Distinct glycoprotein O complexes arise in a post-Golgi compartment of cytomegalovirus-infected cells

Abstract

Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in transfected cells as it does in infected cells. We further determined that gO is modified by O-linked glycosylation and that this terminally processed form is highly enriched in virions. However, during studies of gO processing, novel gO complexes were discovered in CMV virions. The newly identified gO complexes, including gO-gL heterodimers, were not readily detected in CMV-infected cells. Further characterization of the trafficking of gO through the secretory pathway of infected cells localized gH, gL, and gO primarily to the Golgi apparatus and trans-Golgi network, supporting the conclusion that the novel virion-associated gO complexes arise in a post-Golgi compartment of infected cells.

FIG. 1.

Electrophoretic mobilities of different forms of gO. CMV-infected human fibroblasts and transfected cells expressing gO were lysed and analyzed by SDS-PAGE followed by immunoblotting with gO antibody. M, mock-infected cells; I, infected human fibroblasts; T, transfected 293-T cells expressing gO.

FIG. 2.

Endo H digestion of gO. (A) Mock-infected cells (M) or CMV-infected cells were harvested 7 days postinfection and then treated with no enzyme (I), endo H (EH), or PNGase (PNG). Proteins were analyzed by SDS-PAGE and immunoblotting with gO antibody. The diffusely migrating, endo H-resistant form of gO is indicated by brackets. (B) Solubilized CMV virions were mock digested (V), treated with endo H (EH), or digested with PNGase (PNG). Proteins were analyzed as described for panel A. Numbers beside panels show kilodaltons.

FIG. 3.

Analysis of O-linked carbohydrate on gO and gO complexes. Solubilized virions were incubated overnight with jacalin-conjugated agarose beads. After washing was done, bound proteins were eluted from the beads and analyzed by SDS-PAGE and immunoblotting with gO antibody. V, total solubilized virion proteins; A, proteins eluted from control streptavidin-agarose beads; J, proteins eluted from jacalin-agarose beads. (A) Prior to incubation with beads, virion proteins were boiled, reduced, and treated with PNGase to remove N-glycans. (B) Unreduced, fully glycosylated virion proteins were incubated with jacalin beads. Arrows indicate gH-gL-gO complexes, and arrowheads indicate previously unidentified forms of gO. Numbers beside panels show kilodaltons.

FIG. 4.

gO-gL heterodimers identified by immunoblotting. Virion proteins were solubilized and subjected to nonreducing SDS-PAGE followed by immunoblotting. Strips of the same membrane were blotted with antibodies to gO and gL. gH complexes were characterized by blotting with monoclonal antibody AP86.

FIG. 5.

Glycosidase treatment of multimeric complexes. CMV virions were either incubated with neuraminidase (+) or mock digested (−) and then subjected to nonreducing SDS-PAGE and immunoblotting with gO antibody. Numbers beside panels show kilodaltons.

FIG. 6.

Comparison of gO complexes in infected cells and virions. Mock-infected cells (M) and CMV-infected cells (Inf) were directly compared to CMV virions (V) by immunoblotting of solubilized, unreduced samples with gO antibody. Cells and purified virions were solubilized in RIPA medium prior to the addition of SDS-PAGE sample buffer.

FIG. 7.

Immunofluorescence colocalization of gH, gL, and gO. Human fibroblasts were infected with CMV at a multiplicity of infection of approximately 0.5 and harvested 6 days postinfection. Cells were fixed and stained for indirect immunofluorescence; each horizontal series represents a single field. Virus-specific rabbit antibodies (middle columns) were detected with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG). Monoclonal antibody 1G6 (anti-gH) (A), an β-COP monoclonal antibody (B), and a TGN38 monoclonal antibody (C) were detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (left columns). Nuclei were stained with Hoechst dye (blue). The right columns represent the merged images.

FIG. 7.

Immunofluorescence colocalization of gH, gL, and gO. Human fibroblasts were infected with CMV at a multiplicity of infection of approximately 0.5 and harvested 6 days postinfection. Cells were fixed and stained for indirect immunofluorescence; each horizontal series represents a single field. Virus-specific rabbit antibodies (middle columns) were detected with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG). Monoclonal antibody 1G6 (anti-gH) (A), an β-COP monoclonal antibody (B), and a TGN38 monoclonal antibody (C) were detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (left columns). Nuclei were stained with Hoechst dye (blue). The right columns represent the merged images.

FIG. 7.

Immunofluorescence colocalization of gH, gL, and gO. Human fibroblasts were infected with CMV at a multiplicity of infection of approximately 0.5 and harvested 6 days postinfection. Cells were fixed and stained for indirect immunofluorescence; each horizontal series represents a single field. Virus-specific rabbit antibodies (middle columns) were detected with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG). Monoclonal antibody 1G6 (anti-gH) (A), an β-COP monoclonal antibody (B), and a TGN38 monoclonal antibody (C) were detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (left columns). Nuclei were stained with Hoechst dye (blue). The right columns represent the merged images.

FIG. 8.

Model of gH-gL-gO complex assembly and trafficking in CMV-infected cells. The appearance of various gH, gL, and gO complexes is illustrated temporally from top to bottom. Proteins denoted with a slash (e.g., gH/gL) are held together by disulfide linkages. The glycoproteins are cotranslationally inserted into the ER. Subsequently, glycosylated proteins travel to the Golgi apparatus and then to the TGN. gH, gL, and gO accumulate in Golgi apparatus-derived vesicles, although some gH-gL-gO appears on the surface of infected cells. Cell surface tripartite complexes may or may not recycle via endocytosis (?). Multiple TGN-derived complexes likely incorporate into virions during envelopment. Rearrangement of gH-gL-gO complexes results in the emergence of previously undetected forms of gO, including gO-gL heterodimers. Subsequent events in viral egress are not well characterized. Bold letters indicate gO complexes that arise between the Golgi/TGN and the point of virion collection. gH-gL-gO∗, tripartite complex in which gO contains O-glycans.