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In situ structure determination of virus capsids imaged within cell nuclei by correlative light and cryo-electron tomography

Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures at atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (>500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised equipment of limited availability. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions reveal that the capsid associated tegument complex is present on capsids prior to nuclear egress. We show that this approach to cryogenic imaging of cells is suited to both correlative light/electron microscopy and 3D structure determination.


Introduction
Three-dimensional (3D) information on protein structure is key to our understanding of dynamic processes that occur within a cell or tissue at the molecular level. Electron microscopy (EM) has been a critical tool in the study of cellular ultrastructure.
Classical methods for imaging of biological material in the electron microscope involve the use of metal contrasting agents and dehydration of the sample 1 . Such methods limit the preservation and visualisation of fine features. Cryo-EM has recently emerged as a powerful tool in the study of macromolecular structure. Purified protein or nucleoprotein assemblies are embedded in vitreous ice yielding frozenhydrated specimens that are suited to imaging in the transmission electron microscope. This method produces artefact-free, high-resolution images of biological assemblies in near-native conditions that may be processed to determine highresolution structures. Cryo-EM has transformed the field of structural biology providing rapid access to high-resolution macromolecular structure without the need to prepare crystals. These studies have however largely been limited to single-particle analysis -the study of purified homogeneous specimens, which may be computationally averaged to yield high-resolution structure data 2 . Many of the most interesting biological questions involve unique and heterogeneous structures that are not amenable to structural averaging methods employed in cryo-EM single-particle techniques. To address this challenge, considerable efforts have been made in the development of cryo electron tomography (Cryo-ET) as a tool to image preparations of heterogeneous assemblies such as enveloped viruses as well as vitrified cells [3][4][5] .
Electron tomography is a means of determining three-dimensional structures of unique entities by recording multiple images of an object in the transmission electron microscope (TEM), as it is rotated, typically through ~120 o . Owing to the limited penetrating power of electrons, cryo-ET is restricted to samples that are less than 500 nm thick, thus for cellular imaging, only the thin edges of cells are accessible.
Features in the interior of eukaryotic cells, such as the nucleus, are not readily viewed.
To image the cell interior requires thinning of the area of interest to less than 500 nm.
Early attempts to address this problem employed cryogenic microtomy or 'cryosectioning' of vitreous frozen-hydrated cells. While cryo electron microscopy of vitrified sections (CEMOVIS) avoids chemical fixation and staining, it is technically challenging, limited in sample thickness and subject to several artefacts including compression and crevasses. These restrict the potential of CEMOVIS for highresolution structural imaging 6,7 . More recently progress has been made towards artefact-free cell sectioning through milling of cellular specimens using cryo focused ion beam scanning electron microscopy (cryoFIBSEM) 8 . Frozen-hydrated cells, grown on an EM grid are etched into electron-transparent lamellae, using a gallium ion-beam. Lamellae are then imaged by cryo-ET. When successfully applied cryoFIBSEM provides well preserved views of the cell interior. Focussed ion-beam milling of vitreous specimens is, however, a low throughput method that is prone to failure -lamellae are easily broken or contaminated by frost during transfer from the cryoFIBSEM to the cryo-TEM. Moreover, the method involves specialised and expensive equipment.
Artefact-free or low-artefact cryo-microtomy approaches to preparing cellular material for cryo-ET are therefore desirable. Such approaches have the benefit of using less expensive and more widely available equipment for high-resolution 3D structural imaging of cells and tissues, potentially leading to in situ structure determination. Vitrified frozen cell sections (VFS) provide an ideal compromise wherein chemically fixed samples are cut under cryogenic conditions, thawed and then re-vitrified by plunge-freezing for imaging by cryo-EM or cryo-ET 9, 10 . This technique has several advantages for investigators interested in determining macromolecular structures in situ. Many laboratories will have ready access to the necessary equipment; cryo-microtomes are already available in many EM laboratories, or existing microtomes may be modified for low-temperature microtomy. The method is relatively high-throughput; many sections may be prepared at once, with a greater likelihood of successful imaging. To our knowledge however, use of this method has thus far been confined to 3D imaging of tissue specimens [10][11][12][13] .
Sub-tomogram averaging has not been hitherto attempted.
While cryo-ET potentially allows imaging of macromolecular structures in 3D within intact cells, it remains challenging to identify features of interest within the crowded and complex environment of the cell. Such studies are therefore liable to be both challenging and time-consuming especially when structures under investigation are smaller, involved in dynamic or rare events or are not well characterized. To overcome these difficulties methods have been developed that allow investigators to precisely locate features of interest prior to recording tomograms. This is achieved through fluorescence microscopy of cell sections prior to electron imaging and is termed correlative light and electron microscopy (CLEM) 14 .
Here we present a modified strategy that combines correlative light microscopy and cryo-ET to locate regions of interest (ROI) in re-vitrified cell sections with subsequent determination of 3D structure by subtomogram averaging. We demonstrate the feasibility of this technique by determining structures of the herpes simplex virus type 1 (HSV-1) capsid, imaged within the nucleus of intact infected cells.
HSV-1 is an important human pathogen that causes cold sores. Infection with herpesviruses leads to life-long infection owing to their capacity to enter a latent state.
More serious conditions caused by HSV-1 include encephalitis which may be fatal, and keratitis an eye condition that can lead to loss of sight 15 . Herpesviruses have large double-stranded DNA genomes. Virion morphogenesis begins in the nucleus of infected cells with assembly of a T=16 icosahedral procapsid. Viral genomic DNA is pumped into the procapsid through a portal assembly located at a unique five-fold vertex 16 . The resulting mature nucleocapsid then buds through the nuclear envelope via an envelopment/de-envelopment process. In the cytoplasm, nucleocapsids become surrounded by a dense proteinaceous layer known as the tegument, before budding into plasma membrane derived endocytic membranes; giving rise to mature enveloped virions 17,18 . The nucleocapsid structure has been extensively studied and was recently characterised at high resolution, leading to an atomistic model of this large (1250Å diameter) and complex assembly 19 . Of particular relevance to this study is the definition of the structure and composition of an assembly variously known as the capsid-vertex specific component (CVSC) or capsid associated tegument complex (CATC). Comprised of three proteins (pUL17, pUL25 and pUL36), this assembly forms a pentaskelion shaped structure that sits over the five-fold symmetry axes. Each arm of density is composed of a single copy of pUL17, and two copies of pUL25 and pUL36. Furthermore pUL36, described as an inner tegument protein, is understood to be the primary bridge between the capsid and tegument layers. The precise site of pUL36 addition to the capsids, in the nucleus or in the cytoplasm, has been the subject of much debate, with the balance of evidence suggesting that it binds capsids in the cytoplasm 20,21 . This is at odds with structural data that show two copies of pUL36 intimately associated with pUL17 and pUL25 to form a 5-helix bundle in the CATC 19 .
pUL17 and pUL25 are definitively added in the nucleus. Our data reveal the presence of the CATC pentaskelion on HSV nucleocapsids in the nucleus, suggesting that capsids bind the tegument protein pUL36 (VP1/2) prior to nuclear egress.

Correlative imaging of re-vitrified sections shows well-preserved ultrastructure
To image viral infectious processes within the cell, we used the vitreous frozen section (VFS) method. Virus infected cells were aldehyde fixed prior to being scraped and pelleted. The cell pellet was embedded in gelatin and cut into small cubes that were infused with sucrose. Embedded cells were then frozen in liquid nitrogen and ~200 nm thick sections were cut at low temperature (-120ºC). Frozen sections were collected on holey carbon film TEM grids and then washed to both warm the sections and remove the sucrose. Sections were then imaged at room temperature in the confocal microscope to identify regions of interest (ROIs), prior to being re-frozen by plunging into liquid ethane. Areas for cryo-ET were selected based on the presence of many capsids and minimal frost contamination and tomograms were recorded of capsids within the cytoplasm and the nucleus (Supplemental video 4). Our images therefore not only indicate good preservation of ultrastructure but also demonstrate the feasibility of using CLEM on re-vitrified sections to identify macromolecules of interest within the dense complex environment of the cell.

Cellular and capsid 3D ultrastructure revealed by cryo-ET of re-vitrified sections
The inner tegument protein pUL36 of HSV1 plays a vital role in virion assembly, however the site of its recruitment to the capsid, if carried out within the nucleus or outwith is unclear and has been a topic of much debate. To confirm this we imaged refrozen vitreous sections of cells infected with the mutant FRΔUL37 -capsids that lack the pUL37 tegument protein, but have pUL36. Cryo-EM images showed excellent preservation of cell ultrastructure combined with minimal cutting artefacts.
At low magnification the nuclear membrane was readily seen, clearly demarcating the nucleus from the cytoplasm (Figure 2a-b).

3D structure of the intranuclear HSV capsids by subtomogram averaging
To establish whether tomograms of refrozen cell section material are sufficient to determine 3D structures by sub-tomogram averaging, we sought to calculate in-situ reconstructions of HSV-1 capsids.
The inner tegument proteins pUL36 and pUL37 of HSV-1 play a critical role in virion assembly [22][23][24] . There has been uncertainty in the HSV field of how pUL36 and pUL37 are recruited to the capsid, if this happens within the nucleus or after nuclear egress 20,21 .
To shed light on this question, we carried out cryo-ET on the mutant lacking pUL37 (FRΔUL37). As capsids within the nucleus were discrete and well separated ( Figure   3a-b), we performed subtomogram averaging to determine the 3D structures of the  3D structures of the three types of nuclear capsids, A-, B-and C-type were determined by subtomogram averaging. 2D cross-section images clearly indicate density differences in the interior of the capsid between the three types. Density relating to scaffolding proteins and DNA is seen inside the capsids within the B-type and C-type, respectively, while none is present inside the empty capsid shell of the A-type. The 3D structures obtained have been radially coloured at low thresholds of density and are shown along the 5-fold axis of icosahedral symmetry. Although additional pentaskelion density is observed above the pentons in both B and C-type capsids, it is more pronounced in the latter.
In recent high-resolution single particle reconstructions of capsids within purified HSV-1 virions, the pentaskelion structure can be clearly seen above the penton vertices and has been attributed to the proteins pUL17, pUL25 and pUL36 16,19 .
Although at low-resolution, the star-shaped density observed in our C-capsid structure is strongly reminiscent of the penton density in the high-resolution structures determined from purified HSV-1 virions ( Figure 5). and is comparable to the (b) high-resolution structure of purified capsids from within the nucleus 16 . Capsids are radially coloured from the centre.
The CATC density was hardly visible in the B-capsids. We attribute this to low occupancy of CATC in this class of capsids, resulting in its signal being lost during particle and symmetric averaging. The CATC density is completely absent in the Acapsids.
Focussed classification has recently emerged as a means to obtain structural data pertaining to low-occupancy, unique and asymmetric features that are otherwise lost owing to particle averaging upon imposition of higher order symmetry during computational processing 25 . We successfully implemented this method and determined the structure of the unique portal vertex in purified HSV-1 virions 16 . To assess if we might similarly reveal asymmetric features such as the HSV-1 portal or additional density at the penton vertices due to low-occupancy, we carried out focussed classification on a single 5-fold vertex of the 3D in situ structures of the capsids (A, B and C-type) obtained from subtomogram averaging. To our knowledge this is the first attempt to implement this method on sub tomograms. Focussed classification allows for expansion of symmetry, where each virus particle that would normally have a single orientation defined relative to the asymmetric unit, will now encompass 60 symmetry-related orientations of the icosahedral capsid. Thus, each five-fold vertex may be individually masked and classified. Although a total of 10 classes were calculated, only 1 class was identified as containing a distinct star-shaped structure in B-and C-capsids, corresponding to CATC density ( Figure 6, Supplementary Fig. S3). None was observed in the classes calculated from the A-capsids (data not shown).
The pentaskelion density in B-capsids is more prominent than C-capsids; likely owing to far greater numbers of B-capsids (526) used during processing than of C-capsids (125). These data support our suggestion that low occupancy of CATC on B-capsids led to weaker density in icosahedrally averaged density maps. They are clearly visible upon asymmetric reconstruction ( Figure 6), but not during symmetric reconstruction ( Figure 4). Focussed classification of all three capsid subgroups (A, B and C) however did not reveal any structure pertaining to the portal. This is likely owing to low overall numbers of particles used for the calculations.

Discussion
Cryo-EM is fast becoming a critical tool for structural biologists, allowing atomicresolution structures of proteins and viruses to be calculated rapidly 2 . In this work, we have addressed the main bottleneck of obtaining 3D structures of macromolecular assemblies from within the native context of the cell under cryo conditions. By adapting the method of classical Tokuyasu sectioning coupled with CLEM and subtomogram averaging, we offer an easy and attractive alternative for imaging and 3D structure determination of proteins/viruses from within cells 9 . The Tokuyasu method has been previously used to produce sections that were subsequently vitrified for cryo-EM imaging 10 . However, the use of vitrified frozen sections (VFS) has thus far been limited to imaging tissue sections by cryo-EM and cryo-ET 10-12 .
We believe our method offer a less technically challenging and relatively inexpensive alternative for the majority of cryo-EM users to carry out 3D structural characterisation of macromolecular assemblies in cellular and tissue samples. Further, the option of using CLEM on these sections offers the great advantage of precisely locating sub-cellular structures of interest, broadening the applicability of this method to the study of low-frequency events. 3D structure determination of macromolecular assemblies from re-frozen sections via subtomogram averaging provides a starting point to more routine high-resolution structure determination of proteins from within the cell.
The use of chemical fixative may cause some structural artefacts, possibly contributing to the low resolution of capsid structures in our study (5-6 nm), in comparison to resolutions obtained from subtomogram averaging of proteins from unfixed cryo-ET of for example purified virions (0.8-2 nm). In the absence of comparable data from FIB-SEM prepared nuclear capsids, it is not possible to directly compare achievable results and thereby unambiguously assign reasons for the low resolution achieved here.
In this study we chose the highly symmetric and well characterized HSV capsid as a model to test the feasibility of our method to determine the in situ 3D structures of intracellular capsids by subtomogram averaging. Employing a correlative approach using CLEM, allowed us to spatially locate capsids within both the nucleus and cytoplasm. Well-preserved fluorescence signal as well as ultrastructure within the sections observed was consistent with previously published work describing correlative imaging of refrozen Tokuyasu sections 13 .
A complex variable proteinaceous tegument layer, comprising more than 20 proteins surrounds the HSV capsid. Of these, pUL36 and pUL37 are classified as 'innertegument' proteins and are tightly associated with the capsid. The precise location of pUL37 has not been determined. pUL36 is intimately associated with pUL25 and pUL17 to form the CATC at the five-fold vertices [26][27][28] . With the pUL37-null mutant used in this work, accumulation of C-capsids in dense clusters was observed in the cytoplasm of infected cells, confirming that the tegument protein pUL37 is not important for capsid assembly, DNA packaging or nuclear egress, but plays a vital role in tegument addition within the cytoplasm and envelopment. Despite extensive studies on HSV-1 capsid morphogenesis, controversy persists concerning the cellular compartment in which the tegument protein pUL36 is added to the nascent virus capsid. The CATC density observed at capsid five-fold vertices in cryo-EM studies of mature virions, includes a five-helix bundle that is the principal site of interaction for two copies each of pUL36 and pUL25 along with a single copy of pUL17 20, 26, 28-31 . The intranuclear capsid structures obtained from subtomogram averaging here clearly indicate variable pentaskelion CATC density over the pentons between the three subgroups of capsids; B-capsids having weak density compared to C-capsids, likely a consequence of low occupancy of CATC in B-capsids. Upon performing focussed classification for both B-and C-capsids we revealed the presence of CATC density at the penton vertex, comparable to previously reported high-resolution structures of capsids within purified HSV-1 virions 16,19 . Although the presence of putative pUL36 in the pentaskelion density of these low-resolution capsid structures cannot be confirmed, our data strongly argue for a fully assembled CATC prior to nuclear egress. We cannot rule out the proposed incorporation of N-terminally truncated forms of pUL36 however [32][33][34] .
In summary, we have demonstrated a relatively inexpensive and technically straight-

Author Contributions
The CVR has adopted the CRediT taxonomy (https://casrai.org/credit/). Author's contribution is as follows. SV: Conceptualization, Project administration,

Cryo EM/ET and tomogram reconstruction
Sections imaged by light micoscopy were re-vitrified and prepared for cryogenic transmission electron microscopy using an FEI Vitrobot Mk IV. Briefly, 6-8 μl of 15 nm colloidal gold (1:3 dilution, BBI, UK) was applied to the finder grids with sections and blotted for 5 seconds before plunging into liquid ethane 36 . Vitrified samples were viewed at low-temperature (around 98 K) and under low electron dose conditions using a JEOL 2200 FS cryo-microscope operated at 200 KV with zero-loss energy filtered imaging using a slit-width of 30 eV. Samples were held in a Gatan 914 high-tilt cryo-stage. CEM images were typically recorded at zero tilt at low magnifications of 100X-200X as well as medium magnifications of 6000X-8000X using the Gatan Ultrascan 4k × 4k CCD camera to determine the same ROIs and enable superposition with light microscopy images. Tilt series were then recorded using the SerialEM software package 37 at 10,000 × magnification on a Direct-Electron DE20 DDD camera at a sample rate of 5.98 Å/pixel. Images were acquired at twodegree increments with a bi-directional tilt scheme ranging from −20° to +60° for the first tilt set followed by -20° to -60° for the second tilt set. The target defocus was set between 4-10 µm and the electron dose ranged from 50-100 e/Å 2 per tilt-series.
Tomograms were calculated and visualized using the IMOD software package 38 .
Reconstruction was performed using weighted back projection followed by CTF correction available as part of the IMOD package.

Registration of LM and EM images
Alignment of light and electron microscopy images was done with Adobe Photoshop CS5 software. Firstly, the LM image was aligned based on the markings of the finder grid, manually inserted landmarks such as ice contamination and the nuclear DAPI signal and their corresponding signals on the cryo-EM image. After rough alignment, a fine alignment was performed by registering the pattern of holes within the grid square and/or the pattern of cells and their corresponding signals on the cryo-EM image. For data presentation, the light and electron microscopy images were merged with the merge channels option in Adobe photoshop CS5.

Computational subtomogram image processing
Micrograph movies containing 20 frames were processed to calculate a 3D reconstruction using Relion-3.1 39 . Initially movies were corrected for specimen movement and radiation damage using Direct-Electron open source software scripts (DE_combine_references.py and DE_process_frames.py). Processed images were stacked using in-house scripts and tomograms generated with defocus estimation (Ctfplotter) using IMOD 38 . An initial set of 778 particles from 33 tomograms of nuclear capsids was manually picked and extracted using the EMAN program e2boxer.py 40 . Extracted particles were grouped separately based on them being A, B or C-type capsids prior to classification. 3D classification was performed in Relion-3.1 imposing icosahedral symmetry. For the 3D classification, a low resolution starting model of the HSV capsid, obtained after processing and refining ~100 manually extracted particles from our tomogram dataset via EMAN2 was used 39,40 . The subvolumes and the starting model were 2x binned to speed up processing. A subset of particles from each of the three subgroups (A, B, and C-type capsids) was selected from the 3D class that yielded the highest resolution and subjected to further refinement. Resolution assessment was done with Relion, using the postprocessing task to mask the density maps and calculate the 'gold-standard' Fourier shell correlation. The final 3D reconstructions of A, B and C-type capsids were visualized in UCSF Chimera, ChimeraX and IMOD 38,41,42 Movies were created using ChimeraX 42 .
Following 3D reconstruction, focused classification was performed on each of the capsid subgroups (A, B and C-type capsids) using Relion-3.1 to identify unique or additional density on the 5-fold vertex 16 . A cylindrical mask was created in SPIDER covering a single 5-fold vertex 43 . Symmetry expansion of the dataset was performed in Relion by creating a new metadata file that comprised of information pertaining to 60 orientations for each virus particle. The dataset was 2x binned to speed up calculations. The subvolumes were subjected to 3D classification with a T value of 5, to reconstruct a single 5-fold vertex, without refining orientations and origins. A total of 10 classes were calculated with one of them identified to have apparent pentaskelion density over the 5-fold axis, corresponding to CATC, in both B-capsids and C-capsids. All visualization was carried out using ChimeraX 42 .