Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident trans membrane glycoprotein, forms a complex with major histocompatibility comple x (MHC) class I molecules and retains them in the ER, thereby preventing cy tolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association w ith MHC class I molecules, we constructed truncated mutant and chimeric for ms in which US3 domains were exchanged with corresponding domains of CD4 an d analyzed them for their intracellular localization and the ability to ass ociate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the EP, whi le replacement of the US3 luminal domain with that of CD4 led to cell surfa ce expression of the chimera. Thus, the luminal domain of US3 was sufficien t for ER retention. Immunolocalization of the US3 glycoprotein after nocoda zole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER locali zation of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal doma in of US3. Direct interaction between US3 and MHC class I molecules could b e demonstrated after in vitro translation by coimmunoprecipitation. Togethe r, the present data indicate that the properties that allow US3 to be local ized in the ER and bind MHC class I molecules are located in different part s of the molecule.