The human cytomegalovirus US10 gene product delays trafficking of major histocompatibility complex class I molecules

Abstract

Human cytomegalovirus (HCMV) US10 encodes a glycoprotein that binds to major histocompatibility complex (MHC) class I heavy chains. While expression of US10 delays the normal trafficking of MHC class I molecules out of the endoplasmic reticulum, US10 does not obviously facilitate or inhibit the action of two other HCMV-encoded MHC class I binding proteins, US2 and US11.

FIG. 1.

HCMV US10 associates with MHC class I HC. (A) US10-HA-expressing cells and control cells were metabolically labeled for 50 min. Folded MHC class I-β₂m complexes, US10-HA, and free HC were recovered from cell lysates by immunoprecipitation (IP) with W6/32, 12CA5, and anti-HC antibodies (αHC), respectively. A reimmunoprecipitation(re-IP) for HC was performed from a portion of the 12CA5 immunoprecipitates that had been denatured in 1% SDS and boiled (lanes 3 and 7). The positions of prestained molecular size standards (Amersham) are shown on the right. (B) US10-HA and control cells were metabolically labeled for 10 min and chased for up to 45 min in nonradioactive medium. HC and US10-HA molecules were recovered from cell lysates with anti-HC and 12CA5 antibodies, respectively. All samples were analyzed by SDS-PAGE (12.5% polyacrylamide gels).

FIG. 2.

HCMV US10 delays the egress of folded MHC class I molecules from the ER. (A) US10-HA-expressing cells and control cells were metabolically labeled for 10 min. Folded MHC class I molecules were recovered from cell lysates with monoclonal antibody W6/32. Half of the W6/32 immunoprecipitate was digested with Endo H prior to analysis (lanes 9 to 16). Samples were analyzed by SDS-PAGE (12.5% polyacrylamide gels). Fractions of Endo H-sensitive (EH S) and Endo H-resistant (EH R) MHC class I complexes were quantitated by phosphorimage analysis; the graph shows the percentages of Endo H-sensitive complexes at each time point (graph). (B) Cells were treated as described for panel A. Transferrin receptor molecules were recovered from cell lysates by using the monoclonal antibody 66Ig10. Fractions of Endo H-sensitive transferrin receptors (shown in lanes 9 to 16) were quantitated and plotted as described for panel A (graph).

FIG. 3.

US10 does not interfere with US2 or US11 function. Human astrocytoma cells stably transfected with US2, US10-HA and US2, US10-HA and US11, or US11 only were preincubated with 50 μM ZL₃VS, metabolically labeled for 5 min, and chased up to 30 min in nonradioactive medium. HC were recovered from cell lysates and analyzed by SDS-PAGE (10% polyacrylamide gels). HC+CHO and HC-CHO, glycosylated and deglycosylated forms of MHC class I HC, respectively.