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Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures., Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid.

&ldquo, Giant&rdquo, phages have genomes of &gt, 200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them &ldquo, mottled capsids&rdquo, Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a &ldquo, twisted hook&rdquo, conformation. Large patches of a proteinaceous tetragonal lattice with a 67 &Aring, repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids—which develop into infectious virions—are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation damage was used to localize internal proteins of HSV-1, yielding insights into how capsid maturation is regulated. The scaffolding protein, which forms inner shells in the procapsid and B capsid, is exceptionally bubbling-prone. In the mature DNA-filled C capsid, a previously undetected protein was found to underlie the icosahedral vertices: this is tentatively assigned as a storage form of the viral protease. We also observed a capsid species that appears to contain substantial amounts of scaffolding protein as well as DNA, suggesting that DNA packaging and expulsion of the scaffolding protein are coupled processes.

Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions., IMPORTANCE On its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate—the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.

A growing number of reports indicate the frequent presence of DNA sequences and gene products of human cytomegalovirus in various tumors as compared to adjacent normal tissues, the brain tumors being studied most intensely. The mechanisms underlying the tropism of human cytomegalovirus to the tumor cells or to the cells of tumor origin, as well as the role of the host’s genetic background in virus-associated oncogenesis are not well understood. It is also not clear why cytomegalovirus can be detected in many but not in all tumor specimens. Our in silico prediction results indicate that microRNA-34a may be involved in replication of some human DNA viruses by targeting and downregulating the genes encoding a diverse group of proteins, such as platelet-derived growth factor receptor-alpha, complement component receptor 2, herpes simplex virus entry mediators A, B, and C, and CD46. Notably, while their functions vary, these surface molecules have one feature in common: they serve as cellular entry receptors for human DNA viruses (cytomegalovirus, Epstein-Barr virus, human herpes virus 6, herpes simplex viruses 1 and 2, and adenoviruses) that are either proven or suspected to be linked with malignancies. MicroRNA-34a is strictly dependent on its transcriptional activator tumor suppressor protein p53, and both p53 and microRNA-34a are frequently mutated or downregulated in various cancers. We hypothesize that p53 – microRNA-34a axis may alter susceptibility of cells to infection with some viruses that are detected in tumors and either proven or suspected to be associated with tumor initiation and progression.

Herpes simplex virus 1 (HSV-1) was shown to contain catalase, an enzyme able to detoxify hydrogen peroxide by converting it to water and oxygen. Studies with a catalase inhibitor indicated that virus-associated catalase can have a role in protecting the virus from oxidative inactivation. HSV-1 was found to be more sensitive to killing by hydrogen peroxide in the presence of a catalase inhibitor than in its absence. The results suggest a protective role for catalase during the time HSV-1 spends in the oxidizing environment outside a host cell.

In the final stages of the herpes simplex virus 1 (HSV-1) life cycle, a viral nucleocapsid buds into a vesicle of trans -Golgi network (TGN)/endosome origin, acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing a mature virion. Although the process of directed egress has been studied in polarized epithelial cell lines, less work has been done in nonpolarized cell types. In this report, we describe a study of HSV-1 egress as it occurs in nonpolarized cells. The examination of infected Vero cells by electron, confocal, and total internal reflection fluorescence (TIRF) microscopy revealed that HSV-1 was released at specific pocket-like areas of the plasma membrane that were found along the substrate-adherent surface and cell-cell-adherent contacts. Both the membrane composition and cytoskeletal structure of egress sites were found to be modified by infection. The plasma membrane at virion release sites was heavily enriched in viral glycoproteins. Small glycoprotein patches formed early in infection, and virus became associated with these areas as they expanded. Glycoprotein-rich areas formed independently from virion trafficking as confirmed by the use of a UL25 mutant with a defect in capsid nuclear egress. The depolymerization of the cytoskeleton indicated that microtubules were important for the trafficking of virions and glycoproteins to release sites. In addition, the actin cytoskeleton was found to be necessary for maintaining the integrity of egress sites. When actin was depolymerized, the glycoprotein concentrations dispersed across the membrane, as did the surface-associated virus. Lastly, viral glycoprotein E appeared to function in a different manner in nonpolarized cells compared to previous studies of egress in polarized epithelial cells; the total amount of virus released at egress sites was slightly increased in infected Vero cells when gE was absent. However, gE was important for egress site formation, as Vero cells infected with gE deletion mutants formed glycoprotein patches that were significantly reduced in size. The results of this study are interpreted to indicate that the egress of HSV-1 in Vero cells is directed to virally induced, specialized egress sites that form along specific areas of the cell membrane.

The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation., IMPORTANCE This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit “protomers” are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.

Commentary on Ge, P.; Tsao, J.; Schein, S.; Green, T.J.; Luo, M.; Zhou, Z.H. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 2010, 327, 689–693.

Ejection of DNA from the capsid is an early step in infection by all herpesviruses. Ejection or DNA uncoating occurs after a parental capsid has entered the host cell cytoplasm, migrated to the nucleus, and bound to a nuclear pore. DNA exits the capsid through the portal vertex and proceeds by way of the nuclear pore complex into the nucleoplasm where it is transcribed and replicated. Here, we describe use of an in vitro uncoating system to determine which genome end exits first from the herpes simplex virus 1 capsid. Purified DNA-containing capsids were bound to a solid surface and warmed under conditions in which some, but not all, of the DNA was ejected. Restriction endonuclease digestion was then used to identify the genomic origin of the ejected DNA. The results support the view that the S segment end exits the capsid first. Preferential release at the S end demonstrates that herpesvirus DNA uncoating conforms to the paradigm in double-stranded DNA bacteriophage where the last end packaged is the first to be ejected. Release of herpes simplex virus 1 DNA beginning at the S end causes the first gene to enter the host cell nucleus to be alpha4, a transcription factor required for expression of early genes.

All herpesviruses have a layer of protein called the tegument that lies between the virion membrane and the capsid. The tegument consists of multiple, virus-encoded protein species that together can account for nearly half the total virus protein. To clarify the structure of the tegument and its attachment to the capsid, we used electron microscopy and protein analysis to examine the tegument of herpes simplex virus type 1 (HSV-1). Electron microscopic examination of intact virions revealed that whereas the tegument was asymmetrically distributed around the capsid in extracellular virions, it was symmetrically arranged in cell-associated virus. Examination of virions after treatment with nonionic detergent demonstrated that: (i) in extracellular virus the tegument was resistant to removal with Triton X-100 (TX-100), whereas it was lost nearly completely when cell-associated virus was treated in the same way; (ii) the tegument in TX-100-treated extracellular virions was asymmetrically distributed around the capsid as it is in unextracted virus; and (iii) in some images, tegument was seen to be linked to the capsid by short, regularly spaced connectors. Further analysis was carried out with extracellular virus harvested from cells at different times after infection. It was observed that while the amount of tegument present in virions was not affected by time of harvest, the amount remaining after TX-100 treatment increased markedly as the time of harvest was increased from 24 h to 64 h postinfection. The results support the view that HSV-1 virions undergo a time-dependent change in which the tegument is transformed from a state in which it is symmetrically organized around the capsid and extractable with TX-100 to a state where it is asymmetrically arranged and resistant to extraction.

Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358.

The herpes simplex virus type 1 (HSV-1) portal is composed of a dodecamer of UL6 protein molecules whose incorporation into the capsid is mediated by interaction with the HSV-1 UL26.5 scaffold protein. Previous results with an in vitro capsid assembly assay demonstrated that nine amino acids (amino acids 143 to 151) of the UL26.5 protein are required for its interaction with UL6 and for incorporation of the portal complex into capsids. In the present study an HSV-1 mutant, bvFH411, was isolated and contained a deletion that removed the codons for UL26.5 amino acids 143 to 150. The mutant virus failed to produce infectious virus in noncomplementing cells, and only B capsids that contained only minor amounts of portal protein were made. These data corroborate our previous in vitro studies and demonstrate that amino acids 143 to 150 of UL26.5 are required for the formation of portal-containing HSV-1 capsids.

Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the twelve vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid. From a cryo-EM reconstruction of capsids immuno-gold-labeled with anti-UL6 antibodies, we confirmed that UL6 resides at a vertex. To visualize the portal in the context of the assembled capsid, we used cryo-electron tomography to determine the three-dimensional structures of individual A-capsids (empty, mature capsids). The similarity in size and overall shape of the portal and a UL19 pentamer - both are cylinders of ~ 800 kDa - combined with residual noise in the tomograms, prevented us from identifying the portal vertices directly; however, this was accomplished by a computational classification procedure. Averaging the portal-containing subtomograms produced a structure that tallies with the isolated portal, as previously reconstructed by cryo-EM. The portal is mounted on the outer surface of the capsid floor layer, with its narrow end pointing outwards. This disposition differs from that of known phage portals in that the bulk of its mass lies outside, not inside, the floor. This distinction may be indicative of functional divergence at the level of portal-related functions other than its role as a DNA channel.

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.

DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.

Methods of electron cryomicroscopy and three-dimensional image reconstruction have been employed to determine the structure of the herpes simplex virus (HSV-1) capsid at 8.5 A resolution (29, 34). The capsid has been found to be an icosahedral protein shell approximately 125 nm in diameter, 15 nm thick, and composed primarily of VP5 (UL19). The structural components include 162 capsomers (150 hexons and 12 pentons) that are arranged on a T=16 icosahedral lattice. Hexons form the edges and faces of the capsid and are each composed of six VP5 molecules. In contrast, one penton is found at each of the 12 vertices. Eleven of the pentons are VP5 pentamers, while the last vertex is occupied by the portal complex. Adjacent capsomers are linked together in groups of three by triplexes, heterotrimeric complexes composed of one molecule of VP19C and two molecules of VP23. The portal complex is composed of 12 UL6 molecules, which form a ring (23) similar in structure and dimensions to the portals found in the capsids of double-stranded DNA bacteriophages, such as P22 (5), φ29 (9, 30), and SPP1 (26). Like bacteriophage portals, the HSV-1 portal functions as a channel through which the viral DNA enters the preformed capsid (12, 17). The mechanism for introduction of DNA into the HSV-1 capsid is also thought to resemble the mechanism found in bacteriophages, with the portal and the terminase subunits (UL15 and UL28 in HSV-1 [1, 2, 33]) forming a packaging “machine” (4, 7). In addition to the structural proteins, HSV-1 capsid assembly requires the action of a scaffolding protein. Two such proteins have been identified, UL26 and UL26.5. Of these, UL26.5 is found to be the predominant species present in infected cells (25). UL26.5 interacts directly with VP5 and is thought to drive capsid formation by bringing the major capsid molecules together through a mechanism of scaffold self-interaction (12). The regions of UL26.5 required for interaction with VP5 (13, 31), as well as the regions required for intermolecular self-interaction (27, 28), have been identified and shown to be critical for capsid formation. Although the scaffold plays a central role in capsid assembly, no scaffolding protein is found within the mature capsid or the virion. As the procapsid matures, the VP5 binding region (the C-terminal 25 amino acids) of the scaffold is removed by the viral protease (UL26), and the released scaffold is lost from the capsid interior (35). Recently, an interaction between UL26.5 and the portal has been demonstrated by in vitro experiments with purified proteins (24). In addition, evidence suggests that the HSV-1 scaffold-portal interaction plays an important role in recruitment of the portal into the capsid as it is formed. Experiments have shown that the drug WAY-150138 blocks assembly of the portal into HSV-1 capsids, presumably by interfering with the ability of scaffold to interact with the portal (20). To further characterize the role of the scaffolding protein in capsid assembly, we have begun to map the specific regions within the scaffold that are required for interaction with the portal. Deletions in the 329-amino-acid (aa) UL26.5 protein were created and tested for their ability to bind the portal.

Hemagglutinin (HA) is a glycoprotein of influenza virus which mediates fusion of the viral and host membranes. HA binds the virus to a target cell, allowing the virus to be endocytosed. The low-pH environment of the endosome then triggers conformational changes in HA, which cause fusion. The viral genome enters the cytoplasm, and infection proceeds. HA is the most extensively studied viral fusion protein and therefore serves as a paradigm for enveloped virus fusion. Following synthesis and trimerization, each member of the HA homotrimer is cleaved to two subunits, HA1, containing the sialic acid binding site, and HA2, containing the N-terminal fusion peptide and the C-terminal transmembrane domain. Cleavage primes HA for transition from the native, metastable state to the final, lowest-energy state upon acidification (8). The structures of HA in the native form and a fragment of the low-pH form have been solved by X-ray crystallography (Fig. ​(Fig.1)1) (6, 9, 31). In the native state, HA2 trimerizes through the formation of a parallel coiled coil. At the N terminus of the native coiled coil (Fig. ​(Fig.1A,1A, yellow), a loop region (HA2 55-75) (Fig. ​(Fig.1A,1A, dark blue; designated B region in reference 6) connects to a second α-helix (Fig. ​(Fig.1A,1A, green), which runs antiparallel to the first. At the N terminus of the second α-helix is the fusion peptide (Fig. ​(Fig.1A,1A, red), which is buried in the center of the native trimer. The three HA1 subunits (Fig. ​(Fig.1A,1A, gray) cover the HA2 subunits, making trimeric contacts and acting as a clamp. In the fragment of low-pH-treated HA, in which the fusion peptide, transmembrane domain, and most of HA1 have been removed, the structure of HA2 is quite different (Fig. ​(Fig.1B).1B). The B loop region (dark blue) has refolded into an α-helix, connecting the two original helices of the HA2 ectodomain into one coiled coil, with the fusion peptide (red) at its extreme N terminus. This conformational change is referred to as the spring-loaded conformational change (7). Dramatic changes also occur at the C-terminal end of the original coiled coil. A region of six amino acids has unfolded to a loop (purple), and the helix C-terminal to the new loop has flipped to lie antiparallel to the coiled coil (orange). The crystal structure of a recombinant, slightly longer form of HA2 (Fig. ​(Fig.1B)1B) shows that the region between the end of this helical hairpin structure and the transmembrane domain forms an extended chain that lies in the groove between helices of the N-terminal parallel coiled coil (Fig. ​(Fig.1B,1B, light blue). Hence, in the final low-pH state (Fig. ​(Fig.1B),1B), the transmembrane domain lies near the fusion peptide (9). FIG. 1. Location of mutants and model of disrupted coiled coil. (A) Structure of the native HA ectodomain (31) (Protein Data Base [PDB] accession no. 2HMG). A detail of the relevant region of HA2 is boxed. (B) Structure of E. coli-produced HA2 (EHA2) (9) (PDB ... The differences in structure between the native and low-pH forms of HA suggest a mechanism for fusion in which the head groups (Fig. ​(Fig.1A,1A, gray) separate and then the spring-loaded conformational change occurs, moving the fusion peptides toward the target membrane. The fusion peptides embed in the target membrane, and the helix-to-loop conformational change then pulls the fusion peptide, and therefore the attached target membrane, toward the transmembrane domain (14, 29; see also the White laboratory website). Similar models have been advanced for other viral and cellular fusion proteins that contain coiled coils. Several sets of data have, however, been used to argue against the importance of the spring-loaded conformational change. First, head group separation was not detected when low pH was applied at low temperatures (0°C), even though fusion could occur under these conditions (26). Furthermore, fusion at higher temperatures was shown to occur before head group separation was detected by electron microscopy (EM) (25). Because it seems apparent that some head group separation is required for complete coiled-coil formation, it was argued that fusion occurs prior to the spring-loaded conformational change. These and other lines of evidence led to alternate models for HA fusion which do not require the spring-loaded conformational change (3, 5, 26) (see Discussion). We previously showed that a mutant containing proline at residue 55 of the B loop was impaired for fusion, and mutant V55P/S71P, with two B-loop mutations, displayed no fusion (21). Although these findings supported the importance of the spring-loaded conformational change, our prior study did not directly address coiled-coil formation and did not analyze critical conformational changes in HA2. Hence, the root cause for the fusion defects was not uncovered. Because of these limitations and because the role of the spring-loaded conformational change remains controversial (see Discussion), we further explored its function. We first substituted prolines for all of the residues in the B loop that are found at “a” and “d” positions in the final coiled coil; we also made two new double proline substitutions in this region. We asked the following three questions. Are any of the proline-substituted mutants impaired for fusion? If so, at what stage does this occur (i.e., critical conformational changes or target membrane interactions)? Do fusion-impaired mutants form complete coiled coils? None of the double mutants caused any fusion. Double mutant F63P/F70P was analyzed in detail. Like wild-type (WT) HA, F63P/F70P underwent key conformational changes, including head group separation and fusion peptide exposure, and bound tightly to target membranes. Several lines of evidence indicated, however, that, instead of forming a complete coiled coil, F63P/F70P formed a coiled coil that was only about one-half the length of that formed by the WT and that was splayed in its N-terminal half. Our results demonstrate that complete coiled-coil formation is not necessary for fusion peptide exposure and membrane binding but is crucial for fusion to progress from target membrane binding to membrane merger.

During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.

An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C 1 -VP23 2 ) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ∼30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23 2 heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23 2 heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.

An essential step in assembly of herpes simplex virus (HSV) type 1 capsids involves interaction of the major capsid protein (VP5) with the C terminus of the scaffolding protein (encoded by the UL26.5 gene). The final 12 residues of the HSV scaffolding protein contains an A-X-X-F-V/A-X-Q-M-M-X-X-R motif which is conserved between scaffolding proteins found in other alphaherpesviruses but not in members of the beta- or gamma-herpesviruses. Previous studies have shown that the bovine herpesvirus 1 (alphaherpesvirus) UL26.5 homolog will functionally substitute for the HSV UL26.5 gene (E. J. Haanes et al., J. Virol. 69:7375-7379, 1995). The homolog of the UL26.5 gene in the human cytomegalovirus (HCMV) genome is the UL80.5 gene. In these studies, we tested whether the HCMV UL80.5 gene would substitute for the HSV UL26.5 gene in a baculovirus capsid assembly system that we have previously described (D. R. Thomsen et al., J. Virol. 68:2442-2457, 1994). The results demonstrate that (i) no intact capsids were assembled when the full-length or a truncated (missing the C-terminal 65 amino acids) UL80.5 protein was tested; (ii) when the C-terminal 65 amino acids of the UL80.5 protein were replaced with the C-terminal 25 amino acids of the UL26.5 protein, intact capsids were made and direct interaction of the UL80.5 protein with VP5 was detected; (iii) assembly of intact capsids was demonstrated when the sequence of the last 12 amino acids of the UL80.5 protein was changed from RRIFVA ALNKLE to RRIFVAAMMKLE; (iv) self-interaction of the scaffold proteins is mediated by sequences N terminal to the maturation cleavage site; and (v) the UL26.5 and UL80.5 proteins will not coassemble into scaffold structures. The results suggest that the UL26.5 and UL80.5 proteins form a scaffold by self-interaction via sequences in the N termini of the proteins and emphasize the importance of the C terminus for interaction of scaffold with the proteins that form the capsid shell.

Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (∼100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.

Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.

In all herpesviruses, the capsid is icosahedral in shape, composed of 162 capsomers, and assembled in the infected cell nucleus. Once a closed capsid has formed, it is packaged with the virus DNA and transported to the cytoplasm where further morphogenetic events take place. Herpesvirus capsid populations are highly uniform in shape, and this property has made them attractive for structural analysis particularly by cryo electron microscopy followed by three-dimensional image reconstruction. Here we describe what is known about herpesvirus capsid structure and assembly with emphasis on herpes simplex virus and on the contribution of structural studies. The overall analysis has demonstrated that herpesvirus capsids are formed by a pathway resembling that established for dsDNA bacteriophage such as P22 and HK97. For example herpes capsid assembly is found to: (1) involve a scaffolding protein not present in the mature virus; (2) proceed through a fragile, spherical procapsid intermediate; and (3) result in incorporation of a portal complex at a unique capsid vertex.

The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (∼50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.

Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass. The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell. Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA. Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined. Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population. After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction. Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end. In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population. In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined. The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome. Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.

Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins.

Viral B capsids were purified from cells infected with herpes simplex virus type 1 and extracted in vitro with 2.0 M guanidine hydrochloride (GuHCl). Sodium dodecyl sulfate-polyacrylamide gel analyses demonstrated that extraction resulted in the removal of greater than 95% of capsid proteins VP22a and VP26 while there was only minimal (less than 10%) loss of VP5 (the major capsid protein), VP19, and VP23. Electron microscopic analysis of extracted capsids revealed that the pentons and the material found inside the cavity of B capsids (primarily VP22a) were removed nearly quantitatively, but extracted capsids remained otherwise structurally intact. Few, if any, hexons were lost; the capsid diameter was not greatly affected; and its icosahedral symmetry was still clearly evident. The results demonstrate that neither VP19 nor VP23 could constitute the capsid pentons. Like the hexons, the pentons are most likely composed of VP5. When B capsids were treated with 2.0 M GuHCl and then dialyzed to remove GuHCl, two bands of viral material were separated by sucrose density gradient ultracentrifugation. The more rapidly migrating of the two consisted of capsids which lacked pentons and VP22a but had a full complement of VP26. Thus, VP26 must have reassociated with extracted capsids during dialysis. The more slowly migrating band consisted of torus-shaped structures approximately 60 nm in diameter which were composed entirely of VP22a. These latter structures closely resembled torus-shaped condensates often seen in the cavity of native B capsids. The results suggest a similarity between herpes simplex virus type 1 B capsids and procapsids of Salmonella bacteriophage P22. Both contain an internal protein (VP22a in the case of HSV-1 B capsids and gp8 or "scaffolding" protein in phage P22) that can be extracted in vitro with GuHCl and that is absent from mature virions.

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli , can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27–35 nm. The analysis of scanning TEM images revealed particles of mass ∼ 4.3 MDa, representing 101 ± 11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.

By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 Å, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.

The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 U L 6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, U L 26.5. Soluble U L 26.5-portal complexes were found to assemble when purified portals were mixed in vitro with U L 26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter “scaffold particles” formed from purified U L 26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and U L 26.5 combine in various proportions, with the highest observed U L 6 content corresponding to two or three portals per scaffold particle. Association between the portal and U L 26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including U L 6, at a level corresponding to ∼1 portal complex per capsid. The results support the view that U L 6 becomes incorporated into nascent HSV-1 capsids by way of a complex with U L 26.5 and suggest further that U L 6 may be introduced into the growing capsid as an intact portal.

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.

The capsid of herpes simplex virus has an icosahedral surface lattice with a nonskew triangulation number, T=16. Nevertheless, the proteins arrayed on this lattice necessarily have an intrinsic handedness. We have determined the handedness of both the herpes simplex virus type 1 capsid and its precursor procapsid by a cryoelectron microscopic tilting method.

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.

Packaging of DNA into preformed capsids is a fundamental early event in the assembly of herpes simplex virus type 1 (HSV-1) virions. Replicated viral DNA genomes, in the form of complex branched concatemers, and unstable spherical precursor capsids termed procapsids are thought to be the substrates for the DNA-packaging reaction. In addition, seven viral proteins are required for packaging, although their individual functions are undefined. By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process. While the HSV-1 UL6, UL15, UL25, and UL28 packaging proteins are known to associate with different forms of stable capsids, their association with procapsids has not been tested. Therefore, we isolated HSV-1 procapsids from infected cells and used Western blotting to identify the packaging proteins present. Procapsids contained UL15 and UL28 proteins; the levels of both proteins are diminished in more mature DNA-containing C-capsids. In contrast, UL6 protein levels were approximately the same in procapsids, B-capsids, and C-capsids. The amount of UL25 protein was reduced in procapsids relative to that in more mature B-capsids. Moreover, C-capsids contained the highest level of UL25 protein, 15-fold higher than that in procapsids. Our results support current hypotheses on HSV DNA packaging: (i) transient association of UL15 and UL28 proteins with maturing capsids is consistent with their proposed involvement in site-specific cleavage of the viral DNA (terminase activity); (ii) the UL6 protein may be an integral component of the capsid shell; and (iii) the UL25 protein may associate with capsids after scaffold loss and DNA packaging, sealing the DNA within capsids.

Herpes simplex virus type 1 (HSV-1) capsids are initially assembled with an internal protein scaffold. The scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain, and its C-terminal oligomerization and capsid protein-binding domains are identical to those of UL26.5. The UL26 protease cleaves itself, releasing minor scaffold proteins VP24 and VP21, and the more abundant UL26.5 protein, releasing the major scaffold protein VP22a. Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 (containing the protease domain) is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention, we used a mutant virus that fails to express UL26.5 (Δ ICP35 virus). Purified Δ ICP35 B capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B capsids but contained capsid shell proteins in wild-type amounts. Despite C-terminal sequence identity between UL26 and UL26.5, Δ ICP35 capsids lacking UL26.5 products did not contain compensatory high levels of UL26 proteins. Therefore, HSV capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not compensate for the Δ ICP35 growth defect. While indirect, these findings are consistent with the view that UL26 products are restricted from occupying abundant UL26.5 binding sites within the capsid and that this restriction is not controlled by the level of UL26 protein expression. Additionally, Δ ICP35 capsids contained an altered complement of DNA cleavage and packaging proteins, suggesting a previously unrecognized role for the scaffold in this process.

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant ( m 100) deficient in the maturational protease for which it was expected that procapsids—normally, short-lived intermediates—would accumulate in infected cells. Particles isolated from m 100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0°C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-Å resolution from cryoelectron micrographs showed m 100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m 100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant ts Prot.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, ts Prot.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of approximately 2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site.

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.

Condensation/decondensation reactions which effect the packaging of viral genomes into their capsids and their subsequent release constitute one of the basic phenomena which structural biology seeks to explain. The appropriate nucleic acid molecule(s) must be selected; then packaged at a high density to allow efficient utilization of the available space; the mutual electrostatic repulsion of DNA strands must somehow be overcome; and the arrangement of the DNA must allow for its ready release upon initiation of infection. Cryo-electron microscopy has emerged as an incisive tool for visualizing the internal organization of packaged DNA inside viral capsids. We illustrate its effectiveness with observations relating to bacteriophages T4, T7, and herpes simplex virus, type 1 (HSV-1).

Summary High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (ρ = 1.724 g/ml) than the DNA (ρ = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.

The Brookhaven scanning transmission electron microscope was employed to measure the masses of two nucleocapsid species (of light and intermediate densities) of equine herpesvirus 1. These were found to be 196.7 +/- 9.2 and 229.0 +/- 9.5 megadaltons (MDa), respectively. Biochemical assays showed that neither nucleocapsid contained any significant amount of DNA (less than 0.2% [wt/wt]). Taking into account data on protein composition, we conclude that the difference between their masses is essentially contributed by viral protein 22 (46 kDa), which is an integral component of the maturable intermediate nucleocapsid but not of the abortive light nucleocapsid. In view of earlier ultrastructural information on capsomer symmetry, our mass determinations are consistent only with the 150 hexavalent capsomers being hexamers of the 148-kDa major capsid protein.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on aT= 16 icosahedral lattice. The major capsid protein, VP5 (MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862–880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of thetrans-capsomeric channels. We conclude that these sites are occupied by peptide 862–880 in the mature HSV-1 capsid.

The herpes simplex virus type 1 UL12 gene product, alkaline nuclease (AN), appears to be involved in viral DNA processing and capsid egress from the nucleus (Shao, L., Rapp, L. M., and Weller, S. K., Virology 196, 146–162, 1993). Although the HSV-1 AN is not absolutely essential for viral replication in tissue culture, conservation of the AN gene in all herpesviruses suggests an important role in the life cycle of herpesviruses. The counterpart of HSV-1 AN for human cytomegalovirus (HCMV) is the UL98 gene product. To examine whether the HCMV AN could substitute for HSV-1 AN, we performed trans -complementation experiments using a HSV-1 amplicon plasmid carrying the HCMV UL98 gene. Our results indicate (i) HCMV AN can complement the growth of the HSV-1 AN deletion mutant UL12lacZ virus in trans; (ii) a new recombinant virus, UL12laZcUL98/99, appears to be generated by the integration of the HCMV UL98 gene into the HSV-1 UL12lacZ viral genome; (iii) in contrast to its parental HSV-1 UL12lacZ virus, capsids formed in UL12lacZUL98/99 -infected Vero cells were able to transport from the nucleus to the cytoplasm and mature into infectious viruses. Our results demonstrate a functional conservation of AN between HSV-1 and HCMV.