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Cytomegaloviruses (CMV) have developed multiple immunoevasion strategies to compromise host’s immune response. One of the strategies involves downregulation of MHC I molecules from the cell surface to prevent T cell response. Since lack of MHC I could activate NK cells via “missing-self” mechanism, MCMV encodes for m04, a protein that binds to some MHC I molecules, escorts them to the cell surface and enables the engagement to inhibitory NK Ly49 receptors. However, some activating Ly49 receptors specifically recognize MHC I-m04 complex together with another viral component and trigger NK cells. We have narrowed down this missing factor to a novel, spliced most abundant transcript (MAT) that encompasses two viral genes, m168-m169. Furthermore, this transcript codes for at least two proteins and contains a binding site for cellular miR-27 (Juranic Lisnic et al. 2013, Marcinowski, Tanguy et al. 2012). Here we show that 5'-UTR region of MAT (uORF MAT) plays a role in the recognition of infected cells by NK through both activating and inhibitory Ly49 receptors. Our results demonstrate that uORF MAT not only affects the level of MHC I on the surface of infected cells and their maturation, but also regulates quality of peptides that are loaded onto MHC I suggesting its potential role in the evasion of T cell recognition. Furthermore, deletion of uORF MAT region of MCMV results in attenuation of the virus in vivo, affects NK cells maturation and activation and IFNγ production.

Cytomegalovirus (CMV) has developed multiple immunoevasion strategies to compromise host’s immune response and ensure its survival. We have previously shown that the ligation of an inhibitory Ly49A NK cell receptor to complex of MHC-I and viral protein m04/gp34 leads to a protection of infected cells from specific lysis, impaired proliferation of NK cells and loss of control of viral titer in mice of H-2d and H-2k haplotype early upon an infection (Babić et al. J Exp Med, 2010). Moreover, m04 is essential for the recognition of infected cells by several activating Ly49 receptors (Ly49P, Ly49P1, Ly49D2 and Ly49L), which specifically recognize MHC-I, together with m04 and another viral component (Kielczewska et al. J Exp Med, 2009 ; Pyzik et al. J Exp Med, 2011). Subsequently, we have narrowed down this viral factor to recently described most abundant transcript (MAT) encompassing 2 viral genes, m168-m169 (Juranic Lisnic et al. PLoS Pathog, 2013). It has been shown that MAT transcript encodes poorly translated 17 kDa protein of unknown function and contains a binding site for cellular miR-27. Interestingly, neither function of miR-27 sponge nor the produced protein are responsible for recognition by activating Ly49 receptors, but its 5’UTR. Recently, we have detected additional protein in this region. Furthermore, deletion of MAT region of MCMV results in attenuation of the virus in vivo. We have also showed that absence of MAT results in dramatic changes of MHC-I maturation and host and viral peptide repertoire. However, the underlying mechanisms are not completely elucidated due to numerous functions MAT performs in the infected cells. Specifically, our current efforts are aimed to define how 5’UTR mediates Ly49 recognition.

Human cytomegalovirus (HCMV) represents a prototypic pathogenic member of theβ-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.

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 vitae, The 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.