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Bovine herpesvirus 1 glycoprotein M forms a disulfide-linked heterodimer with the U(L)49.5 protein
S X Wu1, X P Zhu, G J Letchworth
1Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison 53706, USA.
Abstract:
Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U(L)10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U(L)10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U(L)49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448-1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U(L)49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.
Insights
Researchers identified a novel 7-kDa protein covalently bound to bovine herpesvirus 1 glycoprotein M (gM). This discovery reveals a previously unknown interaction within herpesviruses, suggesting disulfide bonding in gM homologs across all herpesviruses.
Area of Science:
- Virology
- Molecular Biology
- Protein Chemistry
Background:
- Bovine herpesvirus 1 (BHV-1) possesses nine identified glycoproteins, including gM, which is implicated in membrane fusion in other herpesviruses.
- The precise function and interactions of BHV-1 gM have not been fully elucidated.
Purpose of the Study:
- To express and identify the BHV-1 glycoprotein M (gM) encoded by open reading frame U(L)10.
- To characterize the post-translational modifications and interactions of BHV-1 gM.
Main Methods:
- Corrected the published sequence of BHV-1 open reading frame U(L)10.
- Expressed glutathione S-transferase fusion proteins of BHV-1 gM C-terminal peptides in E. coli.
- Generated antibodies against the fusion protein for immunoprecipitation assays.
- Analyzed protein sizes using reducing and nonreducing SDS-PAGE.
- Performed Western blot analysis with specific antibodies.
Main Results:
- Antibodies against the BHV-1 gM C-terminus immunoprecipitated a 30-kDa protein from in vitro translation.
- In infected cells, immunoprecipitated proteins appeared as 36/43 kDa (reducing) and 43/48 kDa (nonreducing) bands; only the larger band was in virions.
- A 7-kDa protein was released from gM by reducing agents.
- This 7-kDa protein reacted with antibodies against BHV-1 U(L)49.5, not anti-gM antibodies.
- This is the first report of a small protein covalently bound to any herpesvirus gM.
Conclusions:
- BHV-1 gM is covalently linked to a 7-kDa protein, identified as BHV-1 U(L)49.5.
- This disulfide-bonded complex is present in BHV-1 virions.
- Homologs of gM and U(L)49.5 in other herpesviruses likely share similar hydrophobic domains and cysteine patterns, suggesting this disulfide linkage is conserved across all herpesviruses.