The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

Antivirals reduce the formation of key Alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1

Alzheimer's disease (AD) afflicts around 20 million people worldwide and so there is an urgent need for effective treatment. Our research showing that herpes simplex virus type 1 (HSV1) is a risk factor for AD for the brains of people who possess a specific genetic factor and that the virus causes accumulation of key AD proteins (β-amyloid (Aβ) and abnormally phosphorylated tau (P-tau)), suggests that anti-HSV1 antiviral agents might slow AD progression. However, currently available antiviral agents target HSV1 DNA replication and so might be successful in AD only if Aβ and P-tau accumulation depend on viral DNA replication. Therefore, we investigated firstly the stage(s) of the virus replication cycle required for Aβ and P-tau accumulation, and secondly whether antiviral agents prevent these changes using recombinant strains of HSV1 that progress only partly through the replication cycle and antiviral agents that inhibit HSV1 DNA replication. By quantitative immunocytochemistry we demonstrated that entry, fusion and uncoating of HSV1, are insufficient to induce Aβ and P-tau production. We showed also that none of the "immediate early" viral proteins is directly responsible, and that Aβ and P-tau are produced at a subsequent stage of the HSV1 replication cycle. Importantly, the anti-HSV1 antiviral agents acyclovir, penciclovir and foscarnet reduced Aβ and P-tau accumulation, as well as HSV1, with foscarnet being less effective in each case. P-tau accumulation was found to depend on HSV1 DNA replication, whereas Aβ accumulation was not. The antiviral-induced decrease in Aβ is attributable to the reduced number of new viruses, and hence the reduction in viral spread. Since antiviral agents reduce greatly Aβ and P-tau accumulation in HSV1-infected cells, they would be suitable for treating AD with great advantage unlike current AD therapies, only the virus, not the host cell, would be targeted.     

Characterization of two TCR transgenic mouse lines specific for herpes simplex virus

To better understand the T cell-mediated processes involved in the immune response to herpes simplex virus type 1 (HSV-1)infection, two HSV-specific T cell receptor (TCR) transgenic mouse lines were produced. These mice (gBT-I.1 and gBT-I.3) are MHC class I-restricted and specific for the immunodominant peptide from HSV glycoprotein B (gB), gB498-505. Although derived from the same clone, the mice differ in the chromosomal location of the TCR transgenes and show marked differences in TCR alpha/beta expression on both CD4+ and CD8+ cells in the thymus. Despite this, peripheral CD8+ Tcells from both mice express equally high levels of the transgenic TCR and bind the KbgB498-505 tetramer to the same degree. In concordance with this, both were shown to respond equally well in vitro upon stimulation with the gB498-505 peptide or HSV-infected cells. These data show that selection of broadly equivalent peripheral T-cell subsets can occur in the presence of distinctly different thymic T-cell subsets.     

Links Between the Pathology of Alzheimer's Disease and Vascular Dementia

The major neuropathological lesions defining Alzheimer's disease (AD) include neurofibrillary tangles and amyloid plaques, which are mainly composed of abnormally phosphorylated tau and amyloid-beta (A beta), respectively. Numerous neuropathological and neuroimaging studies indicate that at least one-third of AD cases are complicated by some degree of vascular pathology, whereas in a similar proportion of patients clinically diagnosed with vascular dementia, AD pathology is also present. Many classical vascular risk factors such as hypertension, diabetes mellitus, and hypercholesterolemia have recently been shown also to increase the risk of AD. Growing evidence suggests that vascular pathology lowers the threshold for the clinical presentation of dementia at a given level of AD-related pathology and potentially directly promotes AD lesions such as A beta plaques. Cerebral ischemia, chronically up-regulates expression of the amyloid precursor protein (APP), which is the precursor to the amyloid beta peptide and damages the blood-brain barrier (BBB), affecting A beta peptide clearance from the brain. Recognition of the importance of these vascular risk factors for AD-related dementia and their treatment will be beneficial not only for preventing cardiac, cerebral, and peripheral complications of vascular disease, but also will likely have a direct impact on the occurrence of sporadic AD in older subjects. In this paper, we review some of the links between vascular risk factors and AD pathology and present data on the direct effect of ischemia on cognitive function and A beta deposition in a mouse model of AD.     

Intracellular trafficking and maturation of herpes simplex virus type 1 gB and virus egress require functional biogenesis of multivesicular bodies

The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.     

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.     

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.     

Infectious etiology and amyloidosis in Alzheimer's disease: The puzzle continues

Recent studies have renewed the debate on infectious etiology in late-onset Alzheimer''s disease. Bocharova et al. reported that abundant expression of human beta amyloid (Aβ) in the mouse brain (5XFAD animals) failed to protect against acute herpes simplex virus type 1 infection relative to control mice. While this study does not confirm the antiviral actions of Aβ, it neither supports nor disproves the hypothesis that infection with microbial pathogens is the major cause of Alzheimer''s disease.

Alzheimer''s disease (AD) is a devastating and progressive neurodegenerative disorder and the most common form of dementia in the elderly. AD is associated with the pathological deposition of neurofibrillary tau tangles and extracellular amyloid β (Aβ) plaques as well as with chronic neuroinflammation. Numerous studies showed that specific microbial infections, including with herpes simplex virus type 1 (HSV1), Chlamydia pneumoniae, and several types of spirochaete are linked to AD etiology, as these pathogens have been detected in some AD brains, particularly in senile plaques (1). HSV1 is the most common of these and strongly links human pathogens to AD etiology (supported by over 100 publications reporting direct or indirect association), whereas only a handful of reports are contradictory. The following lines of evidence support this connection: (i) the presence of viruses and other microbes in the brain of most elderly individuals; (ii) in AD brains, pathogen signatures like HSV1 DNA specifically colocalize with AD amyloid plaques; (iii) active HSV1 infection can trigger chronic neuroinflammatory responses that lead to herpes simplex encephalitis, known to cause severe damage in brain regions associated with memory and cognitive functions; (iv) circulating levels of anti-HSV antibodies, an indicator of HSV1 reactivation, is positively correlated with AD pathology; (v) HSV1 infection activates neurotoxic pathways and causes an AD-like phenotype in mice, while in clinical studies, treatment with antiherpetic drugs like valacyclovir show improved cognitive functions in patients with AD compared with controls; and (vi) key features of AD pathology are transmissible upon intracerebral injection of AD brain homogenates (1, 2), indicating that microbial infection could represent an important contributor to late-onset AD and supporting “the Aβ antimicrobial protection hypothesis.” This hypothesis proposes that Aβ deposition is an early response against microbial infection, which consequently drives chronic neuroinflammation and neurodegeneration, which is not necessarily contrary to with the established “amyloid hypothesis” that proposes that amyloidosis of Aβ is the root cause of AD (1). Recently, contrasting evidence was provided to continue the debate on the antiviral role of Aβ and the causative role of HSV1 infection in AD and to highlight that chronic neuroinflammation could represent a central mechanism in infectious etiology of late-onset AD (Fig. 1).Open in a separate windowFigure 1Schematic presentation of AD pathogenesis according to the “Aβ antimicrobial protection hypothesis” and the “amyloid β hypothesis.” Chronic microbial infections activate the immune system and lead to sustained inflammatory responses, which allow penetration of microbial pathogens and/or their products to cross the blood–brain barrier. In the brain, they colocalize with Aβ and induce Aβ fibrillation to form senile plaques. Disruption of the blood–brain barrier causes penetration of peripheral inflammatory molecules as well as inflammatory cells into the brain and promotes gliosis. Furthermore, various risk factors, including genetics, stress, sleep, diet, head injury, and aging, influence disease progression. Together, these form a vicious inflammatory response, perpetuated by chronic infections or reactivation, which further stimulate chronic neuroinflammation. This process eventually leads to neuron death and AD pathology. AD, Alzheimer''s disease.Previously, an elegant and high profile study by Eimer et al. (3) reported that human Aβ interacts with viral surface glycoproteins, which mediates Aβ oligomerization and viral entrapment, leading to protection against HSV1 infection in AD mice and 3D human neuronal cell cultures. Others also showed that Aβ amyloidosis protects against bacterial and fungal infections in animal and worm models of AD (4), suggesting that in addition to various genetic, biochemical, and environmental factors, microbial infection could represent one of the possible triggers of amyloidosis in AD neuropathology (Fig. 1).In a recent article in the Journal of Biochemistry, Bocharova et al. (5) questioned the antiviral properties of Aβ and the role of viral infection in AD pathology in contrast to the findings of Eimer et al. (3), demonstrating protective antiherpetic actions mediated by human Aβ-expressing mice (5XFAD mice). Bocharova et al. used three different age groups of 5XFAD mice expressing transgenes for mutant human amyloid precursor protein and human presenilin 1, one of the four core proteins of the gamma secretase complex that generates Aβ from amyloid precursor protein, with five AD-linked mutations. WT control and 5XFAD mice were infected with three different doses of two strains of HSV1 ranging from 5- to 10-fold below or above the dose lethal to 50% of the mice (LD₅₀). Despite the use of different viral strains, doses, and ages of the animals, survival analysis revealed no statistically significant differences between 5XFAD and WT mice, which confirmed the lack of protective effect of the 5XFAD genotype against HSV1-induced encephalitis (5). Bocharova et al. also noted the region-specific or cell-specific tropisms of HSV1 strains that were not affected in 5XFAD mice compared with controls, which suggested that host–pathogen interactions remained unaltered by Aβ overexpression. Also contrary to the study by Eimer et al. (3), Bocharova et al. found no evidence of HSV1-induced Aβ aggregation and Aβ-mediated viral entrapment. This observation was attributed to the microglia transitioning to a chronic reactive stage and subsequently phagocytosing the virus in Aβ aggregate-dense brain regions. Reactive microglia, often seen in human AD brains, might cause chronic neuroinflammation in response to recurrent reactivation or repetitive HSV1 infections (6). This repeated and sustained microglia activation could eventually potentiate deregulated chronic neuroinflammation and development of AD pathology (7).These studies raise obvious fundamental questions, such as what are the targets for antimicrobial actions of Aβ in human? It is noteworthy to mention that mouse models are not natural hosts for HSV1 and hence do not adequately mimic spontaneous viral shedding or recurrent symptomatic diseases in humans (8). Furthermore, antimicrobial proteins show varying activity against different microbial pathogens, and therefore, the lack of protection against HSV1 infection in 5XFAD mice contrasts the strong protective effects against bacterial and fungal infections (5). This could correlate with diverse microbial infections in human brain. The discrepancies between the two studies could also be due to the use of different doses and variations of HSV1 strains. Nevertheless, the current work neither supports nor refutes the hypothesis of the viral etiology of late-onset AD. Indeed, since no protection was found against acute HSV1 infection in 5XFAD mice, it indicates that viral pathogens could even increase the risk of late-onset AD through multiple Aβ-dependent and independent mechanisms.In the future, it would be of interest to examine the antimicrobial actions of Aβ against diverse microbial pathogens, including several types of spirochete, Treponema pallidum, C. pneumoniae, and the protozoan Toxoplasma gondii, implicated in human brain diseases. Certainly, HSV1 is not the sole contributor to late-onset AD, as it is a multifactorial disease with many contributing factors (including other potential pathogens). HSV1 may contribute to a minority of AD cases, and even this etiology may depend on the presence or the absence of other risk factors such as genetic factors (apolipoprotein E4 variant carriers), age, stress, sleep, diet, head injury, cardiovascular disease, and many others (7). Collectively, these contradictory studies question the contributory role of HSV1 to AD development and the antiviral activity of Aβ. Fundamentally, the findings of Bocharova et al. (5) support the notion that chronic infections with viral, bacterial, and fungal pathogens might cause deregulated neuroinflammatory responses, which subsequently increase amyloidosis in the brain and contribute to AD pathogenesis (2, 9, 10). Thus, inflammatory responses against infections might provide the missing link between infectious etiology and late-onset AD, if such a link exists.It is worth mentioning that the infectious etiology in late-onset AD is a puzzle not yet solved, as—at least so far—no specific microbial infection has been conclusively linked to causation of AD in humans. However, interesting data from many laboratories renewed the concern of infectious etiology in late-onset AD, which will provide new opportunities for anti-inflammatory therapy development.     

Alzheimer disease: mercury as pathogenetic factor and apolipoprotein E as a moderator

The etiology of most cases of Alzheimer's disease (AD) is as yet unknown. Epidemiological studies suggest that environmental factors may be involved beside genetic risk factors. Some studies have shown higher mercury concentrations in brains of deceased and in blood of living patients with Alzheimer's disease. Experimental studies have found that even smallest amounts of mercury but no other metals in low concentrations were able to cause all nerve cell changes, which are typical for Alzheimer's disease. The most important genetic risk factor for sporadic Alzheimer's disease is the presence of the apolipoprotein Ee4 allele whereas the apolipoprotein Ee2 allele reduces the risk of developing Alzheimer's disease. Some investigators have suggested that apolipoprotein Ee4 has a reduced ability to bind metals like mercury and therefore explain the higher risk for Alzheimer's disease. Therapeutic approaches embrace pharmaceuticals which bind metals in the brain of patients with Alzheimer's disease. In sum, both the findings from epidemiological and demographical studies, the frequency of amalgam application in industrialized countries, clinical studies, experimental studies and the dental state of AD patients in comparison to controls suggest a decisive role for inorganic mercury in the etiology of AD.     

Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides.

Cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV) polypeptides play an important role in recovery from infection and in preventing latency. We have previously shown that glycoprotein B (gB) is a major target recognized by HSV-specific CTLs in C57BL/6 (H-2b) and BALB/c (H-2d) mice but not in CBA/J (H-2k) mice (L. A. Witmer, K. L. Rosenthal, F. L. Graham, H. M. Friedman, A. Yee, and D. C. Johnson, J. Gen. Virol. 71:387-396, 1990). In this report, we utilize adenovirus vectors expressing gB with various deletions to localize an immunodominant site in gB, recognized by H-2b-restricted anti-HSV CTLs, to a region between residues 462 and 594. Overlapping peptides spanning this region were synthesized and used to further localize the immunodominant site to residues 489 to 515, a region highly conserved in HSV type 1 (HSV-1) and HSV-2 strains. The 11-amino-acid peptide was apparently associated exclusively with the Kb major histocompatibility complex gene product and not the Db gene product. In contrast, H-2d-restricted CTLs recognized an immunodominant site between residues 233 and 379.     

Human alpha- and beta-defensins block multiple steps in herpes simplex virus infection

This study examined the ability of nine human defensins (HD) to protect against herpes simplex virus infection. Noncytotoxic concentrations of all six alpha-defensins (HNP1-4, HD5, and HD6) and human beta-defensin (hBD) 3 inhibited HSV infection. Two other beta-defensins, hBD1 and 2, lacked this protective activity. Synchronized assays revealed that HNP-4, HD6, and hBD3 acted primarily by preventing binding and entry, whereas HNP1-3 and HD5 also inhibited postentry events. Even when added several hours after entry, substantial reduction in viral gene expression ensued. Human cervical epithelial cells incubated with HNP-1 or HD5 accumulated the peptides intracellularly. Surface plasmon resonance studies revealed that HNPs 1, 2, 3, and HD5 bound HSV glycoprotein B (gB) with high affinity, but showed minimal binding to heparan sulfate, the receptor for attachment. In contrast, HNP-4 and HD6 bound heparan sulfate, but not gB. HBD3 bound both gB and heparan sulfate, but hBD1 and hBD2 bound neither. Admixture of HD5 with hydroxyethylcellulose significantly protected mice from a viral challenge lethal to controls receiving an inactive peptide or hydroxyethylcellulose alone. These findings demonstrate that HDs act at multiple steps in the HSV life cycle and support the development of defensins or defensin-like peptides as microbicides.     

Codelivery of CCR7 ligands as molecular adjuvants enhances the protective immune response against herpes simplex virus type 1

Humoral and cellular immunity, associated with long-term protective immunological memory, defines the efficacy of a given vaccine formulation. However, few vaccines achieve this target without the aid of a suitable adjuvant. Molecular adjuvants in vaccination against infectious agents offer a noninvasive means of enhancing the immune response against target antigens. To examine the potency of two beta-chemokines as immunomodulators, plasmid DNA encoding beta-chemokines CCL19 and CCL21 (CCR7L) was codelivered intranasally with plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus (HSV) gB (HSV-gB) in a prime-and-boost vaccination strategy. This vaccination regimen increased serum and vaginal immunoglobulin G (IgG) and IgA, respectively, as well as the numbers of HSV-gB(498-505) peptide-specific gamma interferon-producing CD8(+) T cells. Distinctively, a high number of cytotoxic T lymphocytes was achieved when pCCR7L was applied at both prime and boost as opposed to omission of pCCR7L. A rapid-recall response was induced in the genital tract upon challenge with the HSV McKrae strain, affording a high level of protection and survival of vaccinated mice. Our results demonstrate that high innate immune kinetics and distribution of adaptive response induced in the nasal mucosa appears to be key factors in generating protective memory responses against HSV. Thus CCR7L expressed ectopically may serve as a molecular adjuvant to boost the immune response to a codelivered antigen in mucosal surfaces.     

Genetic Analysis of Interleukin-1A C(-889)T Polymorphism with Alzheimer Disease

Neuroinflammation has been implicated in the etiology of Alzheimer’s disease (AD). Many studies have suggested that C(-889) T promoter polymorphism in one of the proinflammatory cytokine interleukin-1 (IL-1) encoding gene IL-1A may be associated with AD pathogenesis. To determine whether the polymorphism contributes to the risk for late-onset AD (LOAD) in Chinese, we carried out our investigation in 344 sporadic LOAD patients and 224 healthy controls. No statistical significant association was obtained between IL-1A C(-889) T polymorphism and LOAD and no statistical difference was found between cases and controls after stratification for apolipoprotein E allele 4 (APOE ε4) status. The results reveal that it is not likely that the IL-1A C(-889) T polymorphism is involved in AD pathogenesis in the Chinese population. Further studies of the associations between other IL-1A genetic polymorphisms and AD should be performed in a larger population and biologic functional analysis of IL-1A gene is required to verify the underlying roles of IL-IA in LOAD.     

Glycoprotein gB (gII) of pseudorabies virus can functionally substitute for glycoprotein gB in herpes simplex virus type 1.

Glycoproteins homologous to gB of herpes simplex virus (HSV) constitute the most highly conserved family of herpesvirus glycoproteins. All gB homologs analyzed so far have been shown to play essential roles in penetration and direct viral cell-to-cell spread. In studies aimed at assessing whether the high sequence homology is also indicative of functional homology, we analyzed the ability of the gB-homologous glycoprotein (former designation gII) of pseudorabies virus (PrV) to complement a gB- HSV type 1 (HSV-1) mutant and vice versa. The results show that a PrV gB-expressing cell line phenotypically complemented the lethal defect in gB- HSV-1 whereas reciprocal complementation of a gB- PrV mutant by HSV-1 gB was not observed.     

The ectodomain of herpes simplex virus glycoprotein H contains a membrane alpha-helix with attributes of an internal fusion peptide, positionally conserved in the herpesviridae family

Human herpesviruses enter cells by fusion with target membranes, a process that requires three conserved glycoproteins: gB, gH, and gL. How these glycoproteins execute fusion is unknown. Neural network bioinformatics predicted a membrane alpha-helix contained within the ectodomain of herpes simplex virus (HSV) gH, positionally conserved in the gH of all examined herpesviruses. Evidence that it has attributes of an internal fusion peptide rests on the following lines of evidence. (i) The predicted membrane alpha-helix has the attribute of a membrane segment, since it transformed a soluble form of gD into a membrane-bound gD. (ii) It represents a critical domain of gH. Its partial or entire deletion, or substitution of critical residues inhibited HSV infectivity and fusion in the cell-cell fusion assay. (iii) Its replacement with the fusion peptide from human immunodeficiency virus gp41 or from vesicular stomatitis virus G partially rescued HSV infectivity and cell-cell fusion. The corresponding antisense sequences did not. (iv) The predicted alpha-helix located in the varicella-zoster virus gH ectodomain can functionally substitute the native HSV gH membrane alpha-helix, suggesting a conserved function in the human herpesviruses. We conclude that HSV gH exhibits features typical of viral fusion glycoproteins and that this property is likely conserved in the Herpesviridae family.     

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.     

Immunodominant epitopes in herpes simplex virus type 2 glycoprotein D are recognized by CD4 lymphocytes from both HSV-1 and HSV-2 seropositive subjects

In human recurrent cutaneous herpes simplex, there is a sequential infiltrate of CD4 and then CD8 lymphocytes into lesions. CD4 lymphocytes are the major producers of the key cytokine IFN-gamma in lesions. They recognize mainly structural proteins and especially glycoproteins D and B (gD and gB) when restimulated in vitro. Recent human vaccine trials using recombinant gD showed partial protection of HSV seronegative women against genital herpes disease and also, in placebo recipients, showed protection by prior HSV1 infection. In this study, we have defined immunodominant peptide epitopes recognized by 8 HSV1(+) and/or 16 HSV2(+) patients using (51)Cr-release cytotoxicity and IFN-gamma ELISPOT assays. Using a set of 39 overlapping 20-mer peptides, more than six immunodominant epitopes were defined in gD2 (two to six peptide epitopes were recognized for each subject). Further fine mapping of these responses for 4 of the 20-mers, using a panel of 9 internal 12-mers for each 20-mers, combined with MHC II typing and also direct in vitro binding assay of these peptides to individual DR molecules, showed more than one epitope per 20-mers and promiscuous binding of individual 20-mers and 12-mers to multiple DR types. All four 20-mer peptides were cross-recognized by both HSV1(+)/HSV2(-) and HSV1(-)/HSV2(+) subjects, but the sites of recognition differed within the 20-mers where their sequences were divergent. This work provides a basis for CD4 lymphocyte cross-recognition of gD2 and possibly cross-protection observed in previous clinical studies and in vaccine trials.     

The nucleotide sequence of herpes simplex virus type 2 (333) glycoprotein gB2 and analysis of predicted antigenic sites

The gene coding for glycoprotein B2 (gB2) of herpes simplex virus type 2 (HSV-2) strain 333 was mapped and its nucleotide sequence determined. Open reading frame analysis deduced a polypeptide consisting of 902 amino acids and having close homology to gB1 of HSV type 1. Several predicted features of gB2 are consistent with a membrane-bound glycoprotein, i.e., a signal peptide sequence, a hydrophilic extracellular domain containing possible N-linked glycosylation sites, a hydrophobic membrane spanning sequence, and a cytoplasmic domain. Computer analysis on hydrophilicity, accessibility, and flexibility of the gB2 amino acid sequence, produced a composite surface value plot. At least nine major antigenic regions were predicted on the extracellular domain. The amino acids between residues 59-74, 127-139, 199-205, 460-476, and 580-594 exhibited the highest surface values. Comparison of the primary sequence with gB1 revealed localized regions showing amino acid diversity. Several of these locations correspond to major antigenic regions. Chou and Fasman analyses indicated that the amino acid substitutions, between positions 57-66, 461-472, and 473-481, induced changes in the secondary structure of gB. These sites could represent site-specific epitopes in the gB polypeptide.     

A heptad repeat in herpes simplex virus 1 gH, located downstream of the alpha-helix with attributes of a fusion peptide, is critical for virus entry and fusion

Entry of herpes simplex virus 1 (HSV-1) into cells occurs by fusion with cell membranes; it requires gD as the receptor binding glycoprotein and the trigger of fusion, and the trio of the conserved glycoproteins gB, gH, and gL to execute fusion. Recently, we reported that the ectodomain of HSV-1 gH carries a hydrophobic alpha-helix (residues 377 to 397) with attributes of an internal fusion peptide (T. Gianni, P. L. Martelli, R. Casadio, and G. Campadelli-Fiume, J. Virol. 79:2931-2940, 2005). Downstream of this alpha-helix, a heptad repeat (HR) with a high propensity to form a coiled coil was predicted between residues 443 and 471 and was designated HR-1. The simultaneous substitution of two amino acids in HR-1 (E450G and L453A), predicted to abolish the coiled coil, abolished the ability of gH to complement the infectivity of a gH-null HSV mutant. When coexpressed with gB, gD, and gL, the mutant gH was unable to promote cell-cell fusion. These defects were not attributed to a defect in heterodimer formation with gL, the gH chaperone, or in trafficking to the plasma membrane. A 25-amino-acid synthetic peptide with the sequence of HR-1 (pep-gH(wt25)) inhibited HSV replication if present at the time of virus entry into the cell. A scrambled peptide had no effect. The effect was specific, as pep-gH(wt25) did not reduce HSV-2 and pseudorabies virus infection. The presence of a functional HR in the HSV-1 gH ectodomain strengthens the view that gH has attributes typical of a viral fusion glycoprotein.