Characterization of Neutralizing Epitopes of Varicella-Zoster Virus Glycoprotein H

Varicella-zoster virus (VZV) glycoprotein H (gH) is the major neutralization target of VZV, and its neutralizing epitope is conformational. Ten neutralizing human monoclonal antibodies to gH were used to map the epitopes by immunohistochemical analysis and were categorized into seven epitope groups. The combinational neutralization efficacy of two epitope groups was not synergistic. Each epitope was partially or completely resistant to concanavalin A blocking of the glycomoiety of gH, and their antibodies inhibited the cell-to-cell spread of infection. The neutralization epitope comprised at least seven independent protein portions of gH that served as the target to inhibit cell-to-cell spread.     

Role of the Varicella-Zoster Virus gB Cytoplasmic Domain in gB Transport and Viral Egress

To study the function of the varicella-zoster virus (VZV) gB cytoplasmic domain during viral infection, we produced a VZV recombinant virus that expresses a truncated form of gB lacking the C-terminal 36 amino acids of its cytoplasmic domain (VZV gB-36). VZV gB-36 replicates in noncomplementing cells and grows at a rate similar to that of native VZV. However, cells infected with VZVgB-36 form extensive syncytia compared to the relatively small syncytia formed during native VZV infection. In addition, electron microscopy shows that very little virus is present on the surfaces of cells infected with VZV gB-36, while cells infected with native VZV exhibit abundant virions on the cell surface. The C-terminal 36 amino acids of the gB cytoplasmic domain have been shown in transfection-based experiments to contain both an endoplasmic reticulum-to-Golgi transport signal (the C-terminal 17 amino acids) and a consensus YXXphi (where Y is tyrosine, X is any amino acid, and phi is any bulky hydrophobic amino acid) signal sequence (YSRV) that mediates the internalization of gB from the plasma membrane. As predicted based on these data, gB-36 expressed during the infection of cultured cells is transported inefficiently to the Golgi. Despite lacking the YSRV signal sequence, gB-36 is internalized from the plasma membrane; however, in contrast to native gB, it fails to localize to the Golgi. Therefore, the C-terminal 36 amino acids of the VZV gB cytoplasmic domain are required for normal viral egress and for both the pre- and post-Golgi transport of gB.     

Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.     

水痘-带状疱疹病毒糖蛋白E在昆虫细胞中的表达、鉴定及免疫原性分析

目的:利用昆虫-杆状表达系统建立表达和纯化分泌形式的水痘-带状疱疹病毒(varicellazoster virus,VZV)糖蛋白gE的方法,并鉴定其理化性质及免疫原性。方法:利用Gibson assembly同源重组试剂盒快速构建重组质粒pFastbac-VZV gE。在sf9昆虫细胞中鉴定序列优化前后表达量的差异,并在High FiveTM细胞中大量表达。通过Ni-NTA亲和层析方法得到高纯度gE蛋白,之后通过酶联免疫吸附试验等验证了其理化性质,并进行小鼠免疫分析其免疫原性。结果:构建了pFastbac-VZV g E1/2重组质粒,经过PCR及双酶切鉴定后均为阳性克隆。使用BAC/PAC试剂盒提取Bacmid转染sf9细胞制备杆状病毒,经Western blot检测,sf9细胞开始表达gE蛋白且随着病毒代次升高g E蛋白表达量增加。序列优化后表达量明显增加,但是大部分以胞内形式存在。通过Ni-NTA一步亲和层析获得纯度较高的gE蛋白,并与VZV单抗9C8具有较好的反应性。通过免疫小鼠产生高滴度抗体,且免疫荧光结果显示其血清可以与天然病毒上的gE抗原结合。结论:成功获得了杆状表达系统表达的gE蛋白并且纯化和鉴定了蛋白质的性质及免疫原性,为进一步研发具有自主产权的VZV亚单位疫苗研究奠定了基础。     

Inhibition of Varicella-Zoster Virus Glycoprotein Expression by Peripheral Blood Mononuclear Cells

The effect of peripheral blood mononuclear cells (PBMC) on expression of varicella-zoster virus (VZV) glycoproteins (Gps) was analyzed by flow cytometry. PBMC from VZV seropositive and seronegative donors and supernatant of PBMC co-cultured with VZV-infected human embryonic fibroblasts reduced VZV Gp expression. Neutralization of supernatant fluid with mixture of anti-interferons (IFN)-α, -β, -γ, and tumor necrosis factor (TNF)-α partially reduced inhibitory activity of supernatant on VZV Gp expression. Deletion of natural killer (NK) cells and adherent cells from PBMC reduced inhibitory activity of PBMC on VZV Gp expression. These results suggest that IFN-α, -β, -γ, TNF-α and other soluble factors released from NK cells and monocytes by co-cultivation with VZV-infected fibroblasts inhibit VZV Gp expression.     

Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation

Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH₄Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.     

Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication,and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Δ⁴⁹¹RSRR⁴⁹⁴ and point mutation ⁴⁹¹GSGG⁴⁹⁴ in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Δ⁴⁹¹RSRR⁴⁹⁴ or ⁴⁹¹GSGG⁴⁹⁴ mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ or pOka-gB⁴⁹¹GSGG⁴⁹⁴ virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16). Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12, 27, 57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10, 19, 38, 48, 51, 52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N- and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homology-like domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long α helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming the core of the trimer making contacts with the two other subunits. The structural homology and lack of furin cleavage suggest that the herpesvirus gB and VSV G proteins have undergone convergent evolution.Although not proven experimentally, VZV gB is likely to be cleaved by the subtilisin-like proprotein convertase furin as the glycoprotein has a furin recognition motif [R]-X-[KR]-R-|-X (where | indicates the position at which the polypeptide is cleaved) (29). The [R]-X-[KR]-R-|-X motif is conserved in gBs for all of the herpesvirus families (5, 9, 21, 36, 40, 53, 63, 64). This site has been shown to be dispensable for the replication of human cytomegalovirus (HCMV), bovine herpesvirus type 1 (BHV-1), and pseudorabies virus (PRV) in vitro (32, 49, 58). Furin site mutants for BHV-1 and PRV show an altered phenotype in vitro, but effects were not examined in vivo. HSV-1 gB is not cleaved and lacks the [R]-X-[KR]-R-|-X motif at the canonical site, which is of interest because HSV-1 is genetically the most closely related human herpesvirus to VZV.Domain I of HSV gB showed structural conservation of putative fusion loops similar to those found in domain IV of the VSV G protein (28). Despite the lack of conserved amino acids within these loops, the hydrophobicity of the residues appears to be conserved for the Herpesviridae (4). Substitution of hydrophobic residues in Epstein-Barr virus gB and linker insertion mutagenesis close to the putative fusion loops of HSV-1 gB abrogated fusion based on in vitro transfection studies (4, 22, 34). However, the effect of substitutions in these putative fusion loops on viral replication has not been characterized. Since the development of fusion assays for VZV has proven elusive, the effect of substitutions in the putative fusion loop using viral mutagenesis to make recombinant viruses provides an alternative approach for identifying functional residues in VZV gB.In contrast to HSV-1, VZV is a human-restricted pathogen (reviewed in reference 16). To study the pathogenesis of VZV in vivo, well-established human xenograft models have been developed using SCID mice (6, 7, 13, 14, 41, 44, 54, 65). Lesions formed by VZV in the skin are similar to those seen in human subjects following primary infection (15, 43). The relevance of the model was demonstrated by studies with the varicella vaccine virus (vOka) that exhibited decreased growth in skin xenografts in vivo but does not cause disease in the healthy human host. In contrast, the vaccine virus and its parent strain, parental Oka (pOka), have indistinguishable replication kinetics in vitro (15, 43).The present study was designed to investigate the effects of structure-based targeted mutations in VZV gB on viral replication in cultured cells and in human skin xenografts in the SCIDhu mouse model. This was performed in the context of infectious virus recovered using the self-excisable bacterial artificial chromosome (BAC) containing the genome of a clinical isolate, Oka (62). The roles of the conserved residues W180 (gB-W180G) and Y185 (gB-Y185G) in the putative fusion loop were evaluated using glycine substitution, and the role of the furin recognition motif (⁴⁹¹RSRR⁴⁹⁴) was assessed by a complete deletion of the furin motif (gBΔ⁴⁹¹RSRR⁴⁹⁴) or a substitution of the arginine residues with glycine (gB⁴⁹¹GSGG⁴⁹⁴) to conserve the carbon backbone.     

Autophagy and the Effects of Its Inhibition on Varicella-Zoster Virus Glycoprotein Biosynthesis and Infectivity

Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (P < 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (P < 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing.     

Systematic Analysis of Intracellular Trafficking Motifs Located within the Cytoplasmic Domain of Simian Immunodeficiency Virus Glycoprotein gp41

Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.     

Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.Varicella-zoster virus (VZV) causes chicken pox (varicella) upon primary infection. Lifelong latency is established in neurons of the sensory ganglia, and reactivation leads to shingles (herpes zoster) (1). Disease is usually inconsequential in immunocompetent people but can be severe in immunocompromised patients. The current prophylaxis for these high-risk individuals exposed to VZV is high-titer immunoglobulin to VZV administered within 96 h of exposure. This prophylaxis does not always prevent disease, but the severity of symptoms and mortality rates are usually reduced (32).Glycoprotein H (gH) is a type 1 transmembrane protein that is required for virus-cell and cell-cell spread in all herpesviruses studied (12, 15, 24, 26). gH is an important target of the host immune system. Individuals who have had primary infection with VZV or herpes simplex virus (HSV), the most closely related human alphaherpesvirus, have humoral and cellular immunity against gH (1, 56). Immunization of mice with a recombinant vaccinia virus expressing VZV gH and its chaperone, glycoprotein L (gL), induced specific antibodies capable of neutralizing VZV in vitro (28, 37). Immunization of mice with purified HSV gH/gL protein resulted in the production of neutralizing antibodies and protected mice from HSV challenge (5, 44), and administration of an anti-HSV gH monoclonal antibody (MAb) protected mice from HSV challenge (16). Antibodies to HSV and Epstein-Barr virus gH effectively neutralize during virus penetration but not during adsorption in vitro, indicating an essential role for gH in the fusion of viral and cellular membranes but not in initial attachment of the virus to the cell (18, 33).Anti-gH MAb 206, an immunoglobulin G1 (IgG1) antibody which recognizes a conformation-dependent epitope on the mature glycosylated form of gH, neutralizes VZV infection in vitro in the absence of complement (35). MAb 206 inhibits cell-cell fusion in vitro, based on reductions in the number of infected cells and the number of infected nuclei within syncytia, and appears to inhibit the ability of virus particles to pass from the surface of an infected epithelial cell to a neighboring cell via cell extensions (8, 35, 43). When infected cells were treated with MAb 206 for 48 h postinfection (hpi), virus egress and syncytium formation were not apparent, but they were evident within 48 h after removal of the antibody, suggesting that the effect of the antibody was reversible and that there was a requirement for new gH synthesis and trafficking to produce cell-cell fusion. Conversely, nonneutralizing antibodies to glycoproteins E (gE) and I (gI), as well as an antibody to immediate-early protein 62 (IE62), had no effect on VZV spread (46).Like that of other herpesviruses, VZV entry into cells is presumed to require fusion of the virion envelope with the cell membrane or endocytosis followed by fusion. One of the hallmarks of VZV infection is cell fusion and formation of syncytia (8). Cell fusion can be detected as early as 9 hpi in vitro, although VZV spread from infected to uninfected cells is evident within 60 min (45). In vivo, VZV forms syncytia through its capacity to cause fusion of epidermal cells. Syncytia are evident in biopsies of varicella and herpes zoster skin lesions during natural infection and in SCIDhu skin xenografts (34). VZV gH is produced, processed in the Golgi apparatus, and trafficked to the cell membrane, where it might be involved in cell-cell fusion (11, 29, 35). gH then undergoes endocytosis and is trafficked back to the trans-Golgi network (TGN) for incorporation into the virion envelope (20, 31, 42). Since VZV is highly cell associated in vitro, little is known about the glycoproteins required for entry, but VZV gH is present in abundance in the skin vesicles during human chickenpox and zoster (55).Investigating the functions of gH in the pathogenesis of VZV infection in vivo is challenging because it is an essential protein and VZV is species specific for the human host. The objective of this study was to investigate the role of gH in VZV pathogenesis by establishing whether antibody-mediated interference with gH function could prevent or modulate VZV infection of differentiated human tissue in vivo, using the SCIDhu mouse model. The effects of antibody administration at early and later times after infection were determined by comparing infectious virus titers, VZV genome copies, and lesion formation in anti-gH antibody-treated xenografts. In vitro experiments were performed to determine the potential mechanism(s) of MAb 206 interference with gH during VZV replication, virion assembly, and cell-cell spread. The present study has implications for understanding the contributions of gH to VZV replication in vitro and in vivo, the mechanisms by which production of antibodies to gH by the host might restrict VZV infection, and the use of passive antibody prophylaxis in patients at high risk of serious illness caused by VZV.     

Alternative Splicing of the RAGE Cytoplasmic Domain Regulates Cell Signaling and Function

The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling.     

The Membrane-proximal Region of the E-Cadherin Cytoplasmic Domain Prevents Dimerization and Negatively Regulates Adhesion Activity

Cadherins are transmembrane glycoproteins involved in Ca²⁺-dependent cell–cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin–dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.     

水痘-带状疱疹病毒的基因分型与分子进化

水痘-带状疱疹病毒(Varicella-zoster virus,VZV)又称人类疱疹病毒3型,属疱疹病毒科,与单纯疱疹病毒HSV-1、HSV-2一起归入α亚科。人类是其唯一的自然宿主,对其普遍易感。VZV引起的原发感染表现为水痘,并在宿主的感觉神经节内潜伏,再激活时可引起带状疱疹。近年来VZV分子流行病学的研究涉及流行病学、病毒学、生物信息学等相关领域,通过监测、研究VZV的基因变异,区分疫苗株或野生株引起的感染,探讨世界范围内各VZV病毒株的系统发育关系和各遗传支之间的分子进化史。现将近年来有关VZV不同的地理分布和遗传支进化的研究状况综述如下。     

Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.     

Pseudotype Formation of Moloney Murine Leukemia Virus with Sendai Virus Glycoprotein F

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.     

Impact of Varicella-Zoster Virus on Dendritic Cell Subsets in Human Skin during Natural Infection

Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-α) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.Varicella-zoster virus (VZV) is a highly species-specific human herpesvirus that causes the diseases varicella (chicken pox) and herpes zoster (shingles). Varicella results from the primary phase of infection and is characterized by a diffuse rash of vesiculopustular lesions that appear in crops and usually resolve within 1 to 2 weeks (7, 26). Primary infection is initiated by inoculation of mucosal sites, such as the upper respiratory tract and the conjunctiva, with infectious virus, usually contained within respiratory droplets (3, 23). Following inoculation, there is a 10- to 21-day incubation period during which VZV is transported to the regional lymph nodes; however, it remains unclear which cell types are responsible for transport of VZV during natural infection (3). It has been hypothesized that dendritic cells (DC) of the respiratory mucosa may be among the first cells to encounter VZV during primary infection and are capable of virus transport to the draining lymph nodes (1, 45). It is postulated that within lymph nodes, VZV undergoes a period of replication, resulting in a primary cell-associated viremia, during which time virus is transported to the reticuloendothelial organs, where it undergoes another period of replication that results in a secondary cell-associated viremia and virus transport to the skin (3, 23). However, VZV has recently been shown to have tropism for human tonsillar CD4⁺ T lymphocytes (37), and it has been demonstrated that these T lymphocytes express skin homing markers that may allow them to transport VZV directly from the lymph node to the skin during primary viremia (38). Once the virus reaches the skin, it infects cutaneous epithelial cells, resulting in distinctive vesiculopustular lesions.During the course of primary infection, VZV establishes a lifelong latent infection within the sensory ganglia, from which virus may reactivate years later to cause herpes zoster (22, 42, 53). VZV reactivation results in the production of new infectious virus and a characteristic vesiculopustular rash, which differs from that of varicella insofar as the distribution of the lesions is typically unilateral and covers only 1 to 2 dermatomes (8). In both primary and reactivated VZV infection of human skin, VZV antigens are detectable in the epidermis and dermis (2, 30, 46, 47, 49, 52), and although some studies have examined the immune infiltrate present in these lesions, most have focused on T lymphocytes, macrophages, and NK cells (40, 48, 50, 51, 58). The role of DC subsets in VZV infection in human skin has not been previously explored in vivo.Our laboratory provided the first evidence that VZV could productively infect human immature and mature monocyte-derived dendritic cells (MDDC) in vitro (1, 45), and Hu and Cohen (2005) showed that VZV ORF47 was critical for replication of virus in human immature DC but not mature DC (29). However, whether DC become directly infected during natural VZV skin infection and the impact VZV infection may have on DC subsets has yet to be elucidated. The two subsets of DC that are normally present in the skin and which may be involved in the pathogenesis of VZV infection are the Langerhans cells (LC) of the epidermis and dermal DC (DDC) (60). LC are present in an immature state in uninfected skin and in upper respiratory tract epithelium. Upon capture of foreign antigens, LC have the capacity to migrate from the periphery to the lymph nodes, where they seek interaction with T lymphocytes (60). Although the location of cutaneous DC suggests that they are a DC subset likely to be involved in the pathogenesis of VZV infection, other subsets of DC, such as the blood-derived myeloid DC (MDC) and plasmacytoid DC (PDC), are also potentially important in the pathogenesis of VZV infection. Of particular interest are PDC, since these cells are important in innate antiviral immune responses due to their ability to recruit to sites of inflammation and secrete high levels of alpha interferon (IFN-α) (6, 18, 56). PDC also participate in adaptive immune responses through their secretion of cytokines and chemokines that promote activation of effector cells, including NK cells, NKT cells, B lymphocytes, and T lymphocytes, and also through their capacity to present antigen to T lymphocytes (9, 63). Whether PDC and LC can be infected with VZV and their roles during infection have not been previously studied.In this study, we sought to identify and compare the subsets of DC present in human skin lesions following natural VZV infection and to assess DC permissiveness to VZV infection. We utilized immunohistochemical (IHC) and immunofluorescent (IFA) staining to characterize DC subsets within the skin of multiple patients with either varicella or herpes zoster, and identified profound changes in the frequency of LC and PDC as a consequence of cutaneous VZV infection. In addition, some LC and PDC costained with a range of VZV antigens indicative of productive infection. PDC isolated from human blood and LC derived from the MUTZ-3 cells were shown to be permissive to productive VZV infection in vitro. This study defines changes in the type and distribution of DC during natural cutaneous VZV infection and implicates infection of specific DC subsets in VZV pathogenesis.