Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Δ290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Δ290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 × 10⁻⁸ M versus 3.2 × 10⁻⁶ M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Δ290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster k_on rather than to a slower k_off. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 × 10⁻⁵ M) than did gD1(306t) due to a more rapid k_off. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别.表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白.重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.     

Examination of the Kinetics of Herpes Simplex Virus Glycoprotein D Binding to the Herpesvirus Entry Mediator, Using Surface Plasmon Resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (K_D) of 3.2 × 10⁻⁶ M and gD2(306t) had a K_D of 1.5 × 10⁻⁶ M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Δ290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (K_D = 3.3 × 10⁻⁸ M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Δ290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

Ⅰ型单纯疱疹病毒 gD基因片段的高效表达及抗原性分析

目的 获得高表达的Ⅰ型单纯疱疹病毒(HSV)被膜糖蛋白gD（简称gD1）基因的工程菌。方法 通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基-β-D-硫代半乳糖苷诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。　结果　PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建pTrxA-gD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pTrxA-gD1重组质粒的大肠杆菌Rosetta诱导后得到了高效达,SDS-PAGE显示表达产物约Mr48000（Dalton）。免疫印迹结果表明表达产物具有较好的抗原性。结论　成功构建了pTrxA-gD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。     

Three-Dimensional Structure of Herpes Simplex Virus Type 1 Glycoprotein D at 2.4-Nanometer Resolution

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.     

Localization of a Passively Transferred Human Recombinant Monoclonal Antibody to Herpes Simplex Virus Glycoprotein D to Infected Nerve Fibers and Sensory Neurons In Vivo

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.     

单纯疱疹病毒糖蛋白D的表达及免疫学鉴定

单纯疱疹病毒(herpes simplex virus,HSV)是TORCH综合征的病原体之一.新生儿可通过宫内、产道和出生后等多种途径感染,大部分患儿呈现症状,如皮炎、角膜炎、口唇疱疹,也可发生涉及多个器官的播散性感染,严重者出现疱疹性脑膜炎,并常导致婴幼儿死亡.目前尚无全身用的特效药物和有效的防范措施.HSV包膜糖蛋白D(glycoprotein D,gD)是极为保守的免疫原性蛋白,在体内可诱导高滴度的中和抗体,因此gD基因成为近些年来诊断研究的靶基因.本文尝试将gD蛋白在酵母菌中表达,并分析其抗原性,为建立快速易行的重组抗原诊断试剂盒奠定基础.此外,利用该表达系统表达的HSV gD蛋白,可为HSV基因工程重组疫苗的研制提供依据,对优生优育、提高人口出生质量具有重要的理论及实际意义.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

Specific Deposition of Passively Transferred Monoclonal Antibodies against Herpes Simplex Virus Type 1 in Rat Brain Infected with the Virus

The kinetics of human monoclonal antibody (anti-gB) to herpes simplex virus type 1 (HSV-1) were investigated after intravenous injection of anti-gB into an HSV-1 encephalitis animal model. Immunohistochemical study revealed specific deposition of passively tansferred anti-gB in the hippocampus and thalamus of the infected rat brain, and it bound to the same neurons in which HSV-1 antigen was positively stained. To examine the macroscopic distribution of anti-gB in the infected brain, we undertook an ¹²⁵I-labeled anti-gB injection study, and the same distribution of ¹²⁵I-labeled anti-gB deposition was observed by brain semimicroautoradiography as in the immunohistochemical study. These results suggest that anti-gB easily permeates the capillary wall and is deposited in the inflammatory site where HSV-1-specific antigen is detectable. The use of radioisotope-labeled anti-gB injection and external brain imaging could lead to a noninvasive diagnostic tool for the early detection of HSV-1 antigen in cases of suspected HSV-1 encephalitis.     

MEK1-ERKs级联激活方式是调控单纯疱疹病毒Ⅱ型复制的重要机制

本研究通过阐明MEK1和MEK2亚型在单纯疱疹病毒Ⅱ型(herpes simplex virus type 2,HSV2)复制中介导的Raf/MEK/ERK(简称ERK)通路活化中的作用,以期进一步阐明该通路调控病毒复制的机制.研究中应用了MEK抑制剂U0126、针对MEK1和MEK2的特异性小干扰RNA(small ...     

Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.