different domains, rather than through an abrupt large-scale conformational switch (Fig.
3e-p). Despite remaining consistent with its role in organizing the three types of MCP-
MCP interactions (Extended Data Fig. 3e-l), the greatest structural changes were
observed in N-lasso areas. Taken together, the 20 unique conformers of MCP—16
MCPs within an asymmetric unit, 2 P1 and 2 P6 MCPs in CATC-binding penton and
portal vertexes—unveil a remarkable level of structural plasticity of MCP not previously
reported for any herpesviruses.
Supplementary Tables Supplementary Table 1. Statistics of cryoEM imaging, data processing and model refinement.
Supplementary Table 2. Model building statistics.
Supplementary Table 3. RMSD statistics for MCP and Tri1.
References 1. Yu, X., Jih, J., Jiang, J. & Zhou, Z.H. Atomic structure of the human cytomegalovirus
capsid with its securing tegument layer of pp150. Science 356, eaam6892 (2017). 2. Dai, X. & Zhou, Z.H. Structure of the herpes simplex virus 1 capsid with associated
tegument protein complexes. Science 360(2018). 3. Wang, J. et al. Structure of the herpes simplex virus type 2 C-capsid with capsid-vertex-
specific component. Nature Communications 9, 3668 (2018). 4. Yuan, S. et al. Cryo-EM structure of a herpesvirus capsid at 3.1 Å. Science 360(2018). 5. Zhang, Y. et al. Atomic structure of the human herpesvirus 6B capsid and capsid-
associated tegument complexes. Nature Communications 10, 5346 (2019). 6. Dai, X. et al. Structure and mutagenesis reveal essential capsid protein interactions for
KSHV replication. Nature 553, 521 (2018).
1000 Å
Viral envelope
Virus-like vesicles
Virion
Supplementary Figure 1. A representative cryoEM micrograph containing one EBV virionparticle. Two types of particles can be seen: virion containing a nucleocapsid (marked by a green box,middle) and virus-like vesicles39 (upper left). Zoomed-in in the right panel is the nucleocapsid showingfingerprint-like pattern, characteristic of dsDNA.
4
C6 hexon sub‐particle reconstruction
3Å
7Å
3Å
7Å
C5 portal vertex sub‐particle reconstruction (cut‐away view)
CATC‐binding penton vertex sub‐particle reconstruction
3Å
7Å
6Å
10Å
C12 portal vertex sub‐particle reconstruction
CATC‐absent penton vertex sub‐particle reconstruction C1 3‐fold sub‐particle
reconstruction
3Å
7Å
3Å
7Å
C3 3‐fold sub‐particle reconstruction
C2 2‐fold sub‐particle reconstruction
C5 penton sub‐particle reconstruction
3Å
7Å
3Å
7Å
3Å
7Åa
b
FSC = 0.143
0.00.0 0.1 0.2 0.3
0.5
1.0
3.0Å3.4Å
4.0Å4.4Å
5.5 Å
Fourier she
ll correctio
n (FSC) coe
fficie
nt
Resolution (1/Å)
7.8 Å6.7 Å
0.4
C2 2‐fold sub‐particle reconstruction C3 3‐fold sub‐particle reconstruction
CATC‐absent penton vertex sub‐particle reconstructionCATC‐binding penton vertex sub‐particle reconstructionC5 portal vertex sub‐particle reconstruction
C6 hexon sub‐particle reconstruction
Icosahedral capsid reconstruction (bin2)C5 capsid reconstruction with portal vertex (bin2)
C5 penton sub‐particle reconstruction C1 3‐fold sub‐particle reconstruction
C12 portal vertex sub‐particle reconstruction
3.5Å
Supplementary Figure 3. Resolution assessment of 3D reconstructions. (A) Local resolutionevaluation of sub-particle reconstructions by ResMap59. Color bar represents resolution range. (B)“Gold-standard” Fourier shell correlation (FSC) curves of the 3D reconstructions. The resolutions ofthe icosahedral reconstruction and C5 whole virus reconstruction are 5.6 Å and 7.8 Å, respectively;those for the other sub-particles are as follow: 3.0 Å (C6 hexon sub-particle reconstruction), 3.4 Å (C22-fold sub-particle reconstruction), 3.4 Å (C3 3-fold sub-particle reconstruction), 3.5 Å (C5 pentonvertex sub-particle reconstruction), 4.0 Å (CATC-binding sub-particles reconstruction), 4.1 Å (C1 3-fold sub-particles reconstruction), 3.5 Å (CATC-absent penton sub-particle reconstruction) and 4.4 Å(C5 portal vertex sub-particles reconstruction). All resolutions are based on the 0.143 FSC criterion 51.
6
C1C2C3C4
P1P2P3 E3P4
E1E2
PenC5C6
P5P6Pen
P1
P2
P3P4
P5
P6
C1
C2
C3C4
C5
C6E1
E2
E3
Tri1Tri2ATri2B
a.a., 1149-1169MCPa.a., 1256-1267
“up” in Fig. 3, H and I
“down” in Fig. 3, H and I
Un-modeled
E1 E2
E3
E1E2
E3
E1
E2
E3
P1
Pen
PenPen
Pen
P2
P3
P4P5
P6
Ta
Tc
Tb
Te
Tf
Td
Te
MCP within one asymmetric unit
MCP neighboring to one asymmetric unit
Triplex within one asymmetric unit
Triplex neighboring to one asymmetric unit
Supplementary Figure 4. Schematic diagram of plasticity of triplex interactions with MCP buttress domain shown inFigure 3h,i.
7
Tri2ATa
Tc
Tb Td
Te
Tri2BTri2A
Tri2BTri1
Tri2A
P2
Pen1
P1
P6
P5
C6
P4
C1E1
E2 C5
C4
C2
E5
P3
Triplex Ta
Pen1
P6P1Tri1
Tri2BTri2A
Tri1
Tri2B
P6Tri2B
Tri1
Tri2AP1
P1Tri2A
a.a. 1149-1169
a.a. 1256-1267Tri2B
Pen1
a.a. 1256-1267
a.a. 1256-1267
a
b
C6
P5
P2
Tri1
Tri2BTri2A
Triplex Tc
Tri1
Tri1
Tri2B
P2
a.a. 1149-1169
P2
a.a. 1256-1267
c
a.a. 1149-1169missing
Tri2A
P5
C6
Tri2B
a.a. 1256-1267
a.a. 1256-1267
P3
E1
C5
Tri1
Tri2BTri2A
Triplex Tb
dTri1Tri2B
C5Tri1
Tri1
Tri2B
Tri2A
E1
E1
Tri2AP3
Tri2B
a.a. 1149-1169
a.a. 1256-1267
a.a. 1256-1267
a.a. 1149-1169
a.a. 1256-1267
C5
Tri1
Tri2BTri2A
Triplex Td
E5
C1
P4
C1
C1
Tri1
Tri2B
Tri1e
a.a. 1149-1169
a.a. 1256-1267
E5
E5
Tri2A
Tri2B
a.a. 1149-1169
a.a. 1256-1267
P4
P4Tri2A
Tri2A
Tri2B
Tri1 a.a. 1149-1169
a.a. 1256-1267
Triplex Te
Tri1
Tri2BTri2A
C2
E2
C4
E2E2
Tri1
Tri1
Tri2Ba.a. 1149-1169
a.a. 1256-1267
Tri1
Tri2B Tri2A C4
C4Tri2A
a.a. 1256-1267
a.a. 1149-1169
a.a. 1256-1267
Tri2BTri2A
C2
missing
a.a.1149-1169
f
Tri1
Tri2ATri2B
Tri1
Tri2ATri2B
Tri1
Tri1
Tri2B
Supplementary Figure 5. Details of plasticity of triplex interactions with MCP buttress domain shown in Figure3h,i. (A) Distribution of triplexes Ta, Tb, Tc, Td, and Te in the MCP network. (B-F) Magnified profile views of the regionssurrounding triplexes Ta (B), Tb (D), Tc (C), Td (E), and Te (F) as viewed from the outside of the capsid. Away from thecapsid floor, triplex Ta is dominantly stabilized by two long loop-containing segments (a.a. 1149-1169 and a.a. 1256-1267) from the buttress domains of P1 MCP (purple), P6 MCP (orange), and Pen1 MCP (green) (B). Tb (D), Tc (C), Td(E), and Te (F) triplexes are stabilized by their surrounding MCPs in an architecturally similar but locally different manner.
8
SpringerNature_NatMicro_758_ESM.pdfReferencesSfig2-only-largesize.pdfSlide Number 2