Appearance of immunoglobulin G Fc receptor in cultured human cells infected with varicella-zoster virus.

After infection with varicella-zoster virus, HeLa and human embryo lung cells developed a receptor for the Fc portion of human and rabbit immunoglobulin G. The receptor was detected by both hemadsorption and immunofluorescence, using antibody-coated erythrocytes and heat-aggregated immunoglobulin G. However, sheep erythrocytes sensitized with F(ab')2 of anti-sheep erythrocyte antibody did not adsorb to the receptor. When cell-free varicella-zoster virus was inoculated into the HeLa cell monolayer, the Fc receptor appeared at first 6 h after infection; varicella-zoster virus antigen in the cytoplasm and detectable cytopathic effects appeared later.     

Monoclonal antibodies against three major glycoproteins of varicella-zoster virus.

Varicella-zoster virus (VZV) codes for three prominent glycoproteins--gp62, gp98, and gp118--in infected cell cultures. To characterize individually these known immunogens, we first inoculated BALB/c mice with crude VZV extracts, produced hybridoma cultures by Köhler-Milstein cell-fusion technology, and screened culture supernatants by indirect immunofluorescence for reactivity directed against unfixed VZV-infected cells (FAMA assay). Supernatants from five independently derived and subcloned hybridomas with a high VZV-FAMA titer but no reactivity against either uninfected or herpes simplex virus-infected cells were further analyzed by immunoprecipitation of [3H]fucose-labeled and detergent-solubilized VZV antigen preparations. Fractionation of the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that four monoclonal antibodies reacted with both gp62 and gp98, and one precipitated only gp118. The profiles were unchanged whether performed under reducing or nonreducing conditions. When assayed for neutralizing activity, the secretory product of the single anti-gp118 hybridoma, but not the supernatants from the four anti-gp62/gp98 clones, inhibited VZV plaque formation by greater than 80%. Thus, at least one of the glycosylated antigens detected by the FAMA assay is a determinant which elicits neutralizing activity.     

Membrane fusion mediated by herpesvirus glycoproteins: the paradigm of varicella-zoster virus

Varicella-zoster virus (VZV) is well known for its propensity to cause polykaryons (syncytia) in the vesicles within infected skin. Similarly in cultured cells, VZV induces extensive syncytial formation by virus-mediated cell-to-cell fusion. Statistical analyses of fusion parameters demonstrated three-way interactive effects among all three tested variables (incubation temperature, cell type and virus strain). For example, fusion was greatly enhanced at 33 degrees C vs 37 degrees C; also fusion was pronounced in epidermal cells but negligible in fibroblast cells. As with all herpesviruses, VZV gH was a major fusogen. VZV cell fusion was inhibited by antibody to gH, but surprisingly was enhanced by antibody to gE. Other evidence implicating a role for VZV gE in the fusion process was provided by two mutant viruses, in which gE cell surface expression was enhanced. Under transfection conditions, VZV fusion formation occurred after expression of the gH/gL complex; in contrast, pseudorabies virus requires expression of gH, gL and gB, while the herpes simplex virus (HSV) types 1 and 2 require the quartet of gH, gL, gB and gD. VZV has no gD gene and no apparent gD functional homologue. On the other hand, VZV gE exerts a greater effect than HSV gE on membrane fusion. Taken together, the data in this review suggest that VZV has evolved viral glycoprotein machinery more geared toward cell-to-cell fusion (fusion-from-within) than toward virus-to-cell fusion (entry/fusion-from-without), as a means for syncytium formation within the human epidermis.     

Role of the extracellular domain of Fc epsilon RI alpha in intracellular processing and surface expression of the high affinity receptor for IgE Fc epsilon RI

The high affinity receptor for immunoglobulin E, Fc epsilon RI, is a critical component of IgE-mediated allergic reactions. It is expressed as a tetramer (alphabetagamma(2)) made of an IgE-binding alpha chain and a signaling module formed by the beta chain and a dimer of gamma chains. It is expressed in humans and rodents on basophils and mast cells at a high level, and, upon activation, it induces the liberation of allergy mediators. In humans a trimeric form lacking the beta chain also exists (alphagamma(2)). This trimeric form is expressed on antigen presenting cells where it acts to facilitate antigen presentation via IgE. Both the expression and the signaling capacity of the trimer are lower than those of the tetramer. The differences between human (tetrameric and trimeric) and murine (tetrameric only) expression is explained in part by the fact that mouse alpha cannot be expressed at the cell surface in the absence of beta, while human alpha can. Here we demonstrate that the capacity of human alpha to be expressed at the cell surface in the absence of beta is encoded entirely in its extracellular domain. These findings show that the extracellular domain of the type I transmembrane protein Fc epsilon RI alpha plays a role in Fc epsilon RI intracellular processing and expression at the cell surface.     

Differential neutralizing antibody responses to varicella-zoster virus glycoproteins B and E following naked DNA immunization.

The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.     

Absence of IgG Fc receptors on varicella-zoster virus-infected cells

Herpesvirus-infected cells usually express receptors for the Fc portion of immunoglobulin G. Varicella-zoster virus has so far been the sole exception in the family. Both immunehemadsorption and immunofluorescence techniques failed to detect the expression of such receptors. This observation excludes the possibility of false-positive results in serological tests for antibodies to this virus. It is possible that the function of these receptors early in infection is not needed in the subsequent reactivation(s) of the virus. No variation has been shown to occur with different isolates.     

Enhancement of Fc gamma receptor expression in interferon-treated mice

Treatment of C3H mice with partially purified or highly purified virus-induced interferon resulted in a marked increase in the expression of Fcgamma receptors (Fcgamma R) on splenic lymphocytes, mesenteric lymph node cells and thymocytes from cortisone-treated mice. An increase on Fcgamma R on splenic lymphocytes was seen as early as 5 h and lasted at least 72 h after a single injection of interferon. We suggest that enhancement of Fcgamma R is one of the mechanisms by which interferon exerts some immunostimulating effects in vivo.     

Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection,reactivation or immunization

The purpose of this study was to compare the antibody responses to varicella-zoster virus (VZV) gE and gB after natural VZV infection and after vaccination with live attenuated OKA vaccine in order to determine the relative importance of these proteins as components of a subunit vaccine. Anti-VZV antibody titers determined by IFA were of the same order of magnitude in sera from individuals with a history of varicella and in vaccinated children but higher in individuals given booster vaccination. The titers of anti-gE and anti-gB antibodies were measured by ELISA using recombinant gE or gB as capture antigen. From these experiments, it appears that the ratio of anti-gE to anti-gB antibody is highly variable from one individual to another but relatively stable over a long period of time for a particular individual, even after a zoster episode. Neutralizing antibodies directed against gE or gB were also measured by subtracting the neutralization titers obtained before and after depletion of the specific antibodies on immobilized recombinant gE, gB, or both. This showed that, with respect to neutralization, anti-gE and anti-gB are equally prevalent in vaccinated children and that anti-gE is generally, but not always, predominant over anti-gB in VZV-infected individuals. Finally, antibodies to these two glycoproteins appear to be predominant among the neutralizing antibodies directed to other VZV antigens. J. Med. Virol. 53:63–68, 1997. © 1997 Wiley-Liss, Inc.     

Differential B cell expression of mouse Fc receptor homologs

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.     

A vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza A virus

Pseudotyped viruses bearing the glycoprotein(s) of a donor virus over the nucleocapsid core of a surrogate virus are widely used as safe substitutes for infectious virus in virology studies. Retroviral particles pseudotyped with influenza A virus glycoproteins have been used recently for the study of influenza hemagglutinin and neuraminidase-dependent processes. Here, we report the development of vesicular-stomatitis-virus-based pseudotypes bearing the glycoproteins of influenza A virus. We show that pseudotypes bearing the hemagglutinin and neuraminidase of H5N1 influenza A virus mimic the wild-type virus in neutralization assays and sensitivity to entry inhibitors. We demonstrate the requirement of NA for the infectivity of pseudotypes and show that viruses obtained with different NA proteins are significantly different in their transduction activities. Inhibition studies with oseltamivir carboxylate show that neuraminidase activity is required for pseudovirus production, but not for the infection of target cells with H5N1-VSV pseudovirus. The HA-NA-VSV pseudoviruses have high transduction titers and better stability than the previously reported retroviral pseudotypes and can replace live influenza virus in the development of neutralization assays, screening of potential antivirals, and the study of different HA/NA reassortants.     

Herpes simplex virus IgG Fc receptors induced using recombinant adenovirus vectors expressing glycoproteins E and I

Evidence has been presented that herpes simplex virus (HSV) immunoglobulin (IgG) Fc receptors are composed of a complex of two glycoproteins, gE and gI. In previous studies, cells infected with HSV-1 mutants lacking either gE or gI bound lower levels of soluble IgG than cells infected with wild-type viruses suggesting that both gE and gI were required for IgG binding. We have reevaluated the Fc receptor activity of these mutants using a more sensitive assay involving IgG-coated erythrocytes and have found that cells infected with a gE- mutant HSV-1 did not bind IgG-coated erythrocytes whereas cells infected with a gI- mutant retained some Fc binding activity. To further study HSV-induced Fc receptors recombinant adenovirus vectors expressing gE or gI were constructed. Cells expressing gE alone bound both soluble IgG and IgG-coated red cells, although the binding was consistently lower than that observed with HSV-infected cells or cells expressing both gE and gI. Cells expressing only gI were unable to bind either soluble IgG or IgG-coated erythrocytes. These results support the conclusion that both gE and gI are required for full Fc receptor activity, although gE alone can bind IgG to a lesser extent.     

Effect of lymphokine on the activation and Fc receptor expression of rat macrophages

Lymphocyte supernatant (LS) containing lymphokine (LK) activated rat peritoneal macrophages (PM) and at the same time enhanced their Fc receptor (FcR) activity. The signs of the early activation could not be observed on alveolar macrophages (AM) but the augmented FcR expression occurred under the effect of lymphokine. The LK-induced macrophage activation and the enhancement of erythrocyte—antibody (EA) rosette formation were found to be independent of each other. In the presence of soybean trypsin inhibitor (STI) the crude lymphokine-induced augmented EA rosette formation was only slightly diminished. However, it did not seem likely that the LK components of molecular weight over 15,000 could enhance the number of rosettes under the effect of protease inhibitor.These data indicate that the macrophage proteases play an important role in the generation of EA rosette formation enhancing breakbown product(s) during the macrophage—lymphokine interaction.     

Trypanosome variable surface glycoproteins: composite genes and order of expression

Combinatorial processes increase the diversity of variable surface glycoproteins (VSGs) expressed by Trypanosoma equiperdum. We show here that a single telomeric pseudogene provides the 3' portion of three distinct T. equiperdum VSG genes by recombination with different 5' donor pseudogenes. Regions of sequence homology among the pseudogenes determine the sites of recombination in the formation of the expressed copies. This suggests that the recombination between any given basic copy (BC) and the expression-linked copy (ELC) depends on their sharing homology. We present evidence that this is the case and propose that such rules account for the order of expression of the VSGs. These results demonstrate how homologous recombination can generate an ordered sequence of gene expression.     

Cell surface glycoproteins of rabbit lymphocytes: characterization with monoclonal antibodies

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.     

Functional expression and membrane fusion tropism of the envelope glycoproteins of Hendra virus

Hendra virus (HeV) is an emerging paramyxovirus first isolated from cases of severe respiratory disease that fatally affected both horses and humans. Understanding the mechanisms of host cell infection and cross-species transmission is an important step in addressing the risk posed by such emerging pathogens. We have initiated studies to characterize the biological properties of the HeV envelope glycoproteins. Recombinant vaccinia viruses encoding the HeV F and G open reading frames were generated and glycoprotein expression was verified by metabolic labeling and detection using specific antisera. Glycoprotein function and cellular tropism were examined with a quantitative assay for HeV-mediated membrane fusion. Fusion specificity was verified through specific inhibition by anti-HeV antiserum and a peptide corresponding to one of the alpha-helical heptad repeats of F. HeV requires both F and G to mediate fusion. Permissive target cells have been identified, including cell lines derived from cat, bat, horse, human, monkey, mouse, and rabbit. Fusion negative cell types have also been identified. Protease treatments of the target cells abolished fusion activity, suggesting that the virus is employing a cell-surface protein as its receptor.