Human cytomegalovirus interactions with human fibroblast cells: Characterization of a neutralization epitope and identification of cell signalling events.

Abstract

Human cytomegalovirus (HCMV) hyperimmune globulins or intravenous immunoglobulin (IVIG) have been widely used as a means of passive immunoprophylaxis. The efficacy of these blood products has not been clearly demonstrated in terms of the correlation between anti-HCMV titres assayed by enzyme-linked immunoassay (ELISA) and viral neutralizing activity measured in vitro by plaque reduction assay. Recent advances in the characterization of HCMV surface glycoproteins have led to the identification of the immunodominant regions of these molecules. The mouse monoclonal anti-HCMV antibody CMVB1 was utilized to study the properties of a neutralization epitope. Chemical cross-linking experiments with a photoactivable, heterobifunctional and cleavable reagent identified a viral antigen with an apparent molecular weight of approximately 70 kDa. Microtitre plate binding assays verified that the epitope was unique to HCMV and not homologous to herpesvirus-1 or -2 (HSV-1, -2) antigenic sites. The lectin concanavalin A was found to partially block CMVB1 from binding to the viral epitope, suggesting that carbohydrate moieties may be part of the antigen. Lectin specific sugars such as D-glucose, D-mannose and N-acetylglucosamine did not block CMVB1 binding to HCMV. Cell signalling events induced by HCMV particle binding to human foreskin fibroblast cells were also examined. Central molecules involved in three major signal transduction pathways were studied. Tyrosine phosphorylation was investigated with immunoprecipitation and immunoblotting techniques. Intracellular cyclic adenosine monophosphate (cAMP) concentrations were measured by immunoassay and protein kinase C (PKC) activation was established by a peptide radiolabelling assay. With the methodologies utilized, no evidence of HCMV-induced tyrosine phosphorylation or elevated cAMP concentrations was detected. PKC activity was found to increase to levels typically observed in activated cells. (Abstract shortened by UMI.)