engleski

Characterization of murine cytomegalovirus 34 kDa glycoprotein that forms a complex with the cell surface MHC class I molecules

Background and purpose. Infection of embryonal fibroblasts with murine cytomegalovirus (MCMV) leads to early loss of MHC class I molecules from the cell surface. At later times of infection cell surface MHC class I expression is partially restored. With those MHC class I molecules a glycoprotein of 34 kDa (gp34) was coprecipitated. gp34, a product of MCMV m04 gene, forms a complex with MHC class I molecules in the endoplasmic reticulum (ER) which is not retained but transported to the cell surface (Kleijnen M M F, Huppa J B, Lučin P, Mukherjee S, Farrell H, Cambpell A, Koszinowski U H, Hill A B, Ploegh H L 1997 EMBO J 16:684-694). We have produced an antiserum against carboxyterminal peptide of the m04 gene product and used this antiserum for further biochemical characterization of gp34. Materials and methods. Cell surface proteins were labeled by radioiodination using lactoperoxidase-glucose-oxidase method, and intracellular proteins were labeled metabolically with  35S methionine. Proteins were immunoprecipitated from cellular lysates and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or 2-dimensional (2-D) electrophoresis with the isoelectric focusing (NEPHGE) as a first dimension. Results. Synthesis of gp34 in MCMV infected fibroblasts starts between 2 and 3 hours postinfection, reaches maximum at 4-5 hours post infection and continues throughout the replication cycle. In the ER gp34 acquires three N-linked glycans which are not processed during transport through the secretory pathway and reach the cell surface in the endoglycosidase H sensitive form. Cell-surface fraction of gp34, which is in the complex with MHC class I molecules, migrates as a basic protein after isoelectric focusing. Pulse-chase analysis demonstrated that gp34 is very stable glycoprotein with a half-life longer than 6 hours. After electrophoresis under nonreducing conditions gp34 migrates faster, indicating that an intrachain disulfide bridge stabilizes tertiary structure of gp34.

cytomegalovirus; gp34; MHC class I molecules

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