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Control of gene expression is now recognized as a central issue in the field of molecular biology. We now know the sequences of many genomes including that of the human genome, and we know the nature of many pathways involved in control of gene expression. It remains difficult, however, to look at the DNA sequences surrounding a particular gene and tell which methods of regulatory control are in use. I have been pursuing the idea that progress might be made by comparing the regulatory regions of paired gene populations in which one population is strongly expressed and the other weakly. Here I report the results obtained with human genes encoding transcription factors (TF). In this population, broadly expressed genes are strongly expressed while tissue targeted TF expression is suppressed in most tissues. The results demonstrated that the promoter region of broadly expressed TF genes is enriched in binding sites for POLR2A, a component of RNA polymerase II while promoters of tissue targeted genes are enriched in EZH2, a subunit of polycomb repressive complex 2 (PRC2). It was rare to observe promoters with binding sites for both POLR2A and EZH2. The findings are interpreted to indicate that strong expression of broadly expressed TF genes is due to the presence of RNA polymerase II at the promoter while weak expression of tissue targeted promoters results from the presence of PRC2. Finally, transcription factor families were compared in the proportion of broadly expressed and tissue targeted genes they contain. The results demonstrated that most families possess both broadly expressed and tissue targeted members. For instance, this was the case with 16 of 20 TF families examined. The results are interpreted to indicate that while individual TFs such as EZH2 may be specific for broadly expressed or tissue targeted genes, this is not a property of most TF families. HighlightsO_LIHuman transcription factor (TF) genes were noted to be broadly expressed or tissue targeted depending on their breadth of tissue expression. C_LIO_LIThe results of ChIP-seq experiments were used to characterize the promoter regions of broadly expressed and tissue targeted TF genes. C_LIO_LIThe results demonstrated that the promoters of broadly expressed TF genes are enriched in binding sites for RNA polymerase II (pol II) but lack sites for polycomb repressive complex 2 (PRC2) while tissue targeted promoters are the reverse. These have prominent binding sites for PRC2 but are depleted in pol II sites. C_LIO_LIThe results are interpreted to indicate that PRC2 complexes suppress TF expression in tissues where expression is not required. C_LI

BackgroundAs a result of decades of effort by many investigators we now have an advanced level of understanding about several molecular systems involved in the control of gene expression. Examples include CpG islands, promoters, mRNA splicing and epigenetic signals. It is less clear, however, how such systems work together to integrate the functions of a living organism. Here I describe the results of a study to test the idea that a contribution might be made by focusing on genes specifically expressed in a particular tissue, the human testis.Experimental DesignA database of 239 testis-specific genes was accumulated and each was examined for the presence of features relevant to control of gene expression. These include: (1) the presence of a promoter, (2) the presence of a CpG island (CGI) within the promoter, (3) the presence in the promoter of a transcription factor binding site near the transcription start site, (4) the level of gene expression, and (5) the above features in genes of cell types such as spermatocyte and spermatid that differ in their extent of differentiation.ResultsOf the 107 database genes with an annotated promoter, 56 were found to have one or more transcription factor binding sites near the transcription start site. Three of the binding sites observed, Pax-5, AP-2αA and GRα, stand out in abundance suggesting they may be involved in testis-specific gene expression. Compared to less differentiated testis-specific cells, genes of more differentiated cells were found to be (1) more likely to lack a CGI, (2) more likely to lack introns and (3) higher in expression level. The results suggest genes of more differentiated cells have a reduced need for CGI-based regulatory repression, reduced usage of gene splicing and a smaller set of expressed proteins

Background DNA sequence elements in the core promoter can play a central role in regulation of gene expression. Core elements (e.g. INR and TATA box) are located within ~50bp of the transcription start site and both upstream and downstream elements are known. Although all can affect the level of gene expression, their mechanism of action has yet to be fully defined. The studies described here are focused on two core promoter elements, INR and BRE, in the human genome. The locations of the two elements were determined in a large number of human promoters and the results were interpreted in terms of overall promoter function. Results A total of 13,406 promoters were collected from the reference version of the human genome and found to contain 62,891 INR sequences and 32,290 BRE. An INR sequence was found in the core region of 1231 (9.2%) promoters and a BRE in 2592 (19.3%); 121 promoters (0.9%) have both INR and BRE elements. Counts support the view that most human promoters lack an INR or BRE element in the core promoter. Further analysis was carried out with the aligned aggregate of promoters from each chromosome. The results showed distinct INR distributions in separate chromosome groups indicating a degree of chromosome specificity to the way core promoter elements are deployed in the genome. The rare promoters with both INR and BRE elements were found to be enriched among the genes with divergent transcription. Enrichment raises the possibility that core promoter elements can have a function in chromosome organization as well as in initiation of transcription.

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.

The genomes of 39 herpesviruses were examined for the presence of DNA zip codes, sequence elements found in yeast to be involved in gene localization to the nuclear periphery and transcriptional memory. Tests were carried out with three different zip codes and with viruses in all three subfamilies of the Herpesviridae. All genomes were found to have have at least three zip codes with 30 the most observed. Zip codes in many alphaherpesviruses and in human cytomegalovirus were found to be symmetrically arranged within the S genome segment, a region that inverts actively during virus replication. This suggests zip codes may be involved in the segment inversion process. Zip codes located in 16 alphaherpesvirus promoters were examined separately from those in other locations. The genes most likely to have a zip code containing promoter were found to be those encoding glycoprotein B and transcription factor ICP4. Of the two, zip codes associated with ICP4 are of particular interest as their ability to mediate transcriptional memory is well-suited for a role in reactivation of alphaherpesviruses from the latent state.

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.

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.

The herpes simplex virus dsDNA genome is distinguished by an unusually high G+C nucleotide content. HSV-1 and HSV-2, for instance, have GC contents of 68% and 70% respectively, while that of the host (human) genome is 41%. To determine how GC content varies with genome location, GC content was measured separately in coding and intergenic regions of HSV-1 DNA. The results showed that the 75 genes constitute a uniform population with a mean GC content of 66.9 +/- 4.1%. In contrast, intergenic regions were found in two non-overlapping populations, one with a mean GC content (69.3 +/- 4.6% n=32) similar to the coding regions and another where the GC content is lower (56.0 +/- 4.9 n=30). Compared to other regions of the genome, intergenic regions with reduced GC content were found to be enriched in local GC minima, CACACA sequences and a primary target sequence (TTAAAA) for retrotransposition events. The results are interpreted to suggest that a high GC content is part of the way HSV-1 protects its genes from invasion by mobile genetic elements active during cell differentiation in the nervous system.

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.

UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T=16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent, site that is not seen at other quasi-equivalent sites nor on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25-null capsids, and denaturant-extracted capsids, we conclude that (i) the C-capsid-specific component is a heterodimer of UL25 and UL17; and (ii) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.

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.

Herpes simplex virus and other alpha-herpesviruses encode a transcription factor, VP16, able to activate expression of genes containing its binding site (TAATGARAT) in the promoter region. VP16 protein is present inside the infectious virion and it enters the host cell with the virus nucleocapsid. Once inside the cell, VP16 is able to cause the prompt expression of virus genes adjacent to its binding sites. I have examined the possibility that host gene expression may have the potential to be affected by the presence of VP16, a situation that could have important effects on alpha-herpesvirus replication. Bioinformatic methods were used to examine five human genome regions (each 16-73Mb in length) for the presence of genes with upstream TAATGARAT sequences. A total of fourteen characterized genes were identified indicating VP16 has the potential to activate their expression. The identified genes varied considerably in function, and did not appear to support a common theme or goal. The presence of an upstream TAATGARAT sequence was found to be well conserved in the homologous genes of chimpanzee where 11 of 14 homologous genes had upstream TAATGARAT’s. Conservation was poor, however, in three other species examined, mouse (2 of 14 genes), horse (2) and chicken (1). The observed pattern of conservation is interpreted to suggest that alpha-herpesviruses evolved the ability to benefit from expression of TAATGARAT-containing host genes and that this process was complete at or before the time chimpanzees and humans diverged evolutionarily (~7Mya).

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.

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.

Subjects drew lines proportional in length to their subjective valuation of various amounts of money, available immediately and certainly, relative to a standard amount ranging across groups from $1,000,000 to $10. They also drew lines proportional to their subjective valuation of standard amounts with delays ranging from 1 day to 50 years and with probabilities ranging from 1/10 to 1/10,000,000. Amounts of certain‐immediate moneyequivalent (in terms of drawn line length) to delayed or probabilistic money were determined. The delay and probability discount functions thereby obtained were hyperbolic in form, rather than exponential, consistent with previous findings. Large money amounts were valued higherwhentheyweredelayedbyadaythanwhenavailableimmediately.Steepness of delay discounting was not systematically related to standard money amount but probabilistic discounting was steeper for higher standard amounts than for lower amounts. Some of these results diAer from those obtained with choice procedures. Possible reasons for the diAerences are discussed. Copyright #2000 John Wiley & Sons, Ltd.

Participants repeatedly played a self-control game in which choice of the higher of two monetary rewards on the present trial reduced the overall reward (‘alone condition’). Other participants played a prisoner’s dilemma (social cooperation) game in which choices alternated so that overall reward-reducing consequences of choosing the higher current amount were experienced by the other player (‘together condition’). Participants playing the self-control game chose the lower current amount (and higher overall reward) significantly more frequently than did those playing the social cooperation game. In a second phase, half of the subjects who had played the self-control game played the social cooperation game and vice-versa. Little or no transfer was observed between conditions. In a second experiment, raising the amount of the next-trial reward increased self-control but not social cooperation. Some transfer between self-control and social cooperation was observed. The crucial variable responsible for participants’ better performance (closer to optimization) in the self-control game compared to the social cooperation game may have been the higher probability in the former that choice of the lower reward on the present trial would be repeated on subsequent trials.

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.

State University of New York at Stony Brook, USAABSTRACTFemale college students first played a pseudo-prisoner’s dilemma (PPD) gamewith the experimenter, who followed a fixed strategy. In the first experiment theexperimenter’s strategies for di•erent groups of subjects were: (a) play tit-for-tat;(b) play randomly; (c) always cooperate; (d) always defect (‘cooperation’ and‘defection’, defined as in an actual prisoner’s dilemma game). Only the tit-for-tatgroup increased cooperation over trials; other groups decreased cooperation.After playing the PPD with the experimenter, subjects played an actual prisoner’sdilemma (PD) game with each other. In the PD game, subjects began cooperatingmoderately but cooperation deteriorated regardless of what the experimenter’sstrategy had been in the earlier (PPD) game. In a second experiment, subjectsagain played a PPD game with the experimenter and then played a PD game witheach other. Half played one trial at a time as in the first experiment while halfplayed in patterns of four trials at a time. In the PD game, patterning of trialsretarded the development of mutual defection regardless of previous experience.The cooperation-preserving e•ect of patterning of trials in this social task iscompared with similar e•ects on individual tasks involving self-control and risk-aversion. #1998 John Wiley & Sons, Ltd.

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.

The genome of HSV-1 contains 80-85 open reading frames. Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion. The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core. The capsid is an icosahedral shell whose major morphological features are 162 capsomers. It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26. VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers. In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a. DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins. Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques. Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly. More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids. The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads. This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging. Copyright 1997 John Wiley & Sons Ltd.

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.

The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.

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.

Coatomer protein I (COPI) is well known as the protein coat surrounding vesicles involved in returning endoplasmic reticulum (ER)-resident proteins to the ER. COPI coats are also found in vesicles involved in other trafficking processes including endocytosis, autophagy and anterograde transport in the secretory pathway. In view of the diverse functions of COPI proteins, it is expected that they will affect virus replication, and many reports of such COPI involvement have now appeared. The experimental approaches most often employ specific siRNA to deplete COPI subunits or brefeldin A to block COPI activation. Here we briefly describe the results obtained with viruses in which COPI is found to have a role in replication. The results demonstrate that COPI affects viruses quite differently with effects observed in processes such as entry, RNA replication, and intracellular transport of viral proteins.

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.

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.

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.

Fanconi anaemia (FA) is a recessive genetic disorder characterized by bone marrow failure, birth defects and cancer. Cells from FA patients are particularly defective in removing DNA interstrand crosslinks. We have developed a working chromatography purification scheme for FANCD2, a pivotal player in the FA DNA repair pathway, to facilitate identification of FANCD2 interacting partners. In doing so, at least three distinct FANCD2 subcomplexes were found to be present, designated as large, middle, and small complexes. The small complex is composed of tetramer of FANCD2 polypeptides, which may be the building block for other FANCD2 subcomplexes.

The surface shell of the capsid of herpes simplex virus type 1 (HSV-1) is 15 nm thick and 125 nm in outer diameter and has the form of an icosahedral (T = 16) surface lattice, composed of 150 hexons and 12 pentons. Hexons are traversed by axial channels and have six-fold symmetric external protrusions, separated by triangular nodules ("triplexes"). Pentons resemble hexons morphologically, apart from their different order of symmetry. To localize VP5, the major capsid protein, in the shell structure and to investigate whether pentons are composed of the same molecules as hexons, we have performed cryo-electron microscopy and three-dimensional image reconstructions of control HSV-1 B capsids and of B capsids immunoprecipitated with two monoclonal antibodies raised against purified VP5 and purified capsids. The results clearly map the epitope of the anti-VP5 monoclonal antibody to the distal tips of the hexon protrusions. In contrast, no detectable labeling of pentons was observed. We conclude that the hexon protrusions are domains of VP5 hexamers, other parts of these molecules forming the basic matrix of the capsid shell to which the other proteins are attached at specific sites. Conversely, the anti-capsid monoclonal antibody decorates the outer rim of pentons but does not bind to hexons. These observations imply that either pentons are composed of some other protein(s) or that they also contain VP5, but in a conformation sufficiently different from that assumed in hexons as to transform its antigenic character. Other evidence leads us to favor the latter alternative.

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.