Dependence of the localization and function of the human cytomegalovirus protein US6 on the transporter associated with antigen processing

Abstract

Human cytomegalovirus protein US6 inhibits the transporter associated with antigen processing (TAP), which transports peptides into the endoplasmic reticulum (ER) for binding to major histocompatibility complex (MHC) class I molecules. We demonstrate that, in TAP-deficient cells, US6 is retained in the ER and binds to calnexin, but does not inhibit cell-surface expression of HLA-A201, an MHC class I allele that binds to peptides whose import into the ER is TAPindependent. Furthermore, in TAP-positive cells, US6 reduces the cell-surface expression of HLA-B2705, an MHC class I allele that is dependent on TAP for peptide binding, to a greater extent than that of HLA-A201. These data demonstrate that US6 has differential effects on the cell-surface expression of MHC class I alleles and are consistent with TAP being the sole inhibitory target of US6 in the MHC class I antigen-presentation pathway.Major histocompatibility complex (MHC) class I molecules present peptides derived from intracellular proteins to cytotoxic T lymphocytes (CTLs). CTLs monitor MHC class I molecules for peptides derived from viral antigens and kill virus-infected cells. Most peptides presented by MHC class I molecules are generated by the cytosolic proteasome complex (Rock & Goldberg, 1999). These peptides are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen (Abele & Tampe, 2004), which is the site of the chaperone-dependent folding of MHC class I molecules (Antoniou et al., 2003;Peaper & Cresswell, 2008). Peptide binding represents the final step of MHC class I assembly and is facilitated by tapasin, which links the nascent MHC class I molecules and the associated chaperones calreticulin and ERp57 to TAP to form the peptide-loading complex (Antoniou et al., 2003;Peaper & Cresswell, 2008). Once peptide-bound, MHC class I molecules dissociate from this complex and traffic to the plasma membrane.Human cytomegalovirus (HCMV) encodes a cohort of proteins that inhibit the MHC class I antigen-presentation pathway (Hewitt, 2003;Hewitt & Dugan, 2004). These include US6, an ER-localized type I integral membrane glycoprotein that inhibits peptide translocation by TAP (Ahn et al., 1997; Hengel et al., 1997;Lehner et al., 1997). This blocks the supply of peptides for binding to MHC class I molecules and hence impairs recognition by CTLs (Ahn et al., 1997; Hengel et al., 1997;Lehner et al., 1997). The inhibition of ATP binding is an indirect conformational effect, as the ER-luminal domain of HCMV US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide-binding domain of the TAP1 subunit (Hewitt et al., 2001; Kyritsis et al., 2001). Recently, we demonstrated that residues 89-108 of the US6 luminal domain are required for inhibition of TAP, whereas other parts of US6 bind to TAP and stabilize this inhibitory interaction (Dugan & Hewitt, 2008). Yet, whilst US6 binds to TAP, it is not known whether this interaction retains US6 in the ER or whether US6 can inhibit MHC class I expressi...