Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein

Autor: Boyoun Park, Sung-Wook Lee, Kwangseog Ahn
Rok vydání: 2003
Předmět:
Zdroj: Journal of Virology. 77:2147-2156
ISSN: 1098-5514
0022-538X
Popis: The importance of cytotoxic T-lymphocyte (CTL)-mediated immune responses in limiting and clearing viral infections has been well documented for a number of viral systems (7). These processes imply a balance between immune control of the virus and immune escape by the virus (33). Many viruses encode proteins that can inhibit or abolish the surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. Human cytomegalovirus (HCMV), which causes benign but persistent infections in immunocompetent individuals, encodes an endoplasmic reticulum (ER) resident glycoprotein, US3, that prevents intracellular transport of MHC class I molecules (1, 19). HCMV US3 binds physically to MHC class I heterodimers and sequesters them in the ER. This function might play a central role in establishing persistent, latent, and acute viral infections. Therefore, identifying the retention signals and elucidating the structural requirements for US3 to bind to MHC class I molecules might reveal the mechanisms of viral pathogenesis and protein compartmentalization. The ER is a heterologous organelle containing large amounts of newly synthesized polypeptides as well as resident proteins responsible for numerous posttranslational modifications, including glycosylation, folding, and oligomerization reactions. Because of their abundance, ER resident proteins must be efficiently segregated from their substrates by specific retention and retrieval signals in their primary structure. To date, only two systems, both based on a retrieval mechanism, have been characterized. The KDEL tetrapeptide at the extreme carboxyl (COOH) terminus of ER resident proteins is a common signal for a number of luminal chaperones (30). This retrieval mechanism is based on the KDEL receptor (ERD2), which binds escaped proteins in the Golgi complex and returns them to the ER (23, 24). In the other proposed mechanism, double-lysine and presumably double-arginine motifs located in the cytoplasmic domains of several ER membrane proteins also function as retrieval motifs (18, 21). It is known that double-lysine motif-containing proteins bind the complex of cytosolic coat proteins (coatomer), COP I, and that this interaction mediates the retrieval of these proteins from the Golgi for return to the ER (35). Sequences flanking the double-lysine motif also contribute to the steady-state distribution of the proteins between the ER and the Golgi complex (17, 18). The primary structure of the US3 protein (1) consists of a signal sequence of 15 amino acids followed by a luminal domain of 146 amino acids. Twenty membrane-spanning residues separate the luminal portion of the US3 protein from a short, 5-amino-acid cytoplasmic tail. The protein is glycosylated at amino acid 60. US3, unlike most other luminal proteins in the ER, does not contain in its primary structure either the KDEL sequence or any of its close homologues. We have shown previously that the luminal domain of the US3 protein is sufficient for retention in the ER and that the ER localization of US3 involves true retention without recycling through the Golgi (20). To characterize more precisely the sequence or structural requirement of the luminal ER retention signal of US3, we used two different approaches. In the first approach, we constructed fusions of mutated US3 luminal sequences and the green fluorescent protein (GFP) and analyzed the subcellular localization of the resulting chimeric proteins. Second, we investigated whether the sequence elements identified with the first approach could mediate retention of the protein in the ER in the context of homologous US3 glycoproteins. We identified a noncontiguous sequence consisting of three specific amino acids that constitutes the retention signal of the US3 protein. Substitution of alanine for any of these amino acids led to a loss of ER retention of the chimeric reporter constructs and of the homologous US3 glycoproteins. Importantly, these mutant proteins, in contrast to wild-type US3, were unable to prevent class I molecules from reaching the plasma membrane.
Databáze: OpenAIRE
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