ET studies of MHV-68 infected cells were performed at various stages of viral infection. Mock-infected and virus infected fibroblasts (NIH3T3) were fixed by glutaraldehyde at different time points post infection (p.i.), sectioned and visualized in three dimensions. Instead of using ~40 - 70 nm sections common in conventional thin-section 2D TEM, which inevitably cuts the 100 nm-diameter herpesvirus capsid in half and fragments large cellular structures, 200 nm-thick sections were used to enable observations of complete capsid/virion structures, neighboring cellular structures and therefore virus-host interactions in viral infection. Samples were examined using TEM and features of interest were indentified to collect tilt series for 3D construction. In total, 421 single-axis and 63 dual-axis ET data sets were collected. Reconstructed tomograms were further scrutinized to dissect important events in the viral life cycle and selected structures were segmented and colorized in surface rendering for 3D visualization and interpretation.
The major events of the viral life cycle and the cellular compartments where they took place were shown in . A comparison between 3D reconstruction from dual-axis ET and conventional 2D TEM imaging were shown in (an infection-induced intranuclear inclusion body covered by nucleocapsids, see details below): the left panel showed the TEM micrographs; and the right panel showed digital slices extracted from the 3D reconstruction by ET. Overlapped features in 2D images (in ultra-thin sections, the overlap of features still exists, although is less severe than shown here) were clearly resolved in the reconstructed tomogram, indicating the advantage of ET. The angular shape of the nucleocapsid and the viral DNA strands at the beginning of encapsidation visualized in the reconstruction demonstrated the reliability and the resolution of this 3D structural study. Throughout this report, the 1 nm-thick digital slices from reconstructed 3D tomograms were shown in gray-level, 3D structures were shown in color (except for ) or videos (as supplemental materials
) to illustrate various aspects of 3D viral and host structures.
Virus-induced nuclear inclusion bodies
Virus attachment and entry
To visualize initial events of MHV-68 infection, NIH3T3 cells cultured in 37°C were cooled to 4°C to be inoculated with MHV-68 for 2 h (MOI=100). Sections of cells were examined using TEM and areas of interest were further captured by ET for 3D visualization. Reconstructed tomograms revealed that enveloped virus particles and L-particles (virus-like particles comprised of a proteinaceous core without capsid) frequently located in the vicinity of the plasma membranes. A representative tomogram was shown in . Among all cells examined, no virus particle was detected inside cells, suggesting that viral entry was effectively prevented at 4°C and viral attachment was captured in the tomograms.
Viral entry was observed in cells infected at 37°C and fixed at 5 min, 10 min, 20 min, 1 h and 5 h p.i. (MOI=100). Enveloped viral particles were present in endosome-like vesicles inside cytoplasm (). Many virus particles were detected within pits characterized by an electron-dense layer coating the inner surface of the plasma membrane (a representative viral particle was shown in ), resembling clathrin-mediated endocytosis (Doherty and McMahon, 2009
). This was also shown by the 3D colorized surface rendering of an 11 nm-thick digital slab extracted from the tomogram (). Virions and L-particles were also found close to uncoated pits (data not shown). De-enveloped capsids were first observed in cells fixed at 20 min p.i. (). L-particles were found in endosomes-like vesicles inside the cells () indicating that L-particles are entry competent. Notably, no fusion event between the viral envelope and the plasma membrane was seen in any of the cells examined. The results showed that MHV-68 virion enters NIH3T3 cells via endocytosis under the infection conditions used. This observation is consistent with the previous biological data (Gill et al., 2006
Viral DNA injection through nuclear pore complex (NPC) into nucleus
After viral entry, incoming capsids with or without DNA were found at peripheral regions of the nucleus, usually close to nuclear pores. Capsids with viral DNA inside were typically present at larger distances than empty capsids from NPCs. Empty capsids were regularly observed docking in the immediate vicinity of nuclear pores and the absence of highly electron-dense cores (DNA density) suggest that the injections of viral DNA from these particles were completed (). In all tomograms from samples fixed between 20 min and 24 h p.i., 43 capsids in total were found close to nuclear pores in the cytoplasm. Among them, 26 empty capsids were all approximately 40nm from NPCs and 15 capsids with viral DNA inside were all more than 80 nm from NPCs. Interestingly, two capsids with only residual densities of DNA strands both inside and outside of capsids (), which we assumed to be in the process of viral DNA injection, were also at about 40 nm distance from the NPC. The DNA was identified based on its high density and characteristic continuous fibril-shape of 2 - 5 nm in diameter, which is consistent with the diameter of the uranyl acetate stained DNA, 2.5 - 4.0 nm, reported in an SEM study (Inaga et al., 1991
). The results suggest the viral DNA injection occurs within a more restricted distance from the NPC.
Capsids docking at nuclear pore and viral DNA injection
A 3D close-up of the capsid-NPC interaction provided details of the in vivo
viral DNA injection process in herpesvirus infection. A capsid juxtaposed to a nuclear pore with one vertex facing the NPC was shown in . In three 1 nm-thick digital slices from different positions in z-direction, filaments emanating from the NPC were visible () and the viral DNA releasing from the vertex through the NPC was resolved (). A colorized surface rendering () and a supplemental video (Video1)
of the same tomogram illustrated this event in three dimensions.
Filaments emanating from the NPC were observed interacting with all 28 capsids (26 empty and 2 with partial viral DNA) which were about 40 nm from the NPC with one vertex facing the NPC. Therefore, the incoming capsid docked at the nuclear pore possibly through interactions between filaments emanating from the NPC and the capsid; and viral DNA was injected from one vertex (presumably, the “portal”) into the nucleus through the NPC. This observation resembled the in vitro
genome uncoating of HSV-1 capsids following the treatment with trypsin or heating, when the DNA is released as a single double helix at one vertex presumed to be the portal (Newcomb et al., 2007
Assembly of nucleocapsids and encapsidation of viral genome
After viral DNA injection, DNA replication takes place, resulting in the appearance of electron-translucent regions inside the nucleus () where progeny nucleocapsids () were usually observed. We also observed incomplete double-layered spheres with particle radius similar to that of complete capsids () and single-layered spheres with radius similar to that of the scaffolding core (). However, the observation of these particles is rare (only in two tomograms), suggesting that these structures are possible, transient intermediates of capsid assembly.
Maturation of nucleocapsids and encapsidation of viral DNA
The use of dual-axis ET improved the resolving power for filamentous structures and allowed us to identify herpesvirus DNA (based on its density, continuous fibril-shape and diameter of 2 – 5 nm as mentioned above) in the process of being packaged into nascent capsids. Capsids at different stages of DNA encapsidation were detected throughout infected nuclei of cells fixed at 12 h p.i. (MOI=50), 24 h p.i. (MOI=5) and 36 h p.i. (MOI=1). After carefully examining about 200 nucleocapsids, we classified them into six groups and a representative capsid from each group was presented in both 1 nm-thick digital slice and 3D surface-rendered illustration (). A sphere of scaffolding protein existed inside the freshly assembled capsid (pro-capsid) () in accordance with previous studies (Newcomb et al., 1996
; Tatman et al., 1994
). The capsid encapsidating DNA appeared to be more spherical in shape (compare ; also see ). Based on the amount of DNA and the presence or absence of scaffolding protein inside the capsids, we sorted the six groups of capsids and proposed two possible DNA encapsidation mechanisms: DNA encapsidation could take place in both capsids without scaffolding protein density () and capsids with deformed scaffolding protein ().
In the former case (without scaffold), the viral genome entered the capsid from a vertex (the putative portal) and started filling in a random pattern (). As the process went on, more DNA entered and the DNA strand started to line the interior surface of the capsid in circles organized around the “portal” axis (). A section through the axis of the “portal”, shown in , revealed dotted densities decorating the internal surface of the capsid, which we interpreted as cross sectioned DNA strands. In the latter case (with scaffold), deformed scaffolding protein was observed inside the capsid as an elongated sphere (). Circularly organized DNA was found inside the capsids with less electron-dense cylindrical shapes in the middle (). The circular strands of DNA were arranged coaxially with the cylinders that were usually hollow in the middle. In light of the scaffolding protein observed in , we attribute the cylinder density to deformed scaffolding core possibly immediately after the cleavage of its link to the major capsid protein by the viral protease because, to our knowledge, there are no other candidate proteins present in the capsids at this stage. It is noteworthy that at the end of encapsidation, highly electron-dense DNA was organized in the circular pattern described above and no identifiable less-electron dense cylindrical density existed (). A video (supplemental Video2
) was provided to illustrate 3D structures of these possible intermediates stages of DNA encapsidation.
These data provide a 3D observation of herpesvirus encapsidation and suggest that viral DNA may enter the capsid either concurrently with or after the removal of scaffolding. At the end of the encapsidation, there is no scaffolding protein left and the viral genome was packaged into a spool with circles of DNA strands organized coaxially with the “portal”.
Virus-induced intranuclear inclusion bodies
In sections of infected cells harvested at 12 h p.i. (MOI=50), 24 h p.i. (MOI=5) and 36 h p.i. (MOI=1), electron-dense spherical inclusion bodies were observed frequently inside infected nuclei (). The ET reconstructions revealed 3D architectures of these inclusion bodies for the first time and showed that many of them contained nucleocapsids at different stages of maturation at their surfaces (). The sizes of inclusion bodies and the numbers of capsids covering them varied. A low magnification micrograph of an infected cell () showed an inclusion body (about 2 μm in diameter) cross-sectioned through the middle with nucleocapsids surrounding it. Another micrograph () showed the top section of an inclusion body with the capsids present throughout its surface. 1 nm-thick digital slices from reconstructed tomograms () and shaded surface views of entire sections () showed detailed interior architectures and overall morphologies of each inclusion body: both of them are composed of amorphous material as electron-dense as the proteinous capsid shell, but less electron-dense than the DNA cores in the capsids; holes exist inside the first one () and fewer capsids cover it; there are no holes in the second inclusion body (), but capsids are observed at a higher packing density and are present in multi-layers on its surface.
Primary envelopment, invaginations of nuclear membrane(s) and nuclear egress
Enveloped capsids were found in the perinuclear space (PNS) between two nuclear membranes in cells harvested at 12 h p.i. (MOI=50), 24 h p.i. (MOI=5) and 36 h p.i. (MOI=1) (). After assembly and DNA packaging, capsids budded through the inner nuclear membrane (INM) () resulting in the formation of enveloped viral capsids in the PNS (). This process is called primary envelopment (reviewed in Mettenleiter, 2002
). Interestingly, the region on the INM engulfing the capsid () and the primary envelope of the capsid () were thicker and more electron-dense than regular regions of the INM. The envelope is smooth without any spike-like protrusions on the surface. The fusion between the primary envelope and the outer nuclear membrane (ONM) was observed, which served as a de-envelopment process and resulted in the release of unenveloped nucleocapsid into the cytoplasm. As shown in , two primary enveloped viral capsids were seen in the PNS and the envelope of the left one appeared to be fusing with the ONM.
We found that MHV-68 infection induced pronounced invaginations of the INM (). Primary enveloped nucleocapsids usually filled the spaces enclosed by the INM invaginations, as shown in . The arrangements of capsids inside the invaginations were either in layers of ordered array pattern if the space were fully filled () or in rather random patterns if fewer capsids were enclosed (). In addition, less frequently, double-membrane bounded structures were observed within infected nuclei. As shown in , enveloped capsids were seen in the lumen between the two membranes and unenveloped capsids were located in double-membrane bounded regions; ribosomes inside the structures revealed the cytoplasmic origin of the contents enclosed by double membranes. Notably, a NPC-like feature was found on the bounding membranes of the structure showed in . The above observations indicate that those structures are invaginated forms of both nuclear membranes. Another such invagination with two nuclear pores was illustrated in . Three 1 nm-thick slices from the top (), middle () and bottom () of this structure revealed structural details, such as the pit-like structure formed by invaginated membranes (), the connectivity between the contents of the pit and the cytoplasm () and ribosomes inside the pit (). A video demonstrating this double-membrane invagination in three dimensions was provided as the supplemental Video3
. Both single- and double-membrane invaginations were observed either close to the edge () or in the interior () of the nucleus on sectioned samples due to different sectioning orientations. Neither type of invagination was observed in mock infected cells. These observations suggest that nuclear membrane(s) invaginations are induced by viral infection and involved in nuclear egress of progeny capsids.
Tegumentation and secondary envelopment in the cytoplasm
In the cytoplasm of cells harvested at 12 h p.i. (MOI=50), 24 h p.i. (MOI=5) and 36 h p.i. (MOI=1), DNA-containing (i.e., C-capsids) and a few empty (i.e., A-capsids) capsids were observed inside electron-dense deposits that were attributed to tegument proteins (). The capsids were undergoing the tegumentation process, through which they acquired their tegument proteins (reviewed in Mettenleiter, 2002
). This process usually occurred close to Golgi-derived membrane structures (trans-Golgi networks, TGNs) of various sizes (). Tegumented capsids budded into TGNs resulting in complete enveloped virions inside exocytic vesicles (). This process is called secondary envelopment (reviewed in Mettenleiter, 2002
). Each exocytic vesicle could package a single () or multiple () virions; and each virion could contain a single or multiple capsids (). L-particles were also detected in exocytic vesicles (). Spike-like protrusions attributable to glycoproteins were present on bilayer envelopes of all kinds of particles at this stage ().
Tegumentation, secondary envelopment in cytoplasm and extracellular viral particles
Notably, the tegument proteins within the virions had two distinctive layers, consistent with previous observations of purified MHV-68 virions (Dai et al., 2008
). All virions contained an inner, thin spherical layer surrounding the capsid (); and a variable amount of amorphous, mostly asymmetric outer layer was also observed between the inner tegument layer and the envelope ().
Egress and extracellular virions
In MHV-68 infected NIH3T3 cells harvested at late stages of infection (24 h p.i., MOI=5 and 36 h p.i. MOI=1), exocytic vesicles containing mature virions were frequently observed in the vicinity of the plasma membrane () consistent with the notion that other herpesviruses (HSV, PRV & HCMV) egress through exocytosis pathway (reviewed in Mettenleiter, 2004
; Mettenleiter et al., 2006
). Several kinds of extracellular viral particles, including mature virions (with tegumented DNA-filled capsid) (), replication-defective particles (with tegumented A-capsid) () and L-particles (data not shown) were observed in the peripheral regions of cells. In accordance with the observations of enveloped virions in exocytic vesicles, different amounts of amorphous out layer tegument proteins were also found within extracellular virions (data not shown). The architecture of the mature extracellular virion was indistinguishable from that of the secondary enveloped virus particle before cellular egress: they were both composed of a bilayer envelope with spikes, a capsid with viral DNA and tegument densities in between.