NKG2D Ligand MICA Is Retained in thecis-Golgi Apparatus by Human Cytomegalovirus Protein UL142

Abstract

Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the "missing-self" hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) and cis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in its cis-Golgi localization. We also confirm that UL142 downmodulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in the cis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is a ubiquitous human pathogen with seroprevalence ranging between 50 to 100% worldwide (46). Severe morbidity and mortality are associated with HCMV infection in individuals that are immunocompromised or immunologically immature (25), and the severity of HCMV disease in immunocompromised individuals correlates with the level of immune suppression. However, HCMV infection in healthy immunocompetent individuals is usually asymptomatic/subclinical (25). Like all herpesviruses, HCMV establishes lifelong latent infection, HCMV residues, in hematopoietic cells of the myeloid lineage (45). Thus, following primary infection, HCMV persists in the host despite a robust humoral and cellmediated immune response.HCMV down-modulates surface expression of host major histocompatibility complex (MHC) class I molecules in order to evade T-cell recognition (5, 7, 24), but in doing so, the virus risks natural killer (NK) cell activation due to the lack of inhibitory receptor signaling (30). However, there is now ample evidence that HCMV evades NK cell-mediated lysis by a variety of different mechanisms (58). These include the expression of molecules that engage inhibitory NK receptors, as well as the down-modulation of ligands for activating NK receptors.The HCMV UL40 open reading frame encodes a nonameric peptide that ...

2-Deoxy d-Glucose Prevents Cell Surface Expression of NKG2D Ligands through Inhibition of N-Linked Glycosylation

“…The Gateway compatible pCMV-Myc vector was kindly provided by Dr. K. Helin (Biotech Research and Innovation Centre, University of Copenhagen) (30). The GFP-MICA*018 plasmid that contained the fulllength allele MICA*018 downstream of a generic leader, a GFP cassette, and a myc tag was kindly provided by Dr. M. Wills (University of Cambridge, Cambridge, U.K.) (31).…”

Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus

“…N-terminal GFP-tagged UL142 was constructed as described previously (38). All plasmids were prepared using a Nucleobond Maxi Xtra method (Machery Nagel, Düren, Germany) and resuspended in Tris-EDTA or water.…”

“…In addition, we previously showed that UL142 localizes to the ER and cis-Golgi (37,38) and that transduction of UL142 is sufficient to protect cells from NK cell-mediated lysis, whereas reducing UL142 translation with RNA interference during HCMV infection increases NK cellmediated cytotoxicity (37). UL142 can also downregulate the products of full-length alleles of MICA by intracellular retention in the Golgi complex (33,38). Although these effects of UL142 have been proposed as the mechanism by which it exerts protection against NK cells, surface levels of truncated MICA protein (encoded by the p 008 allele) were not downregulated.…”

“…UL142 is a member of the UL18 gene family (36) and is predicted to have structural similarities to MHC class I, with a1 and a2 domains but no a3 domain. In addition, we previously showed that UL142 localizes to the ER and cis-Golgi (37,38) and that transduction of UL142 is sufficient to protect cells from NK cell-mediated lysis, whereas reducing UL142 translation with RNA interference during HCMV infection increases NK cellmediated cytotoxicity (37). UL142 can also downregulate the products of full-length alleles of MICA by intracellular retention in the Golgi complex (33,38).…”

Identification of immediate early gene X-1 as a cellular target gene of hcmv-mir-UL148D

“…Compared with the genome of the prototype laboratory strain, AD169, genomes of low-passage HCMV clinical isolates contain the UL/b' region, including ORFs UL133-UL151, considered as a critical candidate cluster to clinical pathogenesis (3). Although the UL/b' region is not essential to viral growth or replication (4), products of this region, including UL141, UL142 and UL144 have been experimentally identified to aid in viral escape from immune surveillance through interactions with cellular molecules (5)(6)(7)(8)(9)(10).…”