Potential role of cellular ESCRT proteins in the STIV life cycle

We are examining the archaeal virus STIV (Sulfolobus turreted icosahedral virus) in order to elucidate the details of its replication cycle and its interactions with its cellular host, Sulfolobus solfataricus. Infection of Sulfolobus by STIV initiates an unusual cell lysis pathway. One component of this pathway is the formation of pyramid-like structures on the surface of infected cells. Multiple seven-sided pyramid-like structures are formed on infected cells late in the STIV replication cycle. These pyramid-like structures are formed at sites where the Sulfolobus S-layer has been disrupted and through which the cellular membrane protrudes. It is through the pyramid-like structures that virus-induced cell lysis occurs in the final stages of the STIV replication cycle. The pathway and process by which these unusual lysis structures are produced appears to be novel to archaeal viruses and are not related to the well-characterized lysis mechanisms utilized by bacterial viruses. We are interested in elucidating both the viral and cellular components involved with STIV lysis of its infected cell. In particular, we are examining the potential role that Sulfolobus ESCRT (endosomal sorting complex required for transport)-like proteins play during viral infection and lysis. We hypothesize that STIV takes advantage of the Sulfolobus ESCRT machinery for virus assembly, transport and cellular lysis.     

Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures

Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.     

Insights into a Viral Lytic Pathway from an Archaeal Virus-Host System

Archaeal host cells infected by Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. This viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. The STIV protein C92 and the SIRV2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of STIV and SIRV2, respectively. Since SIRV2 and STIV have fundamentally different morphotypes and genome sequences, it is surprising that they share this lysis system. In this study, we have constructed a collection of C92/P98 chimeric proteins and tested their abilities, both in the context of virus replication and alone, to form pyramid lysis structures in S. solfataricus. The results of this study illustrate that these proteins are functionally homologous when expressed as individual chimeric proteins but not when expressed in the context of complete STIV infection.     

Structure of A197 from Sulfolobus turreted icosahedral virus: a crenarchaeal viral glycosyltransferase exhibiting the GT-A fold

Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92 degrees C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the three domains of life. The 17,663-base-pair, circular, double-stranded DNA genome contains 36 potential open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. However, functional and evolutionary information may be suggested by a protein's three-dimensional structure. To this end, we have undertaken structural studies of the STIV proteome. Here we report our work on A197, the product of an STIV open reading frame. The structure of A197 reveals a GT-A fold that is common to many members of the glycosyltransferase superfamily. A197 possesses a canonical DXD motif and a putative catalytic base that are hallmarks of this family of enzymes, strongly suggesting a glycosyltransferase activity for A197. Potential roles for the putative glycosyltransferase activity of A197 and their evolutionary implications are discussed.     

The Architecture and Chemical Stability of the Archaeal Sulfolobus Turreted Icosahedral Virus

Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-Å-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-Å resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.Viruses are valuable biological tools for manipulating the cellular processes of their hosts, and they can also serve as model systems for describing macromolecular interactions through the analysis of their architecture. The Sulfolobus turreted icosahedral virus (STIV) is an archaeal virus that infects Sulfolobus solfataricus (phylum Crenarchaeota). STIV is a lytic virus that was isolated from an acidic hot spring (>80°C and pH of <3) in Yellowstone National Park (27). Hence, STIV is an important model for studying the biochemical requirements to sustain life in extreme physicochemical conditions and has the potential to become a tool for the biochemical and genetic manipulation of its host—much like bacteriophages lambda, P22, and phi29 have done for their respective hosts.Prior structural studies of STIV using cryo-electron microscopy (cryo-EM), X-ray crystallography, and proteomics have described large pentameric turret-like structures, with petal-like protrusions emanating from their central shafts (27). The T=31d capsid shell is composed of trimeric capsomers exhibiting pseudo-hexagonal symmetry, in which each of the three capsomer subunits donates two viral jelly rolls with its β-sheets normal to the capsid surface (15, 27). Capsomers surrounding the icosahedral 3-fold axes, and their neighboring subunits, make direct contact with the viral membrane via a highly basic C-terminal helix of each subunit (15, 23). Surrounding the base of the turrets are proteins that make contact with the capsid shell and a host-derived viral membrane (15). The viral membrane and the enclosed viral genome are referred to as the lipid core.The capsid architecture of STIV and the crystal structure of its major capsid protein (MCP) are strikingly similar to those of the bacteriophages PRD1, Bam35, and PM2, the alga virus PBCV-1, and the mammalian adenovirus. This similarity suggests that these viruses share an ancestral virus (2, 4, 7, 15, 25). Given the evolutionary relationship shared between STIV and PRD1, we postulated that the large turret-like vertices of STIV were used to inject the viral genome into the Sulfolobus host—a genome delivery mechanism employed by PRD1 (27).A recent report by Brumfield et al. (5) describes gross cellular ultrastructural changes induced in the Sulfolobus host during STIV infection and release. The authors identified distinct particles that appear to be assembly intermediates of STIV en route to maturation. From these intermediates the authors proposed a general mechanism of capsid assembly, in which MCP subunits and minor capsid proteins (mCPs) coassemble with the lipid membrane to form a lipid-enclosed protein vesicle. These vesicles are spherical and lack the double-stranded DNA (dsDNA) genome and turret-like appendages at the vertices.While these studies confirm an empty procapsid intermediate, the corresponding molecular mechanism associated with assembly and disassembly remains to be understood. Moreover, little is known about STIV or other archaeal virus genome delivery into the host. To obtain a better understanding of the molecular mechanism of STIV architecture and its role in genome delivery, we characterized three distinct morphologies of STIV particles using cryo-EM. An image reconstruction of one of these revealed the absence of a number of constituents decorating the STIV capsid. Hence, for simplicity, we refer to the previously reported image reconstruction (27) as “decorated” and the new image reconstruction reported here as “undecorated.” Reference-free two-dimensional (2D) class averages of the second identified morphology reveal a partially decorated STIV lipid core. The third identified morphology corresponds to the isolated STIV lipid core. Taken together, our analyses indicate that these morphologies correspond to different disassembly intermediates of STIV that can be isolated in vitro and help provide a picture of the STIV capsid architecture. Additionally, these morphologies allow us to propose an alternative possible mechanism of genome delivery.     

Exceptional virion release mechanism: one more surprise from archaeal viruses

Virion release from the host cell is the final and essential step for completion of the viral life cycle and spread of virions in the environment. Although for eukaryotic and bacterial viruses the egress mechanisms are reasonably well understood, this subject has not been studied in detail for archaeal viruses until recently. Here we summarize available data on the extraordinary egress mechanism exploited by the Sulfolobus islandicus rod-shaped virus SIRV2 and the Sulfolobus turreted icosahedral virus STIV. In addition, we describe features of the virus-induced pyramidal formation, VAP, involved in this process. Being an autonomous structure different from the capsid, the VAP can be considered as a representative of a specific class of virus-coded structures which we suggest to name 'virodomes'.     

In vitro assembly of the herpes simplex virus procapsid: formation of small procapsids at reduced scaffolding protein concentration

The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid. Procapsid assembly requires the capsid shell proteins (VP5, VP19C, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation. While other reaction components (VP5, VP19C, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of approximately 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was approximately 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form. The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.     

Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus

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 (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-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 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 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 m100 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 tsProt.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, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.     

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor.

Empty capsid species formed from the self- and extract-mediated assembly of poliovirus type 1 14S particles in vitro and procapsids isolated from virus-infected cells were subjected to isoelectric focusing in charge-free agarose gels. The empty capsid formed in the self-assembly reaction had an isoelectric point (pI) of 5.0, whereas procapsids and extract-assembled empty capsids focused at pH 6.8. Unreacted 14S particles focused at pH 4.8 to 5.0. The sedimentation coefficient (s20,w) and density of the empty capsid species were also determined. Procapsids had a density in CsCl of 1.31 g/cm3, whereas empty capsids formed by self- or extract-mediated assembly had a density of 1.29 g/cm3. Both extract-assembled empty capsids and procapsids had an s20,w of 75S, whereas self-assembled empty capsids had an s20,w of 71S. Self-assembled empty capsids were not converted to pI 6.8 empty capsids by incubation with poliovirus-infected HeLa cell extracts. The dissociated polypeptides of self-assembled empty capsids (pI 5.0) and procapsids (pI 6.8) behaved identically when analyzed by isoelectric focusing in the presence of 9 M urea and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results suggest that infected cell extracts possess a factor that influences the final conformation of the empty shell (pI 6.8, 75S) formed from 14S particles and that this influences is exerted at the initiation step or during the polymerization reaction. A small amount of this activity (less than or equal to 20% of infected extracts) was detected in uninfected cells; the significance of this remains unknown.     

Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.     

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80α. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80α-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80α and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80α or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80α-encoded structural proteins in 80α and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80α and SaPI1 procapsids.     

Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

ATP-Dependent localization of the herpes simplex virus capsid protein VP26 to sites of procapsid maturation

The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutant tsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.     

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.     

ATP Depletion Blocks Herpes Simplex Virus DNA Packaging and Capsid Maturation

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed.