Domain Of gB Research Articles

New different origins and evolutionary processes of AP2/EREBP transcription factors in Taxus chinensis

BackgroundTaxus spp. produces the anticancer drug, taxol, and hence is planted as an industrial crop in China. APETALA2/ethylene response element binding proteins (AP2/EREBPs) are the key regulators of plant development, growth, and stress responses. Several homologues control taxol biosynthesis. Identifying the AP2/EREBP proteins from Taxus is important to increase breeding and production and clarify their evolutionary processes.ResultsAmong the 90 genes from multi Taxus chinensis transcriptome datasets, 81 encoded full-length AP2-containing proteins. A domain structure highly similar to that of angiosperm AP2/EREBPs was found in 2 AP2, 2 ANT, 1 RAV, 28 dehydration-responsive element-binding proteins, and 47 ethylene-responsive factors contained, indicating that they have extremely conservative evolution processes. A new subgroup protein, TcA3Bz1, contains three conserved AP2 domains and, a new domain structure of AP2/EREBPs that is different from that of known proteins. The new subtype AP2 proteins were also present in several gymnosperms (Gingko biloba) and bryophytes (Marchantia polymorpha). However, no homologue was found in Selaginella moellendorffii, indicating unknown evolutionary processes accompanying this plant’s evolution. Moreover, the structures of the new subgroup AP2/EREBPs have different conserved domains, such as B3, zf-C3Hc3H, and agent domains, indicating their divergent evolution in bryophytes and gymnosperms. Interestingly, three repeats of AP2 domains have separately evolved from mosses to gymnosperms for most of the new proteins, but the AP2 domain of Gb_11937 has been replicated.ConclusionThe new subtype AP2/EREBPs have different origins and would enrich our knowledge of the molecular structure, origin, and evolutionary processes of AP2/EREBP transcription factors in plants.

Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection.

Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.

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.

Mapping regions of Epstein–Barr virus (EBV) glycoprotein B (gB) important for fusion function with gH/gL

Glycoproteins gB and gH/gL are required for entry of Epstein–Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB–gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.

Peptide inhibition of human cytomegalovirus infection

BackgroundHuman cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection.ResultsUsing the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 μM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 μM and 50 μM, respectively, and 60% at a concentration of 2.5 μM. While peptides 264-291 and 297-315, individually failed to inhibit viral infection, when combined, they showed 67% inhibition of HCMV infection at a concentration of 0.125 μM each.ConclusionsPeptides designed to target putative fusogenic domains of gB provide a basis for the development of novel therapeutics that prevent HCMV infection.

The Cytoplasmic Terminus of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein B Is Not Essential for Virion Egress and Infectivity

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted alpha-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linker-insertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state.

Cellular and Humoral Immune Responses to Alphavirus Replicon Vaccines Expressing Cytomegalovirus pp65, IE1, and gB Proteins

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.

Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins

We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384–611 or 384–711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK −gC −, is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119–6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.

Antigenic Domain 1 Is Required for Oligomerization of Human Cytomegalovirus Glycoprotein B

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant virion envelope protein that has been shown to be essential for the infectivity of HCMV. HCMV gB is also one of the most immunogenic virus-encoded proteins, and a significant fraction of virus neutralizing antibodies are directed at gB. A linear domain of gB designated AD-1 (antigenic domain 1) represents a dominant antibody binding site on this protein. AD-1 from clinical isolates of HCMV exhibits little sequence variation, suggesting that AD-1 plays an essential role in gB structure or function. We investigated this possibility by examining the role of AD-1 in early steps of gB synthesis. Our results from studies using eukaryotic cells indicated that amino acid (aa) 635 of the gB sequence represented the carboxyl-terminal limit of this domain and that deletion of aa 560 to 640 of the gB sequence resulted in loss of AD-1 expression. AD-1 was shown to be required for oligomerization of gB. Mutation of cysteine at either position 573 or 610 in AD-1 resulted in loss of its reactivity with AD-1-specific monoclonal antibodies and gB oligomerization. Infectious virus could not be recovered from HCMV bacterial artificial chromosomes following introduction of these mutations into the HCMV genome, suggesting that AD-1 was an essential structural domain required for gB function in the replicative cycle of HCMV. Sequence alignment of AD-1 with homologous regions of gBs from other herpesviruses demonstrated significant relatedness, raising the possibility that this domain may contribute to multimerization of gBs in other herpesviruses.

Pathogenic potentials of glycoprotein C-negative syncytial mutants from rabbit T cells infected persistently with herpes simplex virus type 1

Human T cell lymphotropic virus type I (HTLV-I)-transformed T cells of rabbits were infected persistently with Herpes simplex virus type 1 (HSV-1) strain KOS. These infected cells yielded syncytial mutants, either glycoprotein C (gC)-negative or -positive, which predominated over and replaced the wild-type virus in a long-term culture for 2 years. An alignment of nucleotide sequences showed multiple mutations in glycoprotein B (gB) and gC genes of these mutants, which are or may be responsible for the mutant phenotypes. One of four mutants analyzed produced extensively large syncytia and possessed point mutations within the cytoplasmic domain of gB. All four mutants possessed multiple point mutations in gC and two possessed single insertions which resulted in a frame shift, leading to the premature termination of the gC polypeptide chain. The supernatant of the 2-year culture of cells infected persistently, containing only gC-negative syncytial mutants, induced encephalitic symptoms in B/Jas inbred rabbits, when injected intravenously. One gC-negative syncytial isolate from an encephalitic lesion, together with those from the culture supernatant, were examined for pathogenic potential in vitro and in vivo. All these mutants were more cytotoxic and more susceptible to complement inactivation than the parental virus, and could infect and replicate in adrenal glands when injected intravenously into rabbits. Invasion into the central nervous system appeared to be blocked at the portal of entry, the adrenal gland, i.e., none exhibited neuroinvasive potential by itself. Syncytial gC-negative mutants could thus be pathogenic in rabbits.

Effects of mutations in the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B on intracellular transport and infectivity.

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.

A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate

Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T–C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.

Cytoplasmic domain signal sequences that mediate transport of varicella-zoster virus gB from the endoplasmic reticulum to the Golgi.

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.

An acidic cluster in the cytosolic domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the plasma membrane.

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1–680 of gB, and lacking the fragment encoding amino acids 681–907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.

Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells.

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK-gC-) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO2) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK-gCEPO2), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK-gCEPO2 recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK-gCEPO2 virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK-gCEPO2 showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.