BACKGROUND: All herpesviruses examined to date encode a heterodimeric envelope complex consisting of glycoprotein H (gH) and glycoprotein L (gL); however, co-expression of human cytomegalovirus (HCMV) gH and gL is not sufficient to reconstitute the high molecular weight complex seen in infected cells. Previously, we showed that HCMV encodes a third glycoprotein, gO, which associates with gH and gL to form an unusual tripartite complex. OBJECTIVES: The objective of this study was to reconstitute the HCMV gH-containing complex by co-expression of the gH (UL75), gL (UL115), and gO (UL74) genes. We co-expressed gH, gL, and gO in insect cells using a recombinant baculovirus, and in a mammalian system using triple plasmid transfection. Recombinant complexes from both systems were compared with those expressed in HCMV infected cells by SDS-PAGE and immunoblot or immunoprecipitation with antibodies to gH, gL, or gO. RESULTS: Insect cells infected with the triple gene baculovirus produced gH/gL heterodimers, gH/gL heteromultimers, and gO homomultimers, however, they did not produce detectable tripartite complex. In contrast, co-expression of gH, gL, and gO in mammalian cells produced high molecular weight complexes that closely resemble gH/gL/gO complexes formed in HCMV infected cells. Reduction of disulfide bonds resolved high molecular weight complexes into the three individual glycoproteins. Additionally, cell surface immunofluorescence proved that the complexes are expressed and displayed on the surface of transfected cells. CONCLUSIONS: Triple plasmid transfected cells produced high molecular weight complexes that co-migrated with endogenous HCMV gH/gL/gO complexes as analyzed by SDS-PAGE. In addition, several distinct, novel forms of the three glycoproteins were detected.
BACKGROUND: All herpesviruses examined to date encode a heterodimeric envelope complex consisting of glycoprotein H (gH) and glycoprotein L (gL); however, co-expression of human cytomegalovirus (HCMV) gH and gL is not sufficient to reconstitute the high molecular weight complex seen in infected cells. Previously, we showed that HCMV encodes a third glycoprotein, gO, which associates with gH and gL to form an unusual tripartite complex. OBJECTIVES: The objective of this study was to reconstitute the HCMV gH-containing complex by co-expression of the gH (UL75), gL (UL115), and gO (UL74) genes. We co-expressed gH, gL, and gO in insect cells using a recombinant baculovirus, and in a mammalian system using triple plasmid transfection. Recombinant complexes from both systems were compared with those expressed in HCMV infected cells by SDS-PAGE and immunoblot or immunoprecipitation with antibodies to gH, gL, or gO. RESULTS: Insect cells infected with the triple gene baculovirus produced gH/gL heterodimers, gH/gL heteromultimers, and gO homomultimers, however, they did not produce detectable tripartite complex. In contrast, co-expression of gH, gL, and gO in mammalian cells produced high molecular weight complexes that closely resemble gH/gL/gO complexes formed in HCMV infected cells. Reduction of disulfide bonds resolved high molecular weight complexes into the three individual glycoproteins. Additionally, cell surface immunofluorescence proved that the complexes are expressed and displayed on the surface of transfected cells. CONCLUSIONS: Triple plasmid transfected cells produced high molecular weight complexes that co-migrated with endogenous HCMV gH/gL/gO complexes as analyzed by SDS-PAGE. In addition, several distinct, novel forms of the three glycoproteins were detected.
Authors: James R Netterwald; Thomas R Jones; William J Britt; Shao-Jun Yang; Ian P McCrone; Hua Zhu Journal: J Virol Date: 2004-06 Impact factor: 5.103
Authors: Brent J Ryckman; Barb L Rainish; Marie C Chase; Jamie A Borton; Jay A Nelson; Michael A Jarvis; David C Johnson Journal: J Virol Date: 2007-10-17 Impact factor: 5.103
Authors: Michael P Weekes; Peter Tomasec; Edward L Huttlin; Ceri A Fielding; David Nusinow; Richard J Stanton; Eddie C Y Wang; Rebecca Aicheler; Isa Murrell; Gavin W G Wilkinson; Paul J Lehner; Steven P Gygi Journal: Cell Date: 2014-06-05 Impact factor: 41.582