ABSTRACT The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the

inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent

antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC–capsid interfaces in situ using electron cryo-tomography,

showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC

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ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE HIV CAPSID MIMICS KARYOPHERIN

ENGAGEMENT OF FG-NUCLEOPORINS Article Open access 24 January 2024 MOLECULAR SOCIOLOGY OF VIRUS-INDUCED CELLULAR CONDENSATES SUPPORTING REOVIRUS ASSEMBLY AND REPLICATION Article Open access

06 December 2024 STRUCTURAL BASIS FOR GENOME PACKAGING, RETENTION, AND EJECTION IN HUMAN CYTOMEGALOVIRUS Article Open access 27 July 2021 DATA AVAILABILITY Maps for the following structures

have been deposited in the Electron Microscopy Data Bank: PrV–ΔUS3 capsids under accession codes EMD-17974 (cytosolic), EMD-17975 (perinuclear) and EMD-17976 (nuclear); PrV WT capsids under

EMD-18479 (cytosolic) and EMD-18480 (nuclear); HSV-1 WT capsids under EMD-18481 (cytosolic), EMD-18482 (perinuclear) and EMD-18483 (nuclear); PrV–ΔUS3 NEC under EMD-18474 (spherical form)

and EMD-18473 (helical form); and HSV-1 NEC under EMD-18484 (spherical form). All other data supporting the conclusions of this study can be found within the Article, extended data and

Supplementary Information. Source data are provided with this paper. CODE AVAILABILITY Custom scripts used for this study are available via GitHub at https://github.com/vojtaprazak/NEC.

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Google Scholar  Download references ACKNOWLEDGEMENTS We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy EXC 2155 project

number 390874280 and by the Wellcome Trust through a Collaborative Award (209250/Z/17/Z) as well as Hamburg-X and the Deutsche Forschungsgemeinschaft (DFG, German Research

Foundation)—GRK2771—project number 453548970 (J.B.B. and K.G.), Wellcome Trust grants 107806/Z/15/Z and 209250/Z/17/ Z (K.G.), Wellcome Trust grants 099683/Z/12/Z and 225902/Z/22/Z (M.G.)

and BMBF grants 05K18BHA and 031L0287A (K.G.). This research was funded in part by DFG INST 152/ 772-1, 774-1, 775-1, 777-1 FUGG (CSSB cryoEM facility) and a Wellcome Trust core award to The

Wellcome Centre for Human Genetics (090532/Z/09/Z). V.P. was supported by a Nuffield Dept. of Medicine Prize Studentship. The funders had no role in study design, data collection and

analysis, decision to publish or preparation of the paper. AUTHOR INFORMATION Author notes * These authors contributed equally: Vojtěch Pražák, Yuliia Mironova. AUTHORS AND AFFILIATIONS *

Centre for Structural Systems Biology, Hamburg, Germany Vojtěch Pražák, Yuliia Mironova, Daven Vasishtan, Christoph Hagen, Ulrike Laugks, Yannick Jensen, Saskia Sanders, Jens B. Bosse & 

Kay Grünewald * Leibniz Institute of Virology, Hamburg, Germany Vojtěch Pražák, Yuliia Mironova, Daven Vasishtan, Christoph Hagen, Ulrike Laugks, Yannick Jensen, Saskia Sanders, Jens B.

Bosse & Kay Grünewald * Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK Vojtěch Pražák, Daven

Vasishtan, Christoph Hagen, Michael Grange & Kay Grünewald * Department of Biochemistry, University of Oxford, Oxford, UK Vojtěch Pražák & Daven Vasishtan * Department of Chemistry,

University of Hamburg, Hamburg, Germany Yuliia Mironova, Ulrike Laugks, Yannick Jensen & Kay Grünewald * Hannover Medical School, Institute of Virology, Hannover, Germany Yannick Jensen,

 Saskia Sanders & Jens B. Bosse * Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany Yannick Jensen, Saskia Sanders, Jens B. Bosse & Kay Grünewald *

Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA John M. Heumann * Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut,

Greifswald-Insel Riems, Germany Barbara G. Klupp & Thomas C. Mettenleiter * Structural Biology, Rosalind Franklin Institute, Didcot, UK Michael Grange Authors * Vojtěch Pražák View

author publications You can also search for this author inPubMed Google Scholar * Yuliia Mironova View author publications You can also search for this author inPubMed Google Scholar * Daven

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CONTRIBUTIONS C.H. and K.G. designed the study. J.B.B., K.G., M.G., C.H., J.M.H., Y.J., S.S., V.P. and D.V. designed experiments (laboratory or computational). C.H., Y.J., U.L., B.G.K., Y.M.

and S.S. generated specimens. M.G., C.H., Y.J., B.G.K., U.L., Y.M. and S.S. collected data. M.G., C.H., Y.J., U.L., Y.M., V.P., S.S. and D.V. processed data. J.B.B., M.G., C.H., J.M.H.,

Y.J., Y.M., V.P., S.S. and D.V. analysed data. J.B.B., K.G., M.G., C.H., Y.M., V.P. and D.V. performed critical analysis of the findings. J.B.B., M.G., Y.J., Y.M., V.P., S.S. and D.V. made

figures. M.G., Y.M., V.P. and D.V. wrote the paper. J.B.B., M.G., J.M.H., B.G.K., V.P., D.V. and K.G. performed a critical paper review. CORRESPONDING AUTHORS Correspondence to Michael

Grange or Kay Grünewald. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Microbiology_ thanks Jeremy Kamil,

Phil Pellett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard

to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 WORKFLOW FOR CRYOFIB/SEM AND CRYOET. A, A low magnification SEM image of

PrV-ΔUS3-infected porcine epithelial cells. B, A higher magnification image of the box shown in a. C, An oblique SEM view illuminated by the FIB with yellow boxes indicating the area above

and below the cell for targeting with the focused ion beam. D, SEM image of thinned cellular section (lamella) from the top and side (inset). Transmission electron microscope (TEM) image is

also shown (right), with target area shown in e highlighted by a white box. E, A TEM image shown at 9500x nominal magnification. Details of the cell are visible at this magnification,

allowing targeting of regions of interest for higher magnification tomographic data collection (red box). Shown are representative samples of 113 lamella from 3 biological replicates. cyt =

cytosol, nuc = nucleus. F, A tomographic slice of the region indicated in e) at 35000x magnification, nominal. ONM, outer nuclear membrane; INM, inner nuclear membrane. EXTENDED DATA FIG. 2

QUANTIFICATION OF NUCLEAR EGRESS EVENTS. A, Size and curvature distribution of perinuclear vesicles. Shown are inner luminal diameters of individual vesicles (see Methods) with mean values

represented by red bars. *The mean value and standard deviation of perinuclear vesicles obtained by expression pUL31/pUL3410 are shown for comparison. Vesicles (and the cargo within) were

counted as perinuclear where envelopment was judged to be more than 50% completed (for example Fig. 1c would be considered perinuclear). This is because a significant proportion of vesicles

(~150 nm diameter) were only partially contained within the 150–250 nm thick tomograms (that is cut off during FIB milling). B, Quantification of capsid types observed during nuclear egress

in porcine epithelial (PrV) and African Green monkey cells (HSV-1). C, Quantification of perinuclear vesicles. Classes were assigned to vesicles based on cargo type. In contrast to vesicle

diameter measurements shown in A, vesicles containing procapsids were included in the capsid class, whereas scaffolds were counted as non-capsid cargo (note that there were 6 perinuclear

procapsids). Procapsids contain capsid proteins some of which could be involved in the initiation of envelopment and therefore fit better in the ‘capsid’ class. At the same time, all

perinuclear procapsids were only partially assembled and consequently the perinuclear vesicles were smaller. D, Diagrams indicating capsid classification. A-capsids lack both nucleic acid

and scaffold, B-capsids contain a scaffold, and C-capsids are capsids after completion of DNA packaging. Procapsids (not shown) are C-capsid precursors. Source data EXTENDED DATA FIG. 3

PUTATIVE FLAT NEC DOUBLE-LAYER LATTICE. A, B, C Orthogonal section through average volume of lattice shown in Fig. 1f. There is no density for the membrane bilayers due to the majority of

particles having their six-fold symmetry aligned with the tomogram Z axis (and the missing wedge). A total of 1944 C6 symmetrised particles were included in the average. D, Tangential

sections through the volume at 1.4 nm intervals. E, A slice through the raw tomogram indicating the position of the inner nuclear membranes relative to the lattice. The membrane is not

visible around the majority of the lattice layer due to its orientation to the tomogram missing wedge. The approximate position of the outer nuclear membrane was inferred from the exclusion

zone of cytosolic components (ribosomes, intermediate filaments, microtubules). F, Two NEC hexamers segmented from the spherical lattice (Fig. 5, here shown in yellow) were fitted into one

repeating unit of the lattice. In this orientation, pUL31 would form the interface between the two lattice layers. G, H, I, J, The hexamer centres in the double-layer lattice (yellow) are

spaced 2 nm further apart and are rotated by approximately 20° to the 6-2-6 axis compared to spherical NEC (blue) and flat lattice derived from the HSV-1 crystal structure (purple). K, Slice

through several adjacent type-1 NR, with the lumen of one of these zippered by putative head-head interacting NEC (yellow). Inset shows an orthogonal slice through the centre of the

highlighted area. L, Plotback of individual symmetry units (dodecamers). There is a slight curvature to the lattice with a break in the middle, presumably to accommodate the tighter

curvature of the underlying nucleoplasmic reticulum membrane. EXTENDED DATA FIG. 4 SUBVOLUME AVERAGING GENERATES A PLAUSIBLE MODEL OF NEC LATTICE ORDER. Shown is A top view of a single

perinuclear vesicle (also in Figs. 2, 3 and Supplementary Fig. 8). The surface (generated and visualised using Open3D) was coloured with the intersecting voxel densities of either A, volume

where an NEC average volume was backplotted using subvolume averaging particle positions or B, C, the original data. Orange lines highlight the same areas in A and B where particles were

removed due to their relatively low cross correlation coefficient. A long-range hexagonal order is apparent outside these areas. Assessing the nature of disordered regions is more

challenging. To guide the reader’s eye, some densities in putative disordered regions were highlighted with red lines. A single hexagonal region was highlighted in blue for comparison. Note:

A direct interpretation of tomogram densities on this scale can be misleading and should be used with caution. This example is intended to highlight that there is likely NEC in the

disordered regions. What the structure of this lattice may be is not clear. EXTENDED DATA FIG. 5 QUESTIONABLE CONNECTING DENSITIES BETWEEN NUCLEAR CAPSIDS AND BUDDING NEC. The NEC subvolumes

were classified by the distance to the nearest penton vertex (top panel) and vice versa (bottom panel). Each column shows sections through the resulting class average volume, with the

maximum separation distance and the number of particles included in the average indicated above. All volumes were filtered using the same bandpass filter. The NEC layer is smeared in the

penton-aligned averages and accordingly the capsids are smeared in the NEC-aligned averages, indicating that the two lattices are not aligned. There is a hint of a density originating from

pentons closer than ~7 nm from the nearest NEC surface (orange arrows), but any interpretation of this would be highly questionable due to the small number of particles. EXTENDED DATA FIG. 6

PUTATIVE NEC COAT ON NEGATIVELY CURVED SURFACES. A, B, C, Slices through the same nucleoplasmic reticulum at different depths showing the distribution of the putative NEC layer. D, E, An

enlarged section of panel c, showing top views of ring-like structures (highlighted in blue in e). F Overlay of the surface representation of the average volume of 121 ring-like particles

from two tomograms and the spherical NEC lattice. Each ring could plausibly accommodate two concentric layers of pUL31/34 dimers. Averaging a more exhaustive (but less stringently picked)

set of negatively curved lattice particles did not converge (and is therefore not shown), suggesting a high degree of variability. Notably, the membrane was not included as an alignment

feature. G, The thickness and distance to the membrane of this layer are consistent with the spherical NEC lattice. Sections through the ring average volume with different C symmetries

applied. Visually, C7 is the best match to C1 but it is possible these structures have no strict symmetry, as suggested by D, E. Note that symmetrisation in this case means addition of

subvolumes at defined rotations (for example 5 subvolumes with 60° degree increments for C6 symmetry). Alignment was performed after the addition of symmetry related particles. The bottom

left-most panel is a section through the spherical NEC lattice. EXTENDED DATA FIG. 7 LOCALIZATION OF INDIVIDUAL NUCLEAR AND CYTOPLASMIC MSCARLET-UL25 LABELLED CAPSIDS. A, PK15 cells were

infected with PrV-mScarlet-UL25 or B, PrV-mScarlet-UL25-∆US3 fixed at 7 or 10 hpi, respectively, and imaged using spinning disc microscopy. UL25-mScarlet (red); DNA-Hoechst (blue). One plane

of the acquired volume is shown. C, Viral particles were detected in the 3D volumes using Trackmate in FIJI with an expected blob diameter of 0.4 microns and the quality threshold set to

10.0. c, Fluorescent signal in a single plane of a PK15 cell infected with PrV-mScarlet-UL25-∆US3 and fixed 10 hpi (top) and projection of all detected single particles (purple) of the

volume in a nuclear ROI (yellow) onto one plane (bottom). D, E, The FIJI plugin Trackmate was used to detect and measure individual virus particle fluorescent intensities. For each

condition, the total intensity of more than 4,000 particles was quantified and detected in more than 30 different cells, all using a single biological replicate. Source data EXTENDED DATA

FIG. 8 PRV NUCLEAR EGRESS COMPLEX FORMS TUBES WITH HELICAL SYMMETRY. A, B, Comparison of the NEC curvature in perinuclear vesicles and tubes. B, Shown are sections through the average

volumes of four tubes, with the respective diameters indicated in cyan. The NEC tubes have substantially smaller luminal cavities compared to spherical NEC vesicles. Notably, the two

separate tubes with matching diameters and helical parameters were located within the same nucleoplasmic reticulum and may have originated from the same assembly. C, Tubular NEC forms tubes

with different helical parameters, indicating a flexibility in the direction of largest curvature. Highlighted is the angle of the 6-2-6 axis to the helical symmetry axis. Scale bars

indicate 50 nm. EXTENDED DATA FIG. 9 NEC IN HSV-1. A, Slice through a tomogram depicting primary enveloped nucleocapsid egressing to the cytosol. The NEC coat of WT HSV1 (red) follows the

same pattern as that of ΔUS3 PrV and could be identified on the INM and in perinuclear vesicles. Likewise ring-like structures similar to those in PrV (yellow in i. and iii., Fig. 3c and

Extended Data Fig. 6) was identified near the hexagonal NEC lattice (red in i. and iii.). Red area in the inset ii. shows budding NEC. B, HSV-1 NEC crystal structure (PDB ID 4ZXS) fitted

into the subvolume average of HSV-1 NEC from perinuclear vesicles. ONM, outer nuclear membrane; INM, inner nuclear membrane. C, Raw tomographic slices through C-capsids of WT HSV-1 in

indicated subcellular locations. D, Slices through the average volumes of C-capsids (middle) and their surface representations (bottom). Arrowheads indicate the position of an additional

density present in the cytosolic capsids. 15 tomograms from 5 preparations were used. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–8, Supplementary Table 1 and

sequence notes. REPORTING SUMMARY SUPPLEMENTARY VIDEO 1 Tomographic volume of capsids, perinuclear vesicles and other structures observable in the nucleus. The orange arrowheads indicate

scaffolds found within chromatin-deficient areas. The magenta arrowhead shows a partial capsid that may be undergoing assembly. The yellow arrowhead points towards assembling B-capsids that

are yet to spool DNA. The cyan arrowhead shows a capsid in the process of spooling DNA, which is assumed due to the more loosely packed density within the capsid—individual DNA fibres are

visible. The blue arrowheads show C-capsids in the nucleus containing densely packed DNA. SUPPLEMENTARY VIDEO 2 Tomographic volume of perinuclear vesicles in the lumen between the INM and

ONM. The black arrowheads point towards openings of perinuclear vesicles at the ONM towards the cytosol. SUPPLEMENTARY VIDEO 3 Tomographic volume of NEC-associated structures in type 1

nucleoplasmic reticulum herniations and invagination of the INM by the NEC. The orange arrowheads represent scaffolds found, once again, within chromatin-deficient areas. In one area of the

tomogram, a scaffold is visible in an NEC tube. The yellow arrowhead points towards assembling B-capsids that are yet to spool DNA while the blue arrowheads point towards fully formed

C-capsids. The red arrowhead points to invagination sites caused by the NEC. On one of these occasions, no structures are apparent to cause such an invagination to occur (bottom). The green

asterisk shows a perinuclear vesicle containing a B-scaffold. The blue arrow shows an NEC tube-like structure. SUPPLEMENTARY VIDEO 4 Tomographic volume of perinuclear vesicles in type-1

nucleoplasmic reticula, capsids budding into perinuclear vesicles and tubular NEC-associated structure. Red arrows point toward assembling heterodimeric NEC arrays at the INM that are

associated with capsids and a vesicle-like structure (pink arrowhead). A green asterisk denotes a perinuclear vesicle that has formed and enveloped 2 individual capsids (an A-capsid and a

C-capsid). The blue asterisk denotes an NEC-associated tube, the sub-volume average for which can be seen in Fig. 6. A white arrowhead shows a small opening toward the nucleus in a

perinuclear vesicle that is almost fully formed. SOURCE DATA SOURCE DATA FIG. 1 Source data for Figs. 2 and 5, Extended Data Fig. 2, Extended Data Fig 7 and Supplementary Fig. 6b. RIGHTS AND

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Pražák, V., Mironova, Y., Vasishtan, D. _et al._ Molecular plasticity of herpesvirus nuclear egress analysed in situ. _Nat Microbiol_ 9,

1842–1855 (2024). https://doi.org/10.1038/s41564-024-01716-8 Download citation * Received: 19 July 2023 * Accepted: 29 April 2024 * Published: 25 June 2024 * Issue Date: July 2024 * DOI:

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