Characterization of a panel of insertion mutants in human cytomegalovirus glycoprotein B.
Glycoprotein B (gB; gpUL55) of human cytomegalovirus (HCMV) plays a critical role in virus entry and cell-to-cell spread of infection. To define the structure-function relationships in gB, a panel of linker-insertion mutations was generated throughout the coding region. This strategy yielded a panel of 22 mutants with four amino acid insertions and 3 large truncation mutants. Assessment of the mutant proteins' biosynthetic properties and folding patterns analyzed in context with predicted secondary features revealed novel insights into gB's structure and trafficking properties. All of the insertion mutants were able to assemble into oligomers, suggesting that oligomerization is tolerant of small insertions and/or that multiple regions of the protein may be involved. Computer algorithm predictions of gB's secondary structure indicate that the furin-recognized cleavage site falls within an exposed loop. This loop may be particularly sensitive to structural alterations, since insertions upstream and downstream of the cleavage site rendered the mutant proteins cleavage defective. In addition, a strong correlation existed between terminal folding and cleavage of gB. Interestingly, terminal folding was not correlated with delivery to the cell surface but may influence the rate of transport to the cell surface. Nine mutants, containing insertions in both the extracellular and intracellular portions of gB, retained wild-type structural properties. This panel of characterized gB mutants, the first of this type for an HCMV protein, will be a useful tool in dissecting the role of gB during HCMV infection.
Singh
J
J
Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Compton
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D016254:Mutagenesis, Insertional; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D019286:Endopeptidase K