NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145

The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance

Improved Ribo-seq enables identification of cryptic translation events.

Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translation initiation. We experimentally validated translation using major histocompatibility complex class I (MHC I) peptidomics and observed that sORF-derived peptides efficiently enter the MHC I presentation pathway and thus constitute a substantial fraction of the antigen repertoire

The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

Untersuchung der Struktur und Funktion des Peptidtransporters TAP unter Verwendung herpesviraler Inhibitoren

Cover and Contents Abbreviations Summary Zusammenfassung 1\. Introduction 2\. Materials 3\. Experimental Procedures 4\. Results 5\. Discussion 6\. Appendix I 7\. References 8\. Acknowledgements Publications and Curriculum vitaeThe human pathogenic herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus (HSV) both encode inhibitors of the peptide transporter TAP, i.e. gpUS6 and ICP47, respectively, to block antigen presentation to CD8+ T cells. While gpUS6 is an ER-resident type I transmembrane glycoprotein of 21 kDa, the soluble 8,5 kDa ICP47 inhibitor is located to the cytosol. TAP is a dimeric protein built by the subunits TAP1 and TAP2, each consisting of one transmembrane domain (TMD) and one cytosolic nucleotide binding domain (NBD). The NBDs utilize ATP energy for translocation of the peptide across the ER membrane. The membrane topology of the pore forming TMDs, the number of their transmembrane segments (TMSs) and the functional coupling of the processes by the NBDs with the movements of both TMDs required to complete the transport cycle, are not understood. This thesis comprehends gpUS6 and ICP47 as molecular tools for the investigation of TAP structure and function. Despite their profound biochemical differences, gpUS6 and ICP47 share phenotypical similarities, i.e. a physical interaction with preformed TAP complexes and a species-restricted mode of TAP inactivation. Taking advantage of these characteristics, mixed�species transporters and a large set of human/rat TAP chimeras were constructed, which allowed the identification of explicit and cryptic interaction domains for ICP47 and gpUS6 on TAP1 and TAP2. A dominant binding forwarding the inhibitory function of gpUS6 was delimited to the most C-terminal lumenal loop of the TAP1 TMD. This finding provided for the first time evidence for a model of TAP1 topology with 10 TMSs. Independent gpUS6 interaction with TAP2 is a prerequisite for efficient inhibition of TAP. For initial ICP47 contact to TAP human sequences of the N-terminus of TAP2 were found to be indispensable, but not sufficient for optimal binding. Further sequence requirements to maintain ICP47 interaction were mapped to the C-terminus of TAP2 in addition to undefined parts of TAP1. A second set of human TAP1 and TAP2 constructs mutated in the Walker A domain of the NBDs, which are responsible for ATP hydrolysis, allowed a detailed analysis of the cytosolic NBDs. This approach revealed a direct transmission of conformational changes to the TMDs which are sensored by gpUS6. The findings are integrated into a four step model for gpUS6 interaction with TAP in which initial ATP binding to the NBD of TAP2 results in a conformational change of the TMD. This forms the gpUS6 binding domain on TAP1 before association of gpUS6 to specific sites on TAP2 and TAP1 interrupts the peptide translocation cycle. Unlike gpUS6, ICP47 binding to TAP was found to be ATP independent. However, gpUS6 and ICP47 binding is mutually exclusive, indicating that ICP47 recognizes a distinct cytosolic TAP conformation. Furthermore, while ICP47 binding to TAP is inhibited by gpUS6, whereas peptide binding to TAP is not, this finding indicates that ICP47 acts through conformational constraints rather than competing out peptides for binding as deduced from earlier studies.Zum Zwecke der Immunevasion kodieren sowohl das humane Zytomegalovirus (HCMV) als auch das Herpes Simplex Virus (HSV) für Inhibitoren des Peptidtransporters TAP. Durch spezifische Interaktion von gpUS6 (HCMV) und ICP47 (HSV) mit TAP wird die Peptidtranslokation ins ER-Lumen als Voraussetzung für die Beladung der MHC I-Komplexe und folglich die Antigenpräsentation durch CD8+ T-Zellen gehemmt. Während gpUS6 (21 kDa) zu den Transmembran Typ I Glykoproteinen gehört und im ER lokalisiert ist, handelt es sich bei ICP47 (8,5 kDa) um ein zytosolisch lokalisiertes Protein. TAP bildet ein Dimer aus den Untereinheiten TAP1 und TAP2, welche beide sowohl eine Transmembran- (TMD) als auch eine zytosolische Nukleotid-Binde-Domäne (NBD) umfassen. TAP macht die aus der NBD vermittelten Hydrolyse von ATP resultierende Energie nutzbar, um Peptide über die ER Membran aus dem Zytosol ins ER Lumen zu transportieren. Die Membrantopologie der Pore, die Zahl der Transmembransegmente (TMS) und das funktionelle Zusammenspiel von NBDs und TMDs, unabdingbar hinsichtlich der Funktionalität des Membrantransporters, sind bis zum heutigen Tage weitestgehend unverstanden. In dieser Arbeit wurden gpUS6 und ICP47 als molekulare Instrumente hinsichtlich der Untersuchung der TAP-Struktur und -Funktion nutzbar gemacht. Trotz Unterschieden in ihren biochemischen Eigenschaften sind gpUS6 und ICP47 phenotypische Merkmale gemeinsam, wie die Interaktion ausschliesslich mit preformierten TAP-Komplexen und einen Spezies- spezifischen Wirkungsmechanismus. Die Konstruktion sowohl intermolekularer als auch intramolekularer TAP-Chimären erlaubte die Identifikation ICP47- und gpUS6-relevanter Interaktionsdomänen innerhalb von TAP1 und TAP2. Eine Bindestelle, welche die Hemmung des Peptidtransportes über TAP1 vermittelt, wurde innerhalb des C-terminalen Loops der TAP1-TMD identifiziert, ein erstmaliger Beweis für die Zusammensetzung von TAP1 aus 10 TMSs. Um eine effiziente Hemmung des Peptidtransportes durch TAP zu gewährleisten, ist eine zusätzliche gpUS6-Interaktion mit TAP2 eine unmittelbare Voraussetzung. Für die initiale Interaktion von ICP47 mit TAP sind humane Sequenzen innerhalb des N-Terminus der TAP2-TMD ausreichend. Darüberhinaus wurden Interaktionsdomänen innerhalb von TAP1 und des C-Terminus von TAP2 identifiziert. Weiterere TAP- Konstrukte, die Mutationen innerhalb des Walker A Motifs als Bestandteil der NBD enthielten, erlaubten eine detaillierte Analyse des Wirkungsmechanismuses dieser zytosolischen TAP-Domäne. Es konnte demonstriert werden, dass aus der ATP-Hydrolyse resultierende Konformationsänderungen innerhalb der TMD von gpUS6 erkannt werden. Der Befund wurde in ein mehrstufiges Modell hinsichtlich der gpUS6-Interaktion mit TAP integriert. Nach TAP2-vermittelter ATP-Bindung und �Hydrolyse und den resultierenden Konformationsänderungen innerhalb der TMD wird die gpUS6-Bindungsdomäne exponiert. gpUS6 bindet spezifisch an TAP1 und TAP2 und inhibiert den Peptidtransport über die ER-Membran. Im Gegensatz zu gpUS6 bindet ICP47 TAP unabhängig von ATP. Es konnte gezeigt werden, dass sich gpUS6- und ICP47-Bindung aufgrund unterschiedlicher TAP-Konformationen gegenseitig ausschliessen. Die ICP47-Bindung an TAP kompetiert nicht mit der Peptidbindung an TAP, kann aber von gpUS6 gehemmt werden, was auf einen auf Konformationsveränderungen basierenden Wirkungsmechanismus des ICP47 schliessen lässt. Dieser Befund stellt andere Studien infrage, die ICP47 einen kompetitiven Wirkunsmechanismus attestieren

Human Cytomegalovirus and Autoimmune Disease

Human cytomegalovirus (HCMV) represents a prototypic pathogenic member of the β-subgroup of the herpesvirus family. A range of HCMV features like its lytic replication in multiple tissues, the lifelong persistence through periods of latency and intermitting reactivation, the extraordinary large proteome, and extensive manipulation of adaptive and innate immunity make HCMV a high profile candidate for involvement in autoimmune disorders. We surveyed the available literature for reports on HCMV association with onset or exacerbation of autoimmune disease. A causative linkage between HCMV and systemic lupus erythematosus (SLE), systemic sclerosis (SSc), diabetes mellitus type 1, and rheumatoid arthritis (RA) is suggested by the literature. However, a clear association of HCMV seroprevalence and disease could not be established, leaving the question open whether HCMV could play a coresponsible role for onset of disease. For convincing conclusions population-based prospective studies must be performed in the future. Specific immunopathogenic mechanisms by which HCMV could contribute to the course of autoimmune disease have been suggested, for example, molecular mimicry by UL94 in SSc and UL83/pp65 in SLE patients, as well as aggravation of joint inflammation by induction and expansion of CD4+/CD28− T-cells in RA patients. Further studies are needed to validate these findings and to lay the grounds for targeted therapeutic intervention

Physical and functional interactions of the cytomegalovirus US6 glycoprotein with the transporter associated with antigen processing

The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing (TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8+ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6

A slowly cleaved viral signal peptide acts as a protein-integral immune evasion domain

International audienceStress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains

A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle

The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1-35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation

HLA-B locus products resist degradation by the human cytomegalovirus immunoevasin US11.

To escape CD8+ T-cell immunity, human cytomegalovirus (HCMV) US11 redirects MHC-I for rapid ER-associated proteolytic degradation (ERAD). In humans, classical MHC-I molecules are encoded by the highly polymorphic HLA-A, -B and -C gene loci. While HLA-C resists US11 degradation, the specificity for HLA-A and HLA-B products has not been systematically studied. In this study we analyzed the MHC-I peptide ligands in HCMV-infected cells. A US11-dependent loss of HLA-A ligands was observed, but not of HLA-B. We revealed a general ability of HLA-B to assemble with β2m and exit from the ER in the presence of US11. Surprisingly, a low-complexity region between the signal peptide sequence and the Ig-like domain of US11, was necessary to form a stable interaction with assembled MHC-I and, moreover, this region was also responsible for changing the pool of HLA-B ligands. Our data suggest a two-pronged strategy by US11 to escape CD8+ T-cell immunity, firstly, by degrading HLA-A molecules, and secondly, by manipulating the HLA-B ligandome