The role of herpes simplex virus glycoproteins in immune evasion.
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Animals↗
Biomedical subjects
H M Friedman
Publications and source records attributed to H M Friedman.
Flurbiprofen effect on gingival crevicular fluid prostaglandin and thromboxane levels in humans.
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Cross-Sectional Studies↗
Structural basis of C3b binding by glycoprotein C of herpes simplex virus.
Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.
Amino Acid Sequence↗
Effect of Towne live virus vaccine on cytomegalovirus disease after renal transplant. A controlled trial.
OBJECTIVE: To test the efficacy of vaccination with the Towne live attenuated cytomegalovirus vaccine. DESIGN: A double-blind, randomized, placebo-controlled trial in candidates for renal transplantation. The cytomegalovirus serologic status of both recipients and donors were determined, and the recipients were followed for periods of 6 months to 7 years after transplant. SETTING: A university transplant center. PATIENTS: The analyses were made on 237 patients who were given either vaccine or placebo, received renal transplants, and were followed for at least 6 months. INTERVENTION: Subcutaneous inoculation with Towne live attenuated virus or with placebo. MAIN OUTCOME MEASURES: The presence of cytomegalovirus infection was defined by virus isolation and antibody tests. If infection occurred, a prearranged scoring system for cytomegalovirus disease was used to objectify disease severity. RESULTS: The vaccine was well tolerated, and there were no discernible long-term adverse effects. Recipients who were originally seropositive did not clearly benefit from vaccination. Protective efficacy was analyzed in the group at highest risk for cytomegalovirus disease; recipients who were seronegative at the time of vaccination and who received a kidney from a seropositive donor. Compared with placebo recipients, vaccinated patients in this group had significantly less severe cytomegalovirus disease, with a significant reduction in disease scores (P = 0.03) and 85% decrease in the most severe disease (95% CI, 35% to 96%), although infection rates were similar. Graft survival at 36 months was improved in vaccinated recipients of cadaver kidneys (8 of 16) compared with unvaccinated recipients (4 of 16) (P = 0.04). CONCLUSIONS: Previous vaccination of seronegative renal transplant recipients with live cytomegalovirus results in reduction of disease severity mimicking the action of naturally derived immunity.
Adult↗
Entry of human immunodeficiency virus-1 into glial cells proceeds via an alternate, efficient pathway.
Although the CD4 molecule is the cellular receptor for human immunodeficiency virus-1 (HIV-1) in cells of the lymphocyte/monocyte lineage, a number of investigators have also been able to infect cells, including several of central nervous system (CNS) origin, that do not express CD4 protein or mRNA. These infections are generally nonpermissive. To ascertain whether the nonpermissive nature of infection in glial cells is due to an inefficient entry pathway, we prepared a permanently transfected U373-MG cell line expressing the CD4 molecule and demonstrated that HIV-1 still replicates at a low level. Furthermore, a virus uptake assay indicated that HIV-1 enters glial cells effectively, even in the absence of CD4. These results demonstrate that HIV-1 entry is efficient and that the restrictive nature of the infection in glial cells is due to postentry mechanisms. In addition, these findings support the existence of an alternate, efficient, entry pathway in some glial cells.
Blotting, Western↗
Herpes simplex virus glycoprotein C is a receptor for complement component iC3b.
Herpes simplex virus type 1-infected cells bind C3b and iC3b, but not C3d, at the cell surface. Herpes simplex virus type 2 (HSV-2)-infected cells bind none of these C3 fragments. A transfection assay was used to demonstrate that binding of iC3b was to gC1. Although iC3b did not bind to HSV-2-infected cells, it did bind to mammalian cells transfected with the gC2 gene. Using linker insertion mutants, three domains on gC2 that are important for binding iC3b were mapped; these regions were similar to previously defined regions involved in binding C3b. These results suggest that some of the functions served by gC may be similar to those of CR3, the mammalian receptor for iC3b.
Amino Acid Sequence↗
Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity.
Recent studies indicate that the herpes simplex virus type 1 (HSV-1) Fc receptor (FcR) can bind antiviral immunoglobulin G by participating in antibody bipolar bridging. This occurs when the Fab domain of an immunoglobulin G molecule binds to its antigenic target and the Fc domain binds to the HSV-1 FcR. In experiments comparing cells infected with wild-type HSV-1 (NS) and cells infected with an FcR-deficient mutant (ENS), we demonstrate that participation of the HSV-1 FcR in antibody bipolar bridging reduces the effectiveness of antibody-dependent cellular cytotoxicity.
Animals↗
Viral activation of the coagulation cascade: molecular interactions at the surface of infected endothelial cells.
Herpesviral infection of endothelial cells (ECs) induces arterial injury. We now demonstrate that such infection promoted enhanced monocyte-endothelial adhesion. Enhanced adhesion was blocked by monoclonal antibodies to the viral-encoded cell surface glycoprotein gC but not by antibodies to gD or gE. Adhesion was also blocked by treating ECs with specific thrombin inhibitors or by growing cells in prothrombin-depleted serum. We found that gC bound and promoted activation of factor X on infected ECs, thereby contributing to thrombin generation. Factor X also bound to transfected L cells that were induced to express gC. Cross-linking and immunoprecipitation studies demonstrated factor X-gC complex formation on the surface of these cells. We suggest that gC-dependent thrombin generation by herpes-infected endothelium may be an important mediator of vascular pathology during viral infection.
Blood Coagulation↗
Glycoprotein C of herpes simplex virus type 1 prevents complement-mediated cell lysis and virus neutralization.
Glycoprotein gC1 of herpes simplex virus type 1 (HSV-1) binds complement component C3b. To determine if gC1 modifies the interaction of complement with virus-infected cells or cell-free virus, ns-1, a mutant HSV-1 strain that does not express gC1 at the cell surface and does not bind C3b, was compared with its parental strain, NS. Cells infected with the gC1 mutant were more susceptible to cytolysis mediated by antibody and complement or complement alone. The gC1 or gD1 genes were expressed in mammalian cells under the control of an inducible promoter. Cells induced to express gC1 resisted complement cytolysis, while cells expressing gD1 did not. gC1 modified cytolysis of virus-infected or -transfected cells by blocking alternative complement pathway activation. gC1 also modified complement-dependent virus neutralization, which was mediated by inhibiting the classical complement pathway. These results indicate a protective role for gC1 on the virion and at the cell surface.
Antibody-Dependent Cell Cytotoxicity↗
Cytotoxic T lymphocytes specific for herpes simplex virus (HSV) studied using adenovirus vectors expressing HSV glycoproteins.
In previous work, we observed that H-2k-restricted herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTLs) were effectively able to lyse transfected target cells expressing HSV glycoprotein C (gC), but not cells expressing gB, gD or gE. To confirm and extend our observations on the specificity of anti-HSV CTLs, recombinant adenovirus (Ad) vectors able to express HSV-1 gB or gC (AdgB2 or AdgC) were constructed. Syngeneic target cells infected with AdgB2 were efficiently lysed by primary H-2b and H-2d, but not by H-2k-restricted HSV-specific CTL. Limiting dilution studies indicated that 4 to 10% of H-2b-restricted HSV-specific CTLs recognize gB. H-2k, H-2b and H-2d-restricted anti-HSV-1 CTLs were unable to lyse AdgC-infected syngeneic target cells. To examine the apparent discrepancy between the previous results involving transfected H-2k cells expressing gC and the present results involving AdgC-infected cells, gC-expressing cell lines used in previous experiments were subcloned and retested in CTL assays. DC2 cells which were lysed by HSV-specific CTLs in the previous experiments remained sensitive to anti-HSV CTLs but two other clones derived from the same transfection were not lysed. Further, L cells transfected with the gC or gD gene coupled to the mouse mammary tumour virus promoter and capable of expressing high levels of the glycoproteins following dexamethasone induction were not lysed by H-2k-restricted anti-HSV CTLs. These results suggest that HSV-specific CTLs do not recognize gC, at least when it is expressed using an Ad vector and in most transfected cell lines, whereas a significant proportion of anti-viral CTLs recognize gB presented in some but not all murine haplotypes.
Adenoviridae↗
Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C.
Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.
Amino Acid Sequence↗
Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes.
Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.
Animals↗
Conjugal temporal arteritis.
We report the simultaneous occurrence of biopsy-proven temporal arteritis in husband and wife. Serologic and viral studies were negative, including viral culture of the wife's temporal artery. The concurrent incidence of giant cell arteritis in a married couple would suggest a common exogenous exposure.
Aged↗
Discrepant rotavirus results in two laboratories using the same enzyme immunoassay.
Rotavirus testing was performed on 66 stool samples in two separate laboratories using the same enzyme immunoassay. Sixteen of 46 of the positive results reported by the reference laboratory were negative in the hospital laboratory. In addition, RNA gel electrophoresis had negative results in the 16 discrepant samples. This indicates the need to repeat or confirm positive results.
Child↗
Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge.
To test the protective effect of Towne live attenuated human cytomegalovirus (HCMV) vaccine in normal individuals, we developed a parenteral challenge consisting of a low-passage isolate (Toledo stain) inoculated subcutaneously in graded doses. This challenge virus caused a mild mononucleosis syndrome in seronegative individuals at doses of 10 or 100 pfu. The illness was accompanied by atypical lymphocytosis, raised hepatic enzymes, excretion of HCMV and HCMV-specific immune responses. Naturally seropositive volunteers also developed clinical and laboratory evidence of infection after challenge with 1,000 pfu of Toledo but resisted 10 or 100 pfu. Volunteers who had been vaccinated 1 y earlier also were resistant to disease caused by 10 or 100 pfu of Toledo, although some were asymptomatically infected by the 100 pfu dose. Vaccine-induced immunity to HCMV was as complete as naturally induced immunity when the challenge dose of Toledo was 10 pfu.
Antibodies, Viral↗
A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G.
We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.
Animals↗
Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.
Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.
Animals↗
Progress in the treatment of cytomegalovirus pneumonia.
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Acquired Immunodeficiency Syndrome↗