Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1

Abstract

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as fl-galactosidase fusion proteins, covering about 75 % of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV-and HSV-positive human antisera in immunoblot experiments.

Human cytomegalovirus structural proteins

“…HCMV pUL86 has been prokaryotically expressed in three portions as fl-galactosidase fusion proteins, covering about 75 % of the ORF (Rudolph et al, 1990a). One fusion protein encoded by nucleotides 101 to 1243, a second encoded by nucleotides 1944 to 3089 and a third encoded by nucleotides 2624 to 3793 were tested with human sera and the second protein was found to be immunodominant.…”

“…Rudolph et al (1990a) described rabbit antisera to three recombinant proteins containing portions of pUL86 (amino acids 33 to 414, 648 to 1029 and 874 to 1264). At least two murine MAbs to pUL86 have been obtained (Pereira et al, 1982a;Rudolph et al, 1990a) and one (28-4 produced and characterized by Britt) was shown to react with an epitope localized between amino acids 33 and 414 (Rudolph et al, 1990a). See Table 1.…”

Detection of cytomegalovirus in urine samples by an enzyme-linked immunosorbent assay using a monoclonal antibody against the viral 150-kilodalton protein

“…However, the possibility that the CM3 antibody is directed to the MCP cannot be excluded, be-.roWs E cause the antigen recognized by CM3 was localized mainly IFN Cv i the nucleus of infected cells. The ppl5O antigen has been ACV recently demonstrated to be present in the cytoplasm of CMV-infected cells (19), whereas the MCP antigen is present in the nucleus (17). Further study is required to determine the specificity of the CM3 antibody.…”

Cross-Reactivity of Epstein-Barr Virus-Specific Immunoglobulin M Antibodies with Cytomegalovirus Antigens Containing Glycine Homopolymers

“…A major issue in the development of reliable assays is the identification of viral proteins that react with antibodies induced by other herpesviruses. Since EBV and HCMV show considerable DNA and protein sequence homologies in conserved gene blocks, particular proteins or protein fragments have been excluded from serological assays (2,7,39,51). Pursuing this strategy has allowed the development of recombinant tests for both viruses (15,26,31,45,48,50,51).…”