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. 1998 Oct;72(10):8191-7.
doi: 10.1128/JVI.72.10.8191-8197.1998.

The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex

M T Huber  1 , T Compton
Affiliations

The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex

M T Huber et al. J Virol. 1998 Oct.

Abstract

The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391-5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090-3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.

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Figures

FIG. 1
FIG. 1
N-Glycosidase digestion of the 125-kDa protein. Mock-infected and HCMV-infected IF cells were surface biotinylated and immunoprecipitated with antibody 14-4b. Immunoprecipitated proteins were incubated overnight in the presence or absence of N-glycosidase, subjected to reducing SDS-PAGE, and electroblotted to nitrocellulose. (A) The immunoblot was probed with streptavidin-HRP for detection of biotinylated proteins. (B) The blot in panel A was stripped and reprobed with an anti-gH antibody followed by an HRP-conjugated goat anti-mouse antibody. HC, immunoglobulin heavy chain of the immunoprecipitating antibody.
FIG. 2
FIG. 2
(A) Amino acid sequence of the predicted product of the HCMV UL74 gene. The sequence matching the internal peptide sequence derived from the 125-kDa glycoprotein is boxed in black. The hydrophobic domain is boxed, and potential N-linked glycosylation sites are underlined. The two potential heparin-binding sequences are underlined with a dashed line. Numbers on the right denote amino acid residues. (B) Kyte-Doolittle hydropathy analysis of the UL74 amino acid sequence. Areas above and below the line denote hydrophilic and hydrophobic domains, respectively. The scale above the line graph indicates the amino acid residue.
FIG. 3
FIG. 3
Time course of expression of gO, gH, and gL. IF cells were infected with HCMV (MOI of 3) or mock infected and then lysed at 24-h intervals postinfection as indicated above the lanes. Lysates were resolved by reducing SDS-PAGE and electrotransferred, and the blots were reacted with anti-gO antibody (A), anti-gH antibody AP865 (B) or anti-gL antibody 26388 (C).
FIG. 4
FIG. 4
Immunoblots of purified HCMV virions. Gradient-purified virions were lysed in reducing SDS-PAGE sample buffer, resolved by SDS-PAGE, and electrotransferred to nitrocellulose. The blots was probed with either preimmune serum (as a control) or anti-gO antibody (ab).
FIG. 5
FIG. 5
Immunoprecipitation of the gCIII complex by the gO antibody. HCMV-infected cells were metabolically labeled with [35S]Met-Cys, lysed, and immunoprecipitated with either the anti-gO antibody or anti-gH/gCIII antibody 14-4b. (A) The proteins immunoprecipitated with the indicated antibodies were resolved by nonreducing SDS-PAGE. (B) The ∼240-kDa species precipitated by either the anti-gO or anti-gH immunoprecipitation was excised from the dried gel, reduced, and resolved by a second reducing SDS-PAGE. (C) Duplicates of the gel in panel B were electrotransferred to nitrocellulose and probed with the anti-gO antibody or anti-gH antibody AP865.
FIG. 6
FIG. 6
Schematic representation of betaherpesvirus genomes showing positional homology of the gO genes. Open boxes represent terminal and internal repeat regions of the genomes; striped boxes represent the conserved block of genes including the gN homolog; black boxes represent the gO gene; hatched boxes represent the conserved block of genes including the gH gene. (A) HCMV genome; (B) MCMV, HHV-6A, HHV-6B, and HHV-7 genomes.
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