Cellular expression of gH confers resistance to herpes simplex virus type-1 entry

Entry of herpes simplex virus-1 (HSV-1) into cells requires a concerted action of four viral glycoproteins gB, gD, and gH-gL. Previously, cell surface expression of gD had been shown to confer resistance to HSV-1 entry. To investigate any similar effects caused by other entry glycoproteins, gB and gH-gL were coexpressed with Nectin-1 in Chinese hamster ovary (CHO) cells. Interestingly, cellular expression of gB had no effect on HSV-1(KOS) entry. In contrast, entry was significantly reduced in cells expressing gH-gL. This effect was further analyzed by expressing gH and gL separately. Cells expressing gL were normally susceptible, whereas gH-expressing cells were significantly resistant. Further experiments suggested that the gH-mediated interference phenomenon was not specific to any particular gD receptor and was also observed in gH-expressing HeLa cells. Moreover, contrary to a previous report, gL-independent cell surface expression of gH was detected in stably transfected CHO cells, possibly implicating cell surface gH in the interference phenomenon. Thus, taken together these findings indicate that cellular expression of gH interferes with HSV-1 entry.     

Computer predictions of antigenic domains in herpes simplex virus types 1 and 2 glycoprotein D as compared with experimentally proven domains

The primary amino-acid sequence of the glycoprotein D (gD) of herpes simplex virus types 1 and 2 (HSV-1, HSV-2) was analyzed by computer programs that provided values for hydrophilicity, surface probability, flexibility, and antigenicity, as well as the secondary structure conformation. Putative antigenic domains with a high hydrophilicity, surface probability, and antigenicity index were determined and compared with the reported antigenic domains in HSV-1 and HSV-2 gD protein based on experimental data. The major experimentally proven antigenic domains were detected by the computer analyses. Additional putative antigenic domains with potential for the synthesis of antigenic viral peptides were determined.     

Enhanced resistance to herpes simplex virus type 1 infection in transgenic mice expressing a soluble form of herpesvirus entry mediator

Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). Both human and mouse forms of HVEM can mediate entry of HSV-1 but have no entry activity for pseudorabies virus (PRV). To assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMIg) consisting of an extracellular domain of murine HVEM and the Fc portion of human IgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection when the mice were challenged intraperitoneally with HSV-1, but not to PRV infection. The present results demonstrate that HVEMIg is able to exert a significant antiviral effect against HSV-1 infection in vivo.     

Mapping of linear antigenic determinants on glycoprotein C of herpes simplex virus type 1 and type 2 recognized by human serum immunoglobulin G antibodies

Using membrane-based dekapeptides, the reactivity of human serum antibodies with linear antigenic determinants of herpes simplex virus (HSV) type 1 and type 2 glycoprotein C (gC-1, gC-2) was studied by pep scan and immunodot assay. The entire coding sequences of gC-1 and gC-2 were screened for the presence of linear epitopes by pep scan. Peptides recognized in an HSV-1 type-specific manner were mainly identified within the N-terminal third and at the C-terminus of gC-1, whereas most type-common antibodies were directed against colinear peptides within the central parts of gC-1 and gC-2. The type-specific reaction of human sera with gC-2 peptides in pep scan was poor. Eight peptides identified as immunoreactive by pep scan were further tested in immunodot assay for their reactivity with a human serum panel. None of the eight HSV-negative sera gave positive results by immunodot assay. Positive reactions with gC peptides were found to be strongly age-dependent, i.e., the rate of positive reactions was significantly higher in HSV-positive adults than in HSV-positive children. Antibody reactivity with two type-common gC peptides was demonstrated in 17 out of 28 HSV-positive sera. A putative type-specific gC-2 peptide employed in immunodot assay was inconsistently recognized by human sera. Twenty HSV-positive sera reacted with at least 1 of 5 type-specific gC-1 peptides. Nine sera showing no reactivity with glycoprotein G of HSV-1 (gG-1) by immunoblotting recognized type-specific gC-1 peptides in immunodot assay. Thus, gC-1 peptides might allow the detection of HSV-1-specific antibodies in individuals showing no reactivity with commonly employed HSV-1-specific diagnostic antigens, i.e., purified or recombinant gG-1. J. Med. Virol. 55:281–287, 1998. © 1998 Wiley-Liss, Inc.     

Human antibodies to herpes simplex virus type 1 glycoprotein C are neutralizing and target the heparan sulfate-binding domain

Human antibodies specific for glycoprotein C (gC1) of herpes simplex virus type 1 (HSV-1) neutralized the virus infectivity and efficiently inhibited attachment of HSV-1 to human HaCaT keratinocytes and to murine mutant L cells expressing either heparan sulfate or chondroitin sulfate at the cell surface. Similar activities were observed with anti-gC1 monoclonal antibody B1C1. In addition to HaCaT and L cells, B1C1 antibody neutralized HSV-1 infectivity in simian GMK AH1 cells mildly pre-treated with heparinase III. Human anti-gC1 antibodies efficiently competed with the binding of gC1 to B1C1 antibody whose epitope overlaps a part of the attachment domain of gC1. Human anti-gC1 and B1C1 antibodies extended survival time of mice experimentally infected with HSV-1. We conclude that in HaCaT cells and in cell systems showing restricted expression of glycosaminoglycans, human and some monoclonal anti-gC1 antibodies can target the cell-binding domain of this protein and neutralize viral infectivity.     

Effects of linker-insertion mutations in herpes simplex virus 1 gD on glycoprotein-induced fusion with cells expressing HVEM or nectin-1

Several cell surface molecules, including HVEM and nectin-1, can serve as entry receptors for herpes simplex virus (HSV) and as receptors for virus-induced or viral glycoprotein-induced cell fusion. The viral ligand for these receptors is the HSV envelope glycoprotein gD. A set of linker-insertion and deletion mutants of HSV type 1 (HSV-1) gD was analyzed for effects of the mutations on binding of gD to HVEM and nectin-1, on viral glycoprotein-induced cell fusion with target cells expressing HVEM or nectin-1 and on complementation of infectivity of a gD-null HSV-1 viral mutant. Insertions after amino acid 151 or 225 or deletion of amino acids 234-244 disrupted (i) binding of the mutant forms of gD to both receptors and (ii) functional interactions (cell fusion and complementation) with both receptors, but were without effect on cell surface expression. Insertions in the N-terminal domain of gD (after amino acid 12, 34 or 43) disrupted binding to HVEM and functional activities with HVEM, as expected from a previously reported X-ray structure of a gD-HVEM complex, but were without effect in the case of nectin-1. These and other results indicate that the mutations disruptive of interactions with both receptors probably affect conformations of contact sites that are different for each receptor.     

Mutagenic analysis of herpes simplex virus type 1 glycoprotein L reveals the importance of an arginine-rich region for function

Herpes simplex virus type 1 (HSV-1) glycoproteins H and L (gH and gL) are required for virus-induced membrane fusion. Expression of gH at the virion or infected cell surface is mediated by the chaperone-like activity of gL. We have previously shown that a region between amino acids 155 and 161 is critical for gL chaperone-like activity. Here, we conducted Ala substitution mutagenesis of residues in this region and found that substitution of Cys160, Arg156, Arg158, or Arg156/158/159 with Ala resulted in a gL mutant that bound gH but displayed a reduced ability in gH trafficking and membrane fusion. Substitution of Arg156 with another positively charged amino acid, Lys, restored function. Substitution of Arg158 with Lys restored function in gH trafficking and cell fusion but not virus entry. These results indicate that an arginine-rich region of gL is critical for function.     

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.     

Fusion activity of lipid-anchored envelope glycoproteins of herpes simplex virus type 1

Expression of the herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD, gH, and gL is necessary and sufficient to cause cell fusion. To identify the requirements for a membrane-spanning domain in HSV-1 glycoprotein-induced cell fusion, we created gB, gD, and gH mutants with transmembrane and cytoplasmic domains replaced by a glycosylphosphatidylinositol (gpi)-addition sequence. The corresponding gBgpi, gDgpi, and gHgpi proteins were expressed with wild-type efficiency at the cell surface and were linked to the plasma membrane via a gpi anchor. The gDgpi mutant promoted cell fusion near wild-type gD levels when co-expressed with gB, gH, and gL in a cell-mixing fusion assay, indicating that the gD transmembrane and cytoplasmic domains were not required for fusion activity. A plasma membrane link was required for fusion because a gD mutant lacking a transmembrane and cytoplasmic domain was nonfunctional for fusion. The gDgpi mutant was also able to cooperate with wild-type gB, gH, and gL to form syncytia, albeit at a size smaller than those formed in the wild-type situation. The gBgpi and gHgpi mutants were unable to promote fusion when expressed with the other wild-type viral glycoproteins, highlighting the requirement of the specific transmembrane and cytoplasmic domains for gB and gH function.     

巢式PCR检测龈下菌斑中HCMV、EBV、HSV-1与慢性牙周炎活动性的关系

目的　建立临床检测龈下菌斑标本中人巨细胞病毒 (HCMV)、Epstein Barr病毒 (EBV)、单纯疱疹病毒 1型 (HSV 1 )巢式PCR方法 ,研究这 3种病毒与慢性牙周炎活动性的关系。方法　收集6 2例慢性牙周炎患者 (男性 2 7例、女性 35例 ,平均年龄 5 3岁 )的牙周炎活动部位、牙周炎静止部位的龈下菌斑 ,提取DNA后使用巢式PCR检测HCMV、EBV、HSV 1 ,比较分析其在同一病人不同部位的检出率。结果　牙周炎活动部位的HCMV检出率为 38.7%,EBV的检出率为 5 8%,HSV 1的检出率为30 .6 %,两种以上病毒合并感染的检出率为 4 0 .3%;牙周炎静止部位的HCMV检出率为 1 4 .5 %,EBV为 2 2 .6 %,HSV 1为 1 1 .3%,两种以上病毒合并感染的检出率为 1 1 .3%。这 3种病毒及其合并感染在牙周炎活动部位的检出率均高于牙周炎静止部位 ,差异有统计学意义 (P <0 .0 5 )。结论　提示HC MV、EBV、HSV 1与慢性牙周炎的活动性相关。     

Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger events leading to cell fusion and viral entry

One of the herpes simplex virus envelope glycoproteins, designated gD, is the principal determinant of cell recognition for viral entry. Other viral glycoproteins, gB, gH and gL, cooperate with gD to mediate the membrane fusion that is required for viral entry and cell fusion. Membrane fusion is triggered by the binding of gD to one of its receptors. These receptors belong to three different classes of cell surface molecules. This review summarizes recent findings on the structure and function of gD. The results presented indicate that gD may assume more than one conformation, one in the absence of receptor, another when gD is bound to the herpesvirus entry mediator, a member of the TNF receptor family, and a third when gD is bound to nectin-1, a cell adhesion molecule in the immunoglobulin superfamily. Finally, information and ideas are presented about a membrane-proximal region of gD that is required for membrane fusion, but not for receptor binding, and that may have a role in activating the fusogenic activity of gB, gH and gL.     

Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes

HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2.     

单纯疱疹病毒1型糖蛋白D主要抗原表位区的表达、纯化及鉴定

 目的 原核表达单纯疱疹病毒1型(HSV-1) 糖蛋白D(gD)主要抗原表位区,纯化融合表达蛋白并对其进行鉴定。方法 用Lasergene7.0中Protean软件对HSV-1 gD氨基酸序列进行抗原表位预测和分析,筛选出gD主要抗原表位区(gD MED),人工合成该区域cDNA序列,构建原核表达载体pET-GST-gD MED;将重组质粒转化大肠杆菌(E.coli)BL21(DE3) pLysS,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,通过GST?Bind 纯化试剂盒对gD MED融合蛋白进行纯化;Western blot对gD MED融合蛋白的免疫活性进行分析。结果 HSV-1 gD在266~394区域富含抗原表位,人工合成了该区域的cDNA序列,并成功构建了原核表达载体pET-GST-gD MED;gD MED融合蛋白主要以可溶形式表达,分子质量约为45 ku,纯度能达95%;Western blot结果显示,gD MED融合蛋白能与感染HSV-1患者的血清发生特异结合。结论 成功表达和纯化了具有良好抗原性的HSV-1 gD MED融合蛋白,为HSV免疫诊断试剂盒和基因工程疫苗的研制奠定了基础。     

Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.     

Genomic variation among herpes simplex virus type 1 strains: virus DNA analysis of isolates from Saudi patients.

Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes HindIII and BamHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with HindIII restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BamHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise to the observed DNA polymorphism were mapped to the unique region of the long and short segments of the genome.     

Site-directed mutagenesis in the active site of the herpes simplex virus type 1 thymidine kinase gene

The thymidine kinase (TK) of herpes simplex virus type 1 (HSV-1) contains three regions of homology to other ATP utilizing enzymes. We have altered one region of the protein, which seems to play an important role in phosphorylating substrates by site-directed mutagenesis. When the aspartate 162 was changed to asparagine, the enzyme lost its activity. To identify the inactive protein, expressed by a vaccinia vector in eukaryotic cells, a monospecific antiserum against a bacterial tryptophan E-HSV-1 TK fusion protein was made. These results support the suggestion that aspartate 162 is essential for the enzymatic activity.     

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.     

Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity

Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus-cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.     

Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry

Entry of herpes simplex virus (HSV) occurs either by fusion at the plasma membrane or by endocytosis and fusion with an endosome. Binding of glycoprotein D (gD) to a receptor such as nectin-1 is essential in both cases. We show that virion gD triggered the rapid down-regulation of nectin-1 with kinetics similar to those of virus entry. In contrast, nectin-1 was not constitutively recycled from the surface of uninfected cells. Both the nectin-1α and β isoforms were internalized in response to gD despite having different cytoplasmic tails. However, deletion of the nectin-1 cytoplasmic tail slowed down-regulation of nectin-1 and internalization of virions. These data suggest that nectin-1 interaction with a cytoplasmic protein is not required for its down-regulation. Overall, this study shows that gD binding actively induces the rapid internalization of various forms of nectin-1. We suggest that HSV activates a nectin-1 internalization pathway to use for endocytic entry.     

Reactivation of herpes simplex type 1 in pneumococcal meningitis

BackgroundAcute bacterial meningitis (ABM) and herpes simplex type 1 (HSV-1) encephalitis are two rare but serious infections affecting the central nervous system (CNS). Concurrent bacterial and viral CNS infection has occasionally been reported.ObjectivesTo illustrate the possibility of intrathecal infection with both Streptococcus pneumonia and HSV-1 by presenting a case and to examine whether herpesvirus reactivation is common in ABM.Study designWe report a case diagnosed with HSV-1 reactivation in the cerebrospinal fluid (CSF) during treatment for pneumococcal ABM. A retrospective analysis of CSF samples from 21 patients with ABM was performed, with analysis of DNA from HSV-1 and four other neurotropic herpesviruses.ResultsAll 21CSF samples were negative for HSV-1, HSV-2, varicella zoster-virus, Epstein–Barr virus and human herpesvirus 6 DNA by PCR.ConclusionsAlthough herpesvirus infection does not seem to be a common phenomenon in ABM we suggest that HSV-1 reactivation could be kept in mind if patients with ABM show symptoms or signs compatible with encephalitis.