Passive immunization with immunoglobulin or hyperimmune globulin is used either to prevent infection or as an adjunct to antiviral therapy. Treatment: Infections with herpes simplex virus 1 and 2 and varicella-zoster virus are currently the most amenable to therapy; acyclovir, valaciclovir and famciclovir are all licensed therapeutics.
Ganciclovir is used to treat cytomegalovirus retinitis. B virus appears to respond to either of these drugs. There is as yet no treatment for Epstein-Barr virus or human herpesvirus 6,7 or 8 infections.
Herpes simplex viruses 1 and 2 have only about 50 percent genomic homology. However, they share most other characteristics. Mucocutaneous manifestations of herpes simplex virus infection include gingivostomatitis, herpes genitalis, herpetic keratitis, and dermal whitlows.
Neonatal herpes simplex virus infection and herpes simplex virus encephalitis also occur. The virus replicates initially in epithelial cells, producing a characteristic vesicle on an erythematous base.
It then ascends sensory nerves to the dorsal root ganglia, where, after an initial period of replication, it establishes latency.
Interferon and humoral, mucosal, and cellular immunity are important defenses. Herpes simplex virus infections are more severe in immunocompromised hosts. Herpes simplex virus 1 transmission is primarily oral, and herpes simplex virus 2 primarily genital. Transmission requires intimate contact. Primary varicella-zoster virus infection causes varicella chickenpox. Reactivation of latent virus usually in adults causes herpes zoster shingles , manifesting as vesicular rash with a dermatomal distribution and acute neuritis.
Varicella-zoster virus is usually transmitted by droplets and replicates initially in the nasopharynx. In seronegative individuals, viremia and chickenpox ensue.
Latency is established in dorsal root ganglia, and virus reactivation results in virion transport down sensory nerves. As with herpes simplex virus, interferon and cellular and humoral immunity are important defenses. Reactivated virus can cause disseminated disease in immunocompromised individuals. Varicella-zoster virus is highly contagious; about 95 percent of adults show serologic evidence of infection. Cytomegalovirus causes three clinical syndromes.
Cytomegalovirus replicates mainly in the salivary glands and kidneys and is shed in saliva and urine. Replication is slow, and the virus induces characteristic giant cells with intranuclear inclusions. Transmission is via intimate contact with infected secretions. Cytomegalovirus infections are among the most prevalent viral infections worldwide. Epstein-Barr virus causes classic mononucleosis. In immunocompromised hosts, the virus causes a lymphoproliferative syndrome.
In some families, Epstein Barr virus causes Duncan's syndrome. Human herpes viruses 6 and 7 are associated with exanthem subitem roseola and with rejection of transplanted kidneys. Human herpesvirus 8 has been found associated with Kaposi's sarcoma in AIDS patients as well as intra-abdominal solid tumors.
Virtually nothing is known about the pathogenesis and epidemiology of this newly described herpesvirus. B virus is transmitted to humans by the bite of infected rhesus monkeys and is transported up neurons to the brain. The reservoir for the disease is latent infection in rhesus monkeys, particularly those from Southeast Asia and India. In stressed or unhealthy animals, the virus may reactivate and appear in saliva.
In nature, herpesviruses infect both vertebrate and non-vertebrate species, and over a hundred have been at least partially characterized. Only eight of these have been isolated routinely from humans and are discussed here. They are known as the human herpesviruses and are herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, human herpesvirus 7 and, most recently, Kaposi's Sarcoma herpesvirus.
A primate herpesvirus, namely B virus, is an uncommon human pathogen that may cause life-threatening disease. The human herpesviruses share four significant biologic properties. First, all of the herpesviruses code for unique enzymes involved in the biosynthesis of viral nucleic acids. These enzymes are structurally diverse and parenthetically provide unique sites for inhibition by antiviral agents.
Secondly, the synthesis and assembly of viral DNA is initiated in the nucleus. Assembly of the capsid is also initiated in the nucleus. Third, release of progeny virus from the infected cell is accompanied by cell death. Finally, all herpesviruses establish latent infection within tissues that are characteristic for each virus, reflecting the unique tissue trophism of each member of this family.
Membership in the family Herpesviridae is based on the structure of the virion. These viruses contain double-stranded DNA which is located at the central core. The precise arrangement of the DNA within the core is not known. Herpesvirus DNA varies in molecular weight from approximately 80 to million, or to kilobase pairs, depending on the virus. This DNA core is surrounded by a capsid which consists of capsomers, arranged in icosapentahedral symmetry.
The capsid is approximately to nanometers in diameter. Tightly adherent to the capsid is the tegument, which appears to consist of amorphous material. Loosely surrounding the capsid and tegument is a lipid bilayer envelope derived from host cell membranes.
The envelope consists of polyamines, lipids, and glycoproteins. These glycoproteins confer distinctive properties to each virus and provide unique antigens to which the host is capable of responding. A fascinating feature of herpesvirus DNA is its genomic sequence arrangement. Herpesviruses can be divided into six groups arbitrarily classified A to F. For those herpesviruses which infect humans group C, group D, and group E unique structures are demonstrable.
In the group C genomes, as exemplified by Epstein-Barr virus and the newly identified Kaposi's sarcoma herpesvirus, the number of terminal reiterations divides the genome into several well-delineated domains. The group D genomes, such as varicella-zoster virus, have sequences from one terminus repeated in an inverted orientation internally.
Thus, the DNA extracted from these virions consist of two equal molar populations. For group E viral genomes, such as herpes simplex virus and cytomegalovirus, the genomes are divided into internal unique sequences whereby both termini are repeated in an inverted orientation.
Thus, the genomes can form four equimolar populations which differ in relative orientation of the two unique segments. The grouping of herpesviruses into sub-families serves the purpose of identifying evolutionary relatedness as well as summarizing unique properties of each member.
The members of the alpha herpesvirus sub-family are characterized by an extremely short reproductive cycle hours , prompt destruction of the host cell, and the ability to replicate in a wide variety of host tissues.
They characteristically establish latent infection in sensory nerve ganglia. This sub-family consists of herpes simplex virus 1 and 2 and varicella-zoster virus. In contrast to the alpha herpesviruses, beta herpesviruses have a restricted host range. Their reproductive life cycle is long days , with infection progressing slowly in cell culture systems.
A characteristic of these viruses is their ability to form enlarged cells, as exemplified by human cytomegalovirus infection. These viruses can establish latent infection in secretory glands, cells of the reticuloendothelial system, and the kidneys. Finally, the gamma herpesviruses have the most limited host range. They replicate in lymphoblastoid cells in vitro and can cause lytic infections in certain targeted cells. Latent virus has been demonstrated in lymphoid tissue.
Epstein-Barr virus is a member of this sub-family. In addition, human herpesvirus 6 and 7 are probably best classified as a gamma herpesvirus. However, the latter has host range properties of the beta sub-family. Further studies will need to clarify the most appropriate classification of this virus. Kaposi's sarcoma herpesvirus is most closely related genetically to Epstein-Barr virus. Replication of all herpesviruses is a multi-step process.
Following the onset of infection, DNA is uncoated and transported to the nucleus of the host cell. This is followed by transcription of immediate-early genes, which encode for the regulatory proteins.
Expression of immediate-early gene products is followed by the expression of proteins encoded by early and then late genes. Assembly of the viral core and capsid takes place within the nucleus. This is followed by envelopment at the nuclear membrane and transport out of the nucleus through the endoplasmic reticulum and the Golgi apparatus.
Glycosylation of the viral membrane occurs in the Golgi apparatus. Mature virions are transported to the outer membrane of the host cell inside vesicles.
Release of progeny virus is accompanied by cell death. Replication for all herpesviruses is considered inefficient, with a high ratio of non-infectious to infectious viral particles. A unique characteristic of the herpesviruses is their ability to establish latent infection. Each virus within the family has the potential to establish latency in specific host cells, and the latent viral genome may be either extra-chromosomal or integrated into host cell DNA.
Herpes simplex virus 1 and 2 and varicella-zoster virus all establish latency in the dorsal root ganglia. Epstein-Barr virus can maintain latency within B lymphocytes and salivary glands. Cytomegalovirus, human herpesvirus 6 and 7, Kaposi's sarcoma herpesvirus and B virus have unknown sites of latency.
Latent virus may be reactivated and enter a replicative cycle at any point in time. The reactivation of latent virus is a well-recognized biologic phenomenon, but not one that is understood from a biochemical or genetic standpoint.
It should be noted here that an anti-sense message to one of the immediate-early genes alpha-O may be involved in the maintenance of latent virus.
Stimuli that have been observed to be associated with the reactivation of latent herpes simplex virus have included stress, menstruation, and exposure to ultraviolet light. Precisely how these factors interact at the level of the ganglia remains to be defined. It should be noted that reactivation of herpesviruses may be clinically asymptomatic, or it may produce life-threatening disease. With the exception of cytomegalovirus retinitis, the definitive diagnosis of a herpesvirus infection requires either isolation of virus or detection of viral gene products.
For virus isolation, swabs of clinical specimens or other body fluids can be inoculated into susceptible cell lines and observed for the development of characteristic cytopathic effects. This technique is most useful for the diagnosis of infection due to herpes simplex virus 1 and 2 or varicella-zoster virus because of their relatively short replicative cycles. The identification of cytomegalovirus by cell culture requires a longer period of time due to its prolonged period of replication.
Epstein-Barr virus does not induce cytopathic changes in cell culture systems and, therefore, can only be identified in culture by transformation of cord blood lymphocytes.
Similarly, human herpes virus 6 and 7 have unique growth characteristics which make identification in cell culture systems difficult. Newer and more rapid diagnostic techniques involve the detection of viral gene products.
This can be done by applying fluorescence antibody directed against immediate-early or late gene products to tissue cultures after 24 to 72 hours of incubation. A positive result is the appearance of intranuclear fluorescence.
A method which utilizes monoclonal antibodies to an immediate-early gene has been most useful for the identification of CMV. Alternatively, fluorescence antibodies may be applied directly to cell monolayers or scrapings of clinical lesions, with intranuclear fluorescence again indicating a positive result.
Recently developed diagnostic techniques that have clinical utility include in situ and dot-blot hybridization and, importantly, polymerase chain reaction DNA amplification.
The UCLA researchers used a new electron-counting technology called cryo-electron microscopy, whose inventors won the Nobel Prize in chemistry. The technology enabled the scientists to see the herpes virus with unprecedented resolution, which in turn allowed them to create a 3-D atomic model of the virus.
The virus is composed of approximately 3, proteins, each consisting of roughly 1, amino acids. Dan Gordon January 19, Finally, a comparison between the two gD-receptor structures and the unliganded gD structure provides additional insights into the mechanism of receptor-mediated activation of the HSV fusion machinery.
In other cell adhesion molecules of the Ig-superfamily this same region is involved in homophilic and heterophilic trans-interactions and nectin-1 uses the same surface to homo-dimerize [26]. Consistent with these findings, gD can prevent nectin-1 mediated cell aggregation [32] and HSV infection is favored by prior disruption of cell junctions [54]. Similarly to HSV, these viruses disrupt the homophilic trans-interactions of their receptors [41] , [49].
Binding to cell-adhesion molecules may ultimately favor release of these viruses by opening intercellular junctions [58]. The Ig-like domains of the receptors are shown in similar orientation and colored in red with the region that appears most structurally variable in cyan.
This suggests that the interactions established by single amino acids in this region may not be critical for function. Indeed, the same conclusion can be drawn from the gD side where multiple mutations are needed to abolish nectin-1 usage [36] , [44]. This is quite different from the role of Phe at the tip of the FG loop of the nectin-1 V-domain.
Mutations of Phe to alanine showed that this residue plays an important role for tight gD binding and for HSV entry. Of note, Phe protrudes into a pocket on the gD surface occupied in the unliganded gD by the C-terminal residue Trp Fig. Therefore, nectin-1 Phe effectively substitutes for gD Trp and provides a key contribution to the stable displacement of the gD C-terminal region. This interaction is critical for function.
The side chain of Phe purple located on the FG loop of the nectin-1 V-domain protrudes into the same pocket in the complex with gD. This binding configuration is not compatible with the native position of the gD C-terminus.
The equivalent FG loop in necl-5 has been shown to be at the interface with poliovirus and to be important for poliovirus binding [38] , pointing to a conserved feature in the interaction between HSV and poliovirus with their respective receptors. The latter contacts only residues within the first N-terminal 32 residues of gD folded in a hairpin-like structure and the interaction involves several hydrogen bonds through main and side chain atoms [21].
The nectin-1 V-domain, instead, contacts a large surface on gD formed mostly by residues from the C-terminal extension and some amino acids from the N-terminal region. Thus, despite their considerably different binding sites, each receptor is likely to interfere with binding of the other. Indeed soluble nectin-1 can block virus entry in HVEM expressing cells [60]. A comparison between the gD-receptor complexes and the structure of unliganded gD provides additional insights in the mechanism of receptor-mediated activation of gD.
In the absence of receptors residues from the gD C-terminal region residues — are anchored by Trp on the core of gD.
This is consistent with the increase receptor affinity of the C-terminally truncated form of gD used in this study compared to the full length molecule [25] , [61]. Therefore, for both receptors complex formation requires the displacement of residues from the gD C-terminal region.
Several observations point to a key role of the gD C-terminal region. A soluble gD molecule encompassing the entire ectodomain allows entry of a gDnull virus i. Moreover, mutagenesis data showed that the C-terminal region is essential for virus entry [52] , [62] , [64] , [65] , [66]. They also suggest a critical role for this region for the interaction with other viral glycoproteins. Exposure of the C-terminal region upon receptor binding therefore provides a timely and cell specific trigger for the activation of the HSV entry process.
The conformation of the C-terminus in receptor-bound gD has not been determined due to its high flexibility. Remarkably, gD has been engineered to bind alternate receptors [67] , [68] , [69] , [70]. All these ligands are able to mediate entry of HSV virions carrying the chimeric forms of gD in cells expressing the respective receptors. In the absence of structural data on such chimeras, one can only speculate on their mechanism of action during entry.
Even in the most extreme recombinant [70] , the N-terminal and C-terminal extensions to the Ig core are maintained for activity [71]. Thus, these regions likely contain the necessary sites for binding and activation of the other viral glycoproteins. In the engineered gDs the C-terminus may be exposed upon exogenous receptor binding in a way that is very similar to the model supported by our studies for wt HSV.
Alternatively, this functional region may already be exposed in these molecules so that exogenous receptor binding would solely allow the close proximity of viral and cell membranes. In such a situation, expressing a pre-activated gD may lead to a decrease in viral fitness by diminishing the selectivity for target cells.
It is likely that HSV evolved to unmask the gD C-terminus only upon receptor binding to ensure efficient tropism in the host. Of note, the initial analysis of the structural organization of gD revealed that the Ig-core acts as structural support to the functional C- and N- terminal regions and suggested that it inserted in an ancestor molecule formed by the N- and C- terminal extensions [21]. The results obtained with the above engineered gDs are consistent with such hypothesis.
This new structure shows how nectin-1 and HVEM, albeit belonging to different structural families and establishing different interactions with gD, similarly cause the disruption of intra-molecular contacts between the gD C-terminal region and the rest of the molecule thus leading to activation of the entry process through a conserved mechanism.
Production of gD-1 KOS residues 1 to , gD t and human nectin-1 residues 31—, gD t using recombinant baculoviruses and their purification were described previously [21] , [25]. The complex used for crystallization experiments was formed by mixing purified gD and nectin-1 in a 1. The complex, which eluted as single peak from the SEC step, was concentrated to 3. SDS gels and N-terminal sequencing on washed crystals confirmed the presence of the full-sized proteins.
Nevertheless, after screening more than crystals a 4. Table S1. The HKL suite was used for data integration and scaling [72] and the CCP4 suite was used for further data processing and analysis [73]. Data integration suggested that the crystals belong to the hexagonal P6 2 22 space group, however analysis of the cumulative intensity distribution [73] hinted at the presence of merohedral twinning and as a consequence to a lower symmetry space group.
Due to the difficulty of obtaining similar quality diffraction this data set was used for subsequent structure determination and refinement despite integration in the trigonal space groups resulted in low redundancy Table S1. The initial search was carried out in all trigonal and hexagonal space groups with a gD model PDB entry 2C amino acids 27— and using all data between 12 and 4.
A clear solution for 3 gD molecules was identified in P3 2 21 whereas no solutions were found in all the other space groups tested. Keeping the 3 gD molecules fixed, three solutions for the V domain and then for the C1 domains of nectin-1, consistent with 3 gD-nectin-1 complexes in the asymmetric unit, were clearly identified. First, the nectin-1 V-domain was positioned in proximity of gD residues previously implicated in nectin-1 binding; second, the N- and C-termini of the C1 and V-domain, respectively, were at a distance from each other compatible with the number of missing residues connecting the two domains; and third the model agreed with considerations on packing and symmetry relation between the molecules in the crystal see also Results.
However, only a poorly contrasted solution for one of the C2 domains could be identified by MR. Despite this solution positioned the C2 domain in proximity of a C1 domain with reasonable crystal contacts, its addition to the model did not improve the R free or the quality of the electron density maps and therefore was excluded from the refinement. Simulated annealing composite 2mFo-DFc and mFo-DFc omit electron density maps followed by local three fold NCS averaging for each of the nectin-1 domains were calculated with the program Phenix [75] and used during the initial stages of model building.
The resulting electron density maps clearly revealed the location of some of the loops in the nectin-1 V-domain and some of the glycans both of which had been excluded from the MR model Fig. The electron density of the glycans and of residues with large side chains together with the location of disulfide bonds in V and C1 Ig domains were used to confirm the register of the polypeptide chain. The twinning fraction estimated by Refmac was of 0. Following the initial building the crystal structure of nectin-1 became available [26] and was used to modify the V and C1 domains.
Additional refinement showed that although most of the structure remained the same between the two models, modest differences were present in between the structures in some of the V1 loops and residues involved in the gD-nectin-1 interface.
The final model includes the full V1 domain but does not include some of the loops in the distal part of the nectin-1 C1 domain which did not have interpretable electron density. A DNA fragment corresponding to the variable immunoglobulin domain V-type of nectin-1 was amplified by PCR from a plasmid containing the cDNA form of the full length protein pCK [76] , using primers designed to insert a stop codon after residue This fragment was inserted into the bacterial gene fusion vector pMAL-p New England Biolabs by a blunt-ended ligation into BamH1 restriction site of the plasmid polylinker.
This construct produces a fusion with the maltose-binding protein under the control of the Lac repressor that is exported to the periplasm. Mutants were produced using the Quikchange mutagenesis kit Stratagene. Correctness of all the constructs was verified by sequencing. For protein purification, E. After binding, the resin was washed with Buffer A and proteins were eluted with 20mM maltose in Buffer A.
Proteins were concentrated and purified by size exclusion chromatography on a Superdex S column equilibrated with 40mM Tris pH 8. Plates were blocked with PBS containing 0. Absorbance at nm was read after addition of ABTS as substrate.
Representative electron density gray from a three-fold averaged composite anneal omit map of the nectin-1 V-domain. A region of the final model is shown in the density in stick representation highlighting the presence of two glycosylation sites. Comparison of the elbow angle of necl-5 cyan and nectin-1 purple. An approximate difference of 15 degrees exists between the two structures. Loops not built in the nectin-1 model are shown as a dotted line. We are grateful to Patricia G.
The authors have declared that no competing interests exist. Also, C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
National Center for Biotechnology Information , U. PLoS Pathog. Published online Sep Paolo Di Giovine , 1 Ethan C. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex.
Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses.
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