Herpes virus replication

Herpesviruses are large double stranded DNA animal viruses with the distinguishing ability to establish latent, life-long infections. To date, eight human herpesviruses that exhibit distinct biological and corresponding pathological/clinical properties have been identified. During their life cycles, herpesviruses execute an intricate chain of events geared towards optimizing their replication. This sets an interesting paradigm to study fundamental biological processes. This review summarizes recent developments in herpesvirus research with emphasis on genome transactions, particularly with respect to the prototypic herpes simplex virus type-1.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Simian homologues of human herpesviruses and implications for novel viral introduction to free-living mountain gorillas

The endangered mountain gorilla (Gorilla beringei beringei) in Rwanda, Uganda, and the Democratic Republic of Congo is frequently in contact with humans through tourism, research activities, and illegal entry of people into protected gorilla habitat. Herpesviruses, which are ubiquitous in primates, have the potential to be shared in any setting where humans and gorillas share habitat. Based on serological findings and clinical observations of orofacial ulcerated lesions resembling herpetic lesions, an alpha-herpesvirus resembling human herpes simplex virus type 1 (HSV-1) has long been suspected to be present in human-habituated mountain gorillas in the wild. While the etiology of orofacial lesions in the wild has not been confirmed, HSV-1 has been suspected in captively-housed mountain gorillas and confirmed in a co-housed confiscated Grauer's gorilla (Gorilla beringei graueri). To better characterize herpesviruses infecting mountain gorillas and to determine the presence/absence of HSV-1 in the free-living population, we conducted a population-wide survey to test for the presence of orally shed herpesviruses. DNA was extracted from discarded chewed plants collected from 294 individuals from 26 groups, and samples were screened by polymerase chain reaction using pan-herpesvirus and HSV-1-specific assays. We found no evidence that human herpesviruses had infected free-ranging mountain gorillas. However, we found gorilla-specific homologs to human herpesviruses, including cytomegaloviruses (GbbCMV-1 and 2), a lymphocryptovirus (GbbLCV-1), and a new rhadinovirus (GbbRHV-1) with similar characteristics (i.e., timing of primary infection, shedding in multiple age groups, and potential modes of transmission) to their human counterparts, human cytomegalovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, respectively.     

Genome-wide screen of three herpesviruses for protein subcellular localization and alteration of PML nuclear bodies

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein-Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.     

Fusing structure and function: a structural view of the herpesvirus entry machinery

Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.     

Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication

Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies.     

Serological evidence of herpesvirus infection in gibbons

Background  

Herpesviruses are not only infectious agents of worldwide distribution in humans, but have also been demonstrated in various non-human primates as well. Seventy-eight gibbons were subjected to serological tests by ELISA for herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV) and cytomegalovirus (CMV).     

Ocular herpes: the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Ocular herpes:the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus(HSV), varicella zoster virus(VZV), and cytomegalovirus(CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

Vaccine potential of a herpes simplex virus type 1 mutant with an essential glycoprotein deleted.

Several approaches to the production of vaccines to human herpesviruses have been proposed. Subunit vaccines, subunits delivered by live vectors, and rationally attenuated vaccines have all been shown to be efficacious in animal models but suffer from uncertainties as to the roles of individual genes involved in pathogenesis and the most relevant components of the immune response required for protection in humans and the target antigens involved. With these problems in mind, we examined the vaccine potential of a fully disabled herpes simplex virus type 1 mutant that is capable of only a single round of replication, since a virus of this type should induce the full spectrum of immune responses but has no pathogenic potential. A virus has been described which lacks essential glycoprotein H (gH) and can be propagated in a cell line which supplies gH in trans (A. Forrester, H. Farrell, G. Wilkinson, J. Kaye, N. Davis-Poynter, and T. Minson, J. Virol. 66:341-348, 1992). Infection of normal cells with this mutant is indistinguishable from a wild-type infection, except that the resulting progeny are gH negative and noninfectious: the virus is self-limiting. Infection of mice by the ear pinna route was similarly self-limiting in that input infectivity decreased rapidly at the inoculation site and no infectivity was detected in sensory ganglia. Animals given a wide range of doses of the gH-negative mutant produced both humoral and T-cell responses to herpes simplex virus type 1 and proved solidly resistant to challenge with a high dose of wild-type virus. The gH-negative mutant is presumably capable of establishing a latent infection, but since no infectious virus was detected in numerous attempts to reactivate the mutant, the risk of a pathogenic outcome is minimal.     

Sorting and Transport of Alpha Herpesviruses in Axons

The alpha herpesviruses, a subfamily of the herpesviruses, are neurotropic pathogens found associated with most mammalian species. The prototypic member of this subfamily is herpes simplex virus type 1, the causative agent of recurrent cold sores in humans. The mild nature of this disease is a testament to the complex and highly regulated life cycle of the alpha herpesviruses. The cellular mechanisms used by these viruses to disseminate infection in the nervous system are beginning to be understood. Here, we overview the life cycle of alpha herpesviruses with an emphasis on assembly and transport of viral particles in neurons.     

Alterations in Glycosphingolipid Patterns in a Line of African Green Monkey Kidney Cells Infected with Herpesvirus

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.     

The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences

Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.     

Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.     

Transformation of dog embryo kidney cells by human herpesviruses

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.     

Temperature-sensitive host range mutants of herpes simplex virus type 2.

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblasts cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties.     

Antibody to herpes simplex virus type 2 as serological marker of sexual lifestyle in populations.

OBJECTIVES--To examine the epidemiology of antibody to herpes simplex virus type 2 and to assess its suitability as a serological marker of sexual behaviour in populations with high and low prevalences. DESIGN--Cross sectional survey. SETTING--Department of genitourinary medicine and blood donation centre in central London. SUBJECTS--Representative sample of 869 patients attending department between November 1990 and December 1991, and 1494 consecutive blood donors attending for donation between February and April 1992. METHOD--Participants had a blood sample taken for antibody testing with a novel type specific assay and completed a questionnaire. RESULTS--Prevalence of antibody differed significantly between the two groups (188/833 (22.7%) clinic attenders; 102/1347 (7.6%) blood donors). In both populations antibody was strongly associated with sex, sexual orientation, years of sexual activity, number of lifetime sexual partners, and past infection with sexually transmitted diseases after other factors were controlled for. Only 130 (45%) of all those with antibody had symptoms suggestive of genital herpes, and 79 (27.4%) had had genital herpes diagnosed. Of those without antibody to herpes simplex viruses type 1 and 2, 8.0% reported genital blisters or sores and 1.1% had had genital herpes diagnosed by a doctor. CONCLUSIONS--The strong relation between herpes simplex virus type 2 and sexual lifestyle suggests that the presence of antibody to the virus may be suitable for use as an objective, serological marker of patterns of sexual behaviour in different populations. These data show that only a minority of those infected with herpes simplex virus type 2 have a diagnosis of genital herpes or express clinical symptoms, making serological determinants of infection essential for epidemiological studies.     

Genetically determined resistance to murine cytomegalovirus and herpes simplex virus in newborn mice.

Mice which were infected with the herpesvirus murine cytomegalovirus or herpes simplex virus type 1 on the day of birth exhibited mouse strain-dependent differences in the development of lethal disease. The pattern of resistance among the strains was distinct for each virus and closely resembled that reported in adult mice. However, much lower doses of the viruses were required in newborn mice to reveal these resistance patterns. For murine cytomegalovirus, both H-2-associated and non-H-2 genes conferred resistance, and, as has been shown for adults, there was a 25-fold difference in the dose required to kill 50% of the animals belonging to the most resistant and susceptible strains. The resistance of newborn mice to herpes simplex virus type 1 was conferred by non-H-2 genes in C57BL/6 mice, as has been reported for adults, and newborn C57BL/6 mice were considerably more resistant than mice of susceptible strains. Resistance was also reflected in the titer of these viruses in the spleen or liver early in infection and, with murine cytomegalovirus, in the survival time of infected mice. The resistance of newborn mice to lethal disease was not conferred postnatally by the mother. This appears to be the first report of genetically determined resistance to herpesviruses in newborn mice. Such autonomous virus-specific resistance may provide a significant barrier to naturally acquired infection in genetically resistant strains. Similar genetically regulated mechanisms may protect the newborns of many species, including humans, against infection with herpesviruses.