Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.

The murine gamma-herpesvirus-68 K3 (MK3) is a PHD/LAP finger protein that downregulates major histocompatibility complex (MHC) class I expression. In transfected cell lines, MK3 was expressed in the endoplasmic reticulum (ER) membrane, where it bound the cytoplasmic tail of newly synthesized H-2D(b) glycoproteins and targeted them for degradation. Proteasome inhibitors blocked the degradation and led to an accumulation of ubiquitinated H-2D(b). Because this retained its native conformation, ubiquitination preceded any denaturation or dislocation to the cytosol. The PHD/LAP finger of MK3 was not required for H-2D(b) binding but was essential for its ubiquitination and degradation. Thus, gamma-herpesviruses have adapted the cellular PHD/LAP motif to immune evasion, apparently for the catalysis of MHC class I ubiquitination.

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Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ-inducible, and binds to MHC class I coupled with β2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR: MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/ cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

Intramembrane proteolytic cleavage by signal peptide peptidase is required for the turnover of some ER-resident, tail-anchored membrane proteins.

The assembly of MHC class I molecules is governed by stringent endoplasmic reticulum (ER) quality control mechanisms. MHC class I heavy chains that fail to achieve their native conformation in complex with β2-microglobulin (β2m) and peptide are targeted for ER-associated degradation. This requires ubiquitination of the MHC class I heavy chain and its dislocation from the ER to the cytosol for proteasome-mediated degradation, although the cellular machinery involved in this process is unknown. Using an siRNA functional screen in β2m-depleted cells, we identify an essential role for the E3 ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating enzyme UBE2J1 in the ubiquitination and dislocation of misfolded MHC class I heavy chains. HRD1 is also required for the ubiquitination and degradation of the naturally occurring hemochromatosisassociated HFE-C282Y mutant, which is unable to bind β2m. In the absence of HRD1, misfolded HLA-B27 accumulated in cells with a normal MHC class I assembly pathway, and HRD1 depletion prevented the appearance of low levels of cytosolic unfolded MHC I heavy chains. HRD1 and UBE2J1 associate in a complex together with non-β2m bound MHC class I heavy chains, Derlin 1, and p97 and discriminate misfolded MHC class I from conformational MHC Iβ2m-peptide heterotrimers. Together these data support a physiological role for HRD1 and UBE2J1 in the homeostatic regulation of MHC class I assembly and expression.

The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposi’s sarcoma-associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex (MHC) class I molecules. Here, we show that K5 targets the membrane proximal lysine of MHC I for conjugation with mixed linkage polyubiquitin chains. Quantitative mass spectrometry revealed an increase in lysine-11, as well as lysine-63, linked polyubiquitin chains on MHC I in K5-expressing cells. Using a combination of mutant ubiquitins and MHC I molecules expressing a single cytosolic lysine residue, we confirm a functional role for lysines-11 and -63 in K5-mediated MHC I endocytosis. We show that lysine-11 linkages are important for receptor endocytosis, and that complex mixed linkage polyubiquitin chains are generated in vivo.

Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized. Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice. Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express ,1-galactosidase under control of the human cytomegalovirus iellie2 promoter/ enhancer. The lacZ gene was inserted between the ie2 and sggl genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice. In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50%o lethal dose and all had the capacity to latently infect the spleen. Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers. Plaque assay for virus and 5-bromo-4chloro-3-indolyl-,1-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved. Individual blue-staining cells were readily identified in all infected tissues. These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the