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The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.

Human cytomegalovirus isolates were analyzed, both by restriction fragment-length polymorphism typing and by sequencing for intra- and intergenic variability at 9 sites on the genome, to determine whether genetic variation influenced disease outcome and whether linkage among genes could be identified. Variation at the UL55 (glycoprotein B [gB]), UL74 (gO), UL75 (gH), UL115 (gL), US9, and US28 gene open-reading frames was studied in relationship to outcome of cytomegalovirus disease. Major findings were that (1) on the basis of analysis of only 9 genomic sites, it is apparent that an almost infinite number of genetic combinations are theoretically possible; (2) genetic linkages are rare; (3) intragenic variability may be a complicating factor in molecular epidemiologic studies; and (4) analysis of only a single gene from a clinical isolate may not reveal the presence of either intragenic variants or mixtures of genotypes.

Murine monoclonal antibodies to human cytomegalovirus (CMV) strain AD169 were selected that neutralized virus infectivity. One monoclonal antibody-producing hybridoma, 1G6, was used to produce ascites fluid from which immunoglobulin was isolated. This antibody efficiently new tralized CMV AD169, other laboratory strains (Towne, Davis), and clinical isolates of CMV in early tissue culture passage (<10) in the absence of complement. The antibody immunoprecipitated a single 86,000-dalton protein from both laboratory and clinical strains. This viral protein was demonstrated by indirect immunofluorescence to be localized in the cytoplasm of CMV-infected cells.Human cytomegalovirus (CMV) infection initiates a cascade of protein synthesis in cell culture. There are approximately 30 proteins associated with the virions of CMV and more than 50 proteins specified by the viral genome that are synthesized in infected cells (1-5). The proteins of CMV from both virions and infected cells have been analyzed immunologically by using polyvalent hyperimmune sera (3, 6-8) and monoclonal antibodies (9, 10). These studies have shown that the major immunological determinants are probably glycoproteins and are present both in infected cells and on the viral envelope.The specific proteins that stimulate cell-mediated or humoral immunity have not yet been identified. In order to investigate the viral proteins that provide the target for antibody-mediated virus neutralization, we have selected murine monoclonal antibodies that neutralize CMV infectivity. A single viral protein was identified by immunoprecipitation that may be important for stimulation of neutralizing antibody in vivo. MATERIALS AND METHODSViruses. Human CMV strain AD169 was obtained from S. St. Jeor (University of Nevada). Laboratory strains (Towne, Davis) and clinical isolates from congenitally infected newborn infants were provided by J. Waner (University of Oklahoma). Viruses were grown in cultures of human embryonic lung (HEL) fibroblasts (11) with Dulbecco's minimal essential medium (DME medium) supplemented with 1% fetal calf serum. Cell-free infectious virus stocks that contained 106 plaque-forming units (pfu)/ml for laboratory strains and 102 to 104 pfu/ml for clinical isolates were prepared by sonication of infected cells (11).Assays for Antiviral Antibody. CMV binding antibody was detected by solid-phase RIA (11). Antigen was prepared by pelleting virus from the supernatant of roller bottles (490 cm2) of CMV-infected cells showing 90% cytopathic effect. Approximately 100 ml of supernatant with 106 pfu/ml was clarified at 800 x g for 10 min, then centrifuged for 1 hr at 200,000 x g. The sedimented virus was resuspended in 1 ml of Tris/saline/EDTA (0.05 M Tris/0.15 M saline/0.00i M EDTA, pH 7.4) and diluted 1:100 in the same buffer for binding to plates. Bound antibody was detected with iodinated goat anti-mouse IgG and IgM (Tago, Burlingame, CA).Virus-neutralizing antibody was detected by both microneutralization and plaque reduction. Microneutralization...

Cytomegalovirus isolates can be grouped into 4 gB and 2 gH genotypes. gB genotypes were studied in patients infected with human immunodeficiency virus (HIV) and in allograft transplantation recipients. In allograft recipients, the distribution of gB 1, -2, -3, and -4 in leukocytes and urine, respectively, was 36%, 21%, 43%, and 0% and 39%, 30%, 17%, and 13%. However, in leukocytes of HIV-infected patients with <100/microL CD4 cells, gB1 was found significantly less often than in allograft recipients (11% vs. 36%) but gB2 was more frequent (56% vs. 21%; P < .05). The decreased incidence of gBl and increased incidence of gB2 compared with allograft recipients was also seen in urine of HIV-infected patients and reflected the distribution seen in leukocytes. gB4 was found significantly more often (P < .05) in semen than in leukocytes of HIV-infected patients with < 100/microL CD4 cells. gB1-4 genotypes were similar in patients with < 100/microL CD4 cells with or without retinitis.

Cell surface receptors for the 86-kDa glycoprotein (gp86) of human cytomegalovirus (HCMV) were identified by using two monoclonal anti-idiotype antibodies that bear the internal image of gp86. These antibodies bound to cells permissive for HCMV infection by both ELISA and immunofluorescence assay and inhibited HCMV plaque formation in human embryonic lung (HEL) cells. Immunoblot analysis showed specific binding of both internal image anti-idiotype antibodies as well as gp86 to an HEL cell membrane protein with an approximate molecular mass of 92.5 kDa. In addition, immunoprecipitation of radiolabeled membrane and cell surface proteins from human foreskin tissue, human foreskin fibroblasts, or HEL cells showed specific binding of antiidiotype antibody predominantly to the 92.5-kDa protein.Anti-idiotype antibodies have been used to identify cell surface receptors for both hormones (1) and viruses, specifically reovirus (2), polyoma (3), and measles virus (4). For the identification of viral receptors, anti-idiotype antibodies can be made against neutralizing antibodies that bind to viral proteins. Anti-idiotype antibodies that bind to cell membrane proteins and inhibit either virus binding and/or viral infection of cells usually bear the internal image of the original ligand and therefore are useful for the identification and isolation of the corresponding cell surface receptor.The mechanism of entry of human cytomegalovirus (HCMV) into susceptible cells is unknown. However, it is likely that glycoproteins present in the viral envelope mediate virus entry. At least three glycoprotein complexes are known to exist in the HCMV envelope (5, 6): a 130/55-kDa complex (also called gcI), which is homologous to the gB glycoprotein of herpes simplex virus; a 47/52-kDa complex (also called gcII); and a 145/86-kDa complex (also called gp86 and gcIII), which is homologous to the gH protein of herpes simplex virus.We have described (7) murine monoclonal anti-idiotype antibodies that bear the internal image of an epitope on gp86 and will induce HCMV-neutralizing antibody in mice. Because these anti-idiotype antibodies have characteristics of gp86, we used them to identify a receptor for this viral glycoprotein on cells susceptible to HCMV infection. In this report we identify a 92.5-kDa protein on human fibroblast cells that binds specifically both to anti-idiotype antibodies and gp86 and that can be immunoprecipitated with the anti-idiotype antibody. We suggest that this cell protein may represent a component of a pathway for entry of HCMV into susceptible cells. MATERIALS AND METHODSCell and Virus Propagation. Human embryonic lung (HEL) cells were grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum. Human foreskin (HF) tissue was obtained from routine circumcisions performed at Stanford University Hospital. HF fibroblasts were grown in primary culture from foreskins obtained from routine circumcisions as described (14). These cells were grown in MEM supplemented with 10% fetal calf se...

Chemotherapeutic agents are used widely in clinical medicine for the treatment of conditions where diminution of the host immune response is a goal. The clinical use of immunosuppression is indicated for immunologically mediated disease, lymphoproliferative diseases, and prevention of graft rejection. Five categories of agents are useful for these purposes; they are ionizing irradiation, corticosteroids, biological alkylating agents, antilymphocyte sera and antimetabolites. While the specific molecular action of many of these drugs is known, how they affect cellular events in immune responses is less clear. One of the unfortunate sequelae of chemotherapy induced immunosuppression is an increased susceptibility of the host to opportunistic pathogens or malignancies. Specific methods are described for monitoring the various parameters of both humoral and cellular immunity. Studies of immunologic function in lymphoma patients and cardiac transplant patients treated with immunosuppressive drugs have shown specific defects in cell mediated immunity to herpes viruses which may relate to their increased susceptibility to infection by these agents.

The cellular transcription factors Sp1 and NF-B were upregulated shortly after the binding of purified live or UV-inactivated human cytomegalovirus (HCMV) to the cell surface. The rapid time frame of transcription factor induction is similar to that seen in other systems in which cellular factors are induced following receptor-ligand engagement. This similarity suggested that a cellular receptor-viral ligand interaction might be involved in Sp1 and NF-B activation during the earliest stages of HCMV infection. To focus on the possible role viral ligands play in initiating cellular events following infection, we first used purified viral membrane extracts to demonstrate that constituents on the membrane are responsible for cellular activation. Additionally, these studies showed, through the use of neutralizing antibodies, that the viral membrane mediators of this activation are the major envelope glycoproteins gB (UL55) and gH (UL75). To confirm these results, neutralizing anti-gB and-gH antibodies were used to block the interactions of these glycoproteins on whole purified virus with their cell surface receptors. In so doing, we found that Sp1 and NF-B induction was inhibited. Lastly, through the use of purified viral gB protein and an anti-idiotypic antibody that mimics the image of the viral gH protein, it was found that the engagement of individual viral ligands with their appropriate cell surface receptors was sufficient to activate cellular Sp1 and NF-B. These results support our hypothesis that HCMV glycoproteins mediate an initial signal transduction pathway which leads to the upregulation of host cell transcription factors and suggests a model wherein the orderly sequence of virusmediated changes in cellular activation initiates with viral binding via envelope glycoproteins to the cognate cellular receptor(s).