Herpesviruses include many important human pathogens such as herpes simplex virus, cytomegalovirus, varicella-zoster virus, and the oncogenic Epstein–Barr virus and Kaposi sarcoma–associated herpesvirus. Herpes virions contain a large icosahedral capsid that has a portal at a unique 5-fold vertex, similar to that seen in the tailed bacteriophages. The portal is a molecular motor through which the viral genome enters the capsid during virion morphogenesis. The genome also exits the capsid through the portal-vertex when it is injected through the nuclear pore into the nucleus of a new host cell to initiate infection. Structural investigations of the herpesvirus portal-vertex have proven challenging, owing to the small size of the tail-like portal-vertex–associated tegument (PVAT) and the presence of the tegument layer that lays between the nucleocapsid and the viral envelope, obscuring the view of the portal-vertex. Here, we show the structure of the herpes simplex virus portal-vertex at subnanometer resolution, solved by electron cryomicroscopy (cryoEM) and single-particle 3D reconstruction. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex. Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses. Finally, we show that the viral genome is consistently packed in a highly ordered left-handed spool to form concentric shells of DNA. Our data provide new insights into the structure of a molecular machine critical to the biology of an important class of human pathogens.
Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) are important human pathogens. It is estimated that approximately 90% of the world’s population are infected with one or both viruses [1]. HSV-1 is the primary cause of cold sores and HSV-2 of genital herpes. These conditions are both highly contagious, and HSV-2 is amongst the most common sexually transmitted infections. Infection with HSV is lifelong, owing to the ability of herpesviruses to enter a latent state with periodic reactivations [2]. HSV can also cause more serious conditions including keratitis, which may lead to loss of sight [3], and a potentially fatal encephalitis [4]. The herpesvirus family includes many other important human pathogens, such as varicella-zoster virus, the cause of chicken pox and shingles; cytomegalovirus, a notable cause of congenital abnormalities; Kaposi sarcoma–associated herpesvirus, which causes cancer in immune-compromised individuals; and Epstein–Barr virus, the cause of infectious mononucleosis that has also been linked to several cancers. Herpesviruses are large double-stranded DNA viruses, having genomes up to 240 kbp. The viral DNA is packaged in a complex T = 16 icosahedral capsid that is 1,250 Å in diameter [5,6]. The DNA-containing capsid, or nucleocapsid, is embedded in a proteinaceous layer known as the tegument that is in turn surrounded by a host-derived lipid envelope. The viral envelope is studded with glycoproteins that mediate viral attachment and entry. HSV virions enter host cells by fusing their envelopes with the host cell plasma membrane, allowing the nucleocapsid and tegument to enter the cytoplasm [7]. The nucleocapsid traffics along microtubules to the microtubule-organising centre, and from there, to the nucleus [8]. The nucleocapsid then docks to a nuclear pore complex, through which it injects its genome into the nucleus [9,10]. DNA egress from the capsid is through a unique portal-vertex, located at an icosahedral 5-fold symmetry axis. The portal-vertex is also the means by which the viral DNA is packaged into capsids within the nucleus [11]. Virion morphogenesis commences in the nucleus with the formation of the procapsid; an icosahedrally symmetrical spherical shell assembly of capsomeres that are hexamers (hexons) and pentamers (pentons) of the major capsid protein pUL19 (VP5) [12]. Heterotrimers of pUL38 (VP19C) and pUL18 (VP23) —termed triplexes—along with the scaffold protein pUL26, direct procapsid assembly, which nucleates around the dodecameric portal formed by pUL6 [13]. Procapsid maturation—angularisation and expulsion of the scaffold protein—occurs as the viral genome is pumped into the shell [14]. Replication of the viral genome results in formation of a concatemer, from which unit-length genomes are packaged into procapsids by the portal (pUL6) and terminase complex consisting of pUL33, pUL28, and pUL15 [15,16]. It has been noted that herpesviruses are structurally and biologically similar to DNA-containing tailed bacteriophages, and it has been suggested that these two viral groups share a common ancestry. This is based on the observation of fold conservation in the major capsid protein [17] and their similar capsid assembly and DNA-packaging strategies. Sequence analysis also indicates that pUL15 is a homolog of the phage terminase large subunit. By analogy, pUL15 is predicted to have ATPase activity, powering the translocation of the genome into the capsid through the portal, and endonuclease activity cleaving the DNA when a cleavage signal is detected [18]. pUL28 is known to bind viral DNA and is equivalent to the small terminase subunit of bacteriophages [19]. The capsid-associated tegument complex (CATC—previously termed CCSC and CVSC), is composed of pUL17, pUL25, and pUL36 and binds to the triplexes and hexons about the icosahedral 5-fold vertices of mature capsids within the nucleus [5,20–25]. Notably, it has been observed that pUL25 is essential for retention of DNA within the nucleocapsid [26,27]. Moreover, pUL25 has also been shown to be important for genome release [28]. The mature nucleocapsid leaves the nucleus by budding through the nuclear membrane via an envelopment/de-envelopment step [29]. Cytoplasmic capsids acquire further tegument proteins in the cytoplasm and are enveloped by budding into plasma membrane–derived lipid vesicles, from which they are released by exocytosis at the cell surface [30]. The structure of the HSV particle has been the subject of investigation for over 30 years [31], using cryoEM and icosahedral 3D reconstruction to determine the high-resolution features of the nucleocapsid [5,25,32,33] and lower-resolution tomography to investigate the nature of asymmetric features such as the portal-vertex and viral envelope [34,35]. Attempts to resolve the structure of the portal-vertex have been largely unsuccessful, however. This is because the herpesvirus portal-vertex is similar in size and mass to the penton-vertex, unlike bacteriophages, in which the portal-vertex is marked by the presence of a substantial tail assembly. Moreover, in herpes virions, the subtle differences between the portal-vertex and penton-vertices are obscured by the tegument layer. Finally, the high symmetry of the viral capsid dominates attempts to align particle images for asymmetric reconstruction. Here, we show the structure of the portal-vertex of HSV-1 at 8 angstroms resolution, revealed by focussed-classification [36,37] and 3D reconstruction of cryoEM images of purified virions. These data reveal that the usual pUL19 penton is replaced by a unique 5-fold symmetrical assembly. This feature displays five well-defined coiled-coil motifs, each made up of two α-helices, arranged perpendicular to the capsid surface about the 5-fold symmetry axis. It appears to be anchored to the virion by interactions with triplex-like structures that occupy the position normally taken up by peripentonal Ta triplexes, immediately about the 5-fold vertex. The CATC assembly is still present and, similarly to penton associated CATC, is bound to the Tc triplexes, forming a bridge across the periportal triplex-like structures towards the 5-fold axis. We interpret our data as showing that the pUL25 C-terminal domains are positioned differently to those seen at penton-vertices, giving rise to a small tail-like assembly that crowns the unique 5-fold vertex. Strong density was seen to extend through the portal-vertex structures that we interpret as DNA. This suggests that the trailing end of the packaged genome remains engaged in the portal-vertex, ready for release through the nuclear pore. The portal itself is not well resolved owing to a mismatch between the C5 symmetry imposed in calculating our reconstruction and the C12 symmetry of that feature. Finally, our reconstruction also reveals the arrangement of packaged DNA within the virion, which is clearly resolved as a left-handed spool arranged in concentric layers. We provide the highest-resolution view to date of a critical component of the herpesvirus virion. The portal-vertex is a molecular machine responsible for both packaging and release of the viral genome, in one of the most important groups of viral pathogens to infect humans. Furthermore, our focussed classification approach demonstrates the power of modern image-processing algorithms to break the shackles of symmetry that have limited our understanding of virus structural biology for so long.
A relaxed symmetry (C5) reconstruction was calculated yielding a map at a resolution of 7. 7 Å (Fig 1b, S4 Fig, S3 Data). This revealed a uniquely structured capsid-associated tegument assembly at the portal-vertex (Fig 1f–1h, S1 Movie), giving rise to the tail-like features previously described at lower resolution, termed the PVAT [35]. At intermediate resolution, we see that the portal-vertex CATC density closely resembles that seen at the penton-vertices. The five-helix bundle, comprising pUL17, pUL25, and pUL36 is well resolved but is rotated approximately 6° counter-clockwise relative to that of the penton-vertex. In penton-vertices, this part of the CATC is anchored to the capsid by binding to two triplexes, the peripentonal Ta triplex, and the Tc triplex. At the portal-vertex, the CATC still binds to the Tc triplex; however, the Ta triplex is replaced with a globular density that appears almost twice the size of a normal triplex (pink arrow, Fig 1e and 1h). At this resolution, it is unclear whether this feature is made up of a heterotrimer of pUL38 and pUL18, plus an additional component, or whether the triplex has been entirely substituted by another protein. A candidate for this additional protein density is pUL36. This gene product was recently shown to be a component of the CATC, contributing two copies of its C-terminal domain to the five-helix bundle; however, only a small proportion of that large protein has thus far been accounted for (47 of 3,164 amino acids) [5,21]. As well as the novel triplex-like density, we see major differences in the arrangement of the C-terminal domains of pUL25 at the portal-vertex. Peripentonal CATC complexes have two globular densities that bind to penton pUL19: one on top and one to the side of each copy of the major capsid protein (black arrows, Fig 1c). Portal-vertex CATC also shows 10 globular densities that we attribute to the pUL25 C-terminal domains. These are however arranged to form two star-shaped tiers to make up the tail-like PVAT. The distal (outermost) tier being rotated approximately 36° relative to the proximal one (Fig 2, S2 Movie). As was seen in penton vertices of our icosahedral reconstruction and published structures [5,21], the C-terminal domains of pUL25 are less well resolved than the CATC five-helix bundle; in particular, the distal tier is only visible at lower threshold levels and is more clearly seen in unsharpened maps, suggesting that this feature is not rigidly constrained. Sharpening is a process of weighting data across different resolution ranges to compensate for the loss of high-resolution information during imaging and data-processing—effectively, it down-weights low-resolution features to reveal finer details. Sharpened maps are typically shown at a higher isosurface threshold level, however density arising from flexible regions may only present low-resolution features and can disappear in sharpened maps. Atomic coordinates for the CATC five-helix bundle (extracted from PDB 6CGR [5]) and the C-terminal domain of pUL25 (PDB 2F5U [38]) were docked to the PVAT density. The CATC bundle fitted the density well, suggesting that the assembly, although slightly displaced, is not radically different from that at penton vertices (Fig 1h). Given the limited resolution in the density that we attribute to the pUL25 C-terminal domains, however, docking of these coordinates is only shown to illustrate our interpretation; there is insufficient detail to produce a reliable fit (Fig 2, S2 Movie). A central slice through the 5-fold symmetrical reconstruction of the herpes simplex virion reveals the internal features of the portal-vertex and the packaged DNA (Fig 3a, S3 Movie). Lying just inside the capsid shell, we can see poorly defined density that we attribute to the portal protein pUL6 (Fig 3c and 3d). Single-particle 3D reconstruction of this assembly has previously shown it to have cyclic symmetry, forming oligomers ranging from undecamers to tetradecamers. Authentic pUL6 portals assemble as dodecamers [39]. Thus, a symmetry mismatch between this structure and the C5 capsid leads to incoherent averaging and explains the lack of high-resolution features seen in our reconstruction. Nonetheless, we can segment this feature from our density map to highlight its position and gross morphology (Fig 3f and 3g). Running through the centre of the portal-vertex along the 5-fold axis, we see strong density that extends through the portal and up against the inner surface of the PVAT. This is highlighted with a white arrow in Fig 3a and in S3 Movie. We interpret this as being the trailing end of the viral DNA, retained in the portal-vertex and ready to be ejected upon infection of a new host cell. A similar feature has been shown in the bacteriophages Ø29 and Spp1, in which specific interactions between stopper proteins and the DNA hold the end of the DNA within the tail assembly [40,41]. Lying between the portal and the pUL25 PVAT density, a novel 5-fold symmetrical assembly replaces the usual pUL19 penton. This structure is composed of five protein subunits that are largely α-helical. In each subunit, a well-resolved two-helix coiled coil extends radially and is approximately 10 nm in length (Fig 3e). This structure is anchored to the capsid through an interaction with the Ta triplex–like structures that are arranged about the portal-vertex. At this resolution, we see no evidence of an interaction with the major capsid protein. The tapered end of the portal inserts into this assembly; however, owing to the symmetry mismatch, we are unable to identify specific contacts between the two structures. The identity of the pentameric portal-vertex protein is unclear. However, as the locations of all the components of the icosahedral capsid shell and the pUL6 portal protein are known, together with the CATC proteins, pUL17, and most of pUL25, a candidate for this density is also the inner tegument protein, pUL36. As noted above, pUL36 is known to be a CATC component; however, this represents only a small proportion of this large protein, leaving the rest unaccounted for. Another possible candidate for this novel assembly is the terminase protein pUL33. In tailed phages, terminase complexes usually comprise two proteins: a small subunit that mediates binding of the terminase to the viral DNA and a large subunit (having both endonuclease and ATPase activity) that powers the translocation of the genome into the capsid through the portal and then cleaves the DNA when the head is full or a cleavage signal is detected. The HSV-1 terminase is made up of three proteins: pUL15, pUL28, and pUL33 [16]. pUL28 is functionally equivalent to the small terminase subunit [19], and pUL15 shows sequence similarity to the large terminase subunit of phage. The function of pUL33 is rather less well defined. It is predicted to be largely α-helical in structure [42], and thus we do not rule out the possibility that the novel pentameric portal-vertex protein could be pUL33. By analogy to bacteriophage terminase complexes, however, the terminase assembly is expected to detach from the portal following cleavage of the concatamer, remaining associated with the unpackaged DNA, ready to engage the portal in another empty procapsid. Furthermore, proteomics analysis has failed to detect terminase components in purified HSV-1 virions [43]. Further biochemical and structural analysis of isolated nucleocapsids and virions is therefore required to unambiguously identify this protein. We have applied a novel image-processing approach to determining the structure of an important asymmetric feature of the icosahedral herpesvirus nucleocapsid. Our data provide new insights into the organisation and composition of this important molecular machine. The herpesvirus portal-vertex is the unique site through which the viral genome both enters and exits the nucleocapsid. It is therefore a critical assembly in both virion morphogenesis and initiation of infection. We show that the PVAT, a tail-like structure previously described at low resolution, is likely composed of the C-terminal domain of pUL25, a protein known to be critical to DNA retention. We have also described two novel structures. The first, a hitherto unknown pentameric assembly, lays between the portal and PVAT in place of the pUL19 penton and has a clear two-helix coiled-coil structure. The second is a large assembly that occupies the space usually taken up by the Ta triplex in peripentonal vertices. We have suggested possible identities for both structures; however, further biochemical analysis, coupled with higher-resolution structure determination, are required to provide a definitive description. In agreement with previous lower-resolution studies that employed electron tomography, we show that the portal motor, composed of 12 copies of pUL6, lays against the inner edge of the capsid shell. Finally, our data show that HSV-1 genomic DNA is packaged as a left-handed spool arranged in concentric layers. This suggests that the process of genome translocation combined with interactions between the inner surface of the capsid and successive layers of DNA lead to a highly reproducible packing process. This is perhaps not as remarkable as one might initially surmise. Herpesviruses package their genomes to extremely high density. It is critical, then, that the genome should be able to reliably achieve this high density and, with equal reliability, eject the complete genome through the nuclear pore to initiate infection.
Herpes simplex virus was propagated in BHK cells grown in roller bottles containing growth medium (GMEM, 10% FCS, 10% tryptose phosphate broth). Confluent cells were infected with HSV-1 strain 17syn+ at a m. o. i. of 0. 002 pfu/cell. Cells were incubated at 37 °C for 3 days, then harvested by shaking into the medium. Cells and media were centrifuged at 1600× g for 10 minutes to remove cell debris. The supernatant was then transferred to a new tube and spun at 17,000× g for 2 hours. The pellet was then gently resuspended on ice overnight by overlaying with 2 ml GMEM. The resuspended material was moved to a new tube and clarified by spinning at 200× g for 10 minutes. The supernatant was layered onto the top of a 5%–15% Ficoll gradient prepared in GMEM and centrifuged at 26,000× g for 2 hours. The opaque band containing the virions was collected by side puncture of the tube using an 18-gauge needle. Collected virions were diluted in GMEM and pelleted at 40,000× g for 1 hour. The pellet was washed gently in PBS and then allowed to resuspend in 50–100 μl PBS by incubating on ice for at least 1 hour. HSV virions were prepared for cryoEM by plunge freezing into liquid ethane. Four μl of purified virions was loaded onto a freshly glow-discharged holey-carbon support film (R2/2 Quantifoil) in an FEI Vitrobot mk. IV vitrification robot. The grid was immediately blotted for 3 seconds and then plunged into liquid nitrogen–cooled liquid ethane. Grids were imaged at the United Kingdom national cryoEM facility (electron bioimaging centre—eBIC) at Diamond Light Source, Harwell, in an FEI Titan Krios cryotransmission electron microscope at a nominal magnification of 81,000×. Images were recorded as ‘movies’ on a Falcon III camera operated in integrating mode with sampling of 1. 78 Å/pixel. Movies were recorded as 12-second exposures, giving a total of 480 detector frames that were integrated into 40 fractions, with a dose rate of 1. 95 e/Å2/fraction. All image processing was performed using Relion 2. 1 [46] on a GPU workstation running Linux CentOS 7. 3,702 micrograph movies were processed to correct for particle movement using Motioncor2 [47], and the defocus for each motion-corrected micrograph was estimated using GCTF [48]. A small subset of particles was picked and subjected to 2D classification to produce a template for automated particle picking, which yielded a total of 12,431 virion images for further processing. Particles were initially extracted with 2× binning. After 2D classification of this dataset 7,476 particles were selected for ab initio calculation of a starting model, followed by 3D classification (imposing full icosahedral symmetry in both cases). This led to the definition of a final dataset of 6,069 virion images that were taken forward for 3D refinement with full icosahedral symmetry. Once completed, a new dataset of particles with 1. 5× binning was extracted and used to calculate the final icosahedral reconstruction. Following icosahedral reconstruction, focused classification was performed to identify the unique portal-vertices [36,37]. A cylindrical mask was prepared in SPIDER [49] to cover a single 5-fold symmetry axis. A metadata file (STAR file) was generated to expand the symmetry for our dataset, i. e. , for each virion image, 60 orientations were defined, corresponding to the 60 symmetry-related views of the icosahedral object. To speed up the calculation, a new dataset was extracted from the raw micrographs with 5× binning. These data were then subjected to masked 3D classification, with a T value of 20, to reconstruct a single 5-fold vertex and classify the data into self-similar classes. During this process, orientations and origins were not refined. A total of 10 classes were calculated, one of which was identified as containing the unique portal-vertex. To calculate our final C5 symmetric reconstruction, we used 1. 5×-binned particle images. Resolution assessment was performed in Relion, using the postprocessing task to mask the density maps and calculate the ‘gold-standard’ Fourier shell correlation. A B-factor was estimated and applied to each reconstruction [50], which was then interpreted by visualization in UCSF Chimera [51]. Docking of atomic coordinates was performed using the ‘fit model in map’ function of UCSF Chimera. Segmentation was performed using the ‘Segger’ plugin in UCSF Chimera. The C5 reconstruction has been deposited in the EM databank with accession code EMD-4347. Motion-corrected micrographs (raw data) are deposited in EMPIAR with accession code EMPIAR-10189.
The herpesvirus family includes many important human pathogens such as herpes simplex viruses that cause cold-sores and human cytomegalovirus, a major cause of congenital abnormalities. Several herpes viruses are known to cause cancer. Herpes viruses assemble enveloped virus particles (virions) that incorporate a large DNA-containing icosahedral capsid. Virion assembly commences in the nucleus of an infected cell, where the viral genome is pumped into preassembled capsids by a portal motor that is located at a unique 5-fold symmetry axis: the portal-vertex. We have used cryogenic electron microscopy and 3D image reconstruction to solve the structure of the portal-vertex of herpes simplex virus type 1 at 8 Å resolution. Our structure reveals the presence of several previously unknown features including a novel pentameric assembly that exhibits a coiled-coil motif comprising two α-helices. This substitutes the penton, a pentamer of the major capsid protein that is seen at other 5-fold symmetry axes (penton-vertices). Our data allowed us to postulate an identity for the small tail-like structure previously identified at the portal-vertex and termed the portal-vertex–associated tegument (PVAT). Finally, our 3D reconstruction reveals that the viral DNA is packaged within the capsid as a left-handed spool that is arranged in concentric shells. Our data present a structural view of a molecular machine that plays a critical role in the replication cycle of an important family of human pathogens.
herpes simplex virus
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Structure of the herpes simplex virus portal-vertex