Die Bildung infektiöser Viruspartikel des humanen Zytomegalievirus (HCMV) ist ein mehr-stufiger Prozess. Sie beginnt mit der Verpackung der DNA in die Kapside im Kern, gefolgt von weiterer Reifung während des Transports durch das Zytoplasma und der abschließenden Freisetzung aus der Zelle. Im Zuge dieser Arbeit wurden zwei Proteine, die Einfluss auf die ebengenannten Prozesse haben, analysiert. Der erste Teil der Arbeit befasst sich mit der funktionellen Charakterisierung des HCMV Pro-teins pUL77. Es ist bekannt, dass das homologe Protein pUL25 in alpha-Herpesvirinae essentiell für die DNA-Verpackung ist. Zunächst konnte das Protein als Kapsid-assoziiertes strukturelles Protein identifiziert werden. Es wurden Interaktionen von pUL77 mit DNA-Verpackungs- und Kapsidproteinen gezeigt. Weiterhin wurde die DNA-Bindungsfähigkeit von pUL77 in verschiedenen „in vitro“-Experimenten untersucht. Zusammengefasst weisen unsere Ergebnisse auf eine Funktion von HCMV pUL77 bei der DNA-Verpackung hin. Im zweiten Teil der Arbeit wurde das HCMV Protein pUL71 charakterisiert, das in allen Herpesviren konserviert vorkommt, dessen Funktion jedoch nicht charakterisiert ist. Zunächst wurde das Protein als strukturelles Tegumentprotein mit “earlylate“ Expressionskinetik klassifiziert. Weiterhin wurden die subzelluläre Lokalisation sowie virale und zelluläre Interaktionspartner untersucht. Die Ergebnisse weisen auf eine Funktion von HCMV pUL71 bei der Reifung und beim Transport der Virionen im Zytoplasma hin. „In silico“-Vorhersagen zeigten ein „Leuzin Zipper“-Motiv in pUL71, das als mögliche Oligomerisationsdomäne dienen könnte. Mutationen wurden in dieses Motiv eingebracht und die resultierenden Proteine auf ihre Oligomerisationsfähigkeit mit „in vitro“-Methoden und in rekombinanten Viren untersucht. Zusammenfassend konnten wir zeigen, dass das „Leuzin Zipper“-Motiv wichtig für die Funktion von pUL71 ist und diese mit einer unbeeinträchtigten Oligomerisation des Proteins zusammen hängt. / The morphogenesis of Human cytomegalovirus (HCMV) virions starts with the capsid assem-bly and DNA insertion in the nucleus followed by maturation during transport through the cytoplasm prior to release of virus progeny. In this study we are functionally characterising two proteins that are involved in those steps. The function of essential HCMV protein pUL77 is characterised in the first part of the study. HCMV pUL77 was shown to be a structural protein associated with capsids. Furthermore, our experiments demonstrated that HCMV pUL77 interacts with DNA packaging motor compo-nents and capsid proteins. The ability of HCMV pUL77 to bind double-stranded DNA was studied in “in vitro” assays designed for this study. The homologue α-Herpesvirinae protein pUL25 is described to be involved in processes connected with DNA packaging. Data ob-tained in this study demonstrates that HCMV pUL77 might serve a similar function. In the second part of the study HCMV pUL71, conserved throughout the Herpesvirus family but to date unclassified, was functionally characterised. HCMV pUL71 was defined a struc-tural tegument protein with early-late expression kinetics. We studied the sub-cellular local-isation and interactions of pUL71 with a subset of cellular and viral proteins. Thereby we could show that HCMV pUL71 function might be connected with processes of viral egress. By in silico analyses we identified a leucine zipper motif in pUL71 that might serve as a puta-tive oligomerisation domain. In order to investigate the function of the leucine zipper motif, we performed in vitro assays and investigated the alterations of the motif in the viral context. Taken together we can conclude that (i) an intact leucine zipper motif is crucial for the func-tion of pUL71 and (ii) this function is dependent upon undisturbed oligomerisation of the pro-tein.

This work is centered on the study of the NKG2 C-type lectin-like receptors on NK and CD4+T cells. We provide evidence supporting that CD4+T cells specific for Human Cytomegalovirus (HCMV) may express different NK cell receptors, and demonstrate that the C-type lectin-like receptor NKG2D is expressed on cytotoxic CD4+T cells with an effector/memory phenotype, enhancing their TCR-dependent proliferation and cytokine production. A second part of the work is centered on the study of the CD94/NKG2 receptors on NK cells. We show that NKG2A can be induced on NKG2C+ NK cells upon activation with rIL-12 or when cocultured with HCMV-infected dendritic cells, and that NKG2A expression inhibits the response of NKG2C+NK clones against HLA-E-expressing targets, providing a potential regulatory feedback mechanism to control cell activation. Altogether, our results support that expression of NKG2 C-type lectin like receptors may be shaped during the course of viral infections, providing mechanisms to finely regulate both NK and CD4+T cell functions. / Aquesta tesi es centra en l'estudi dels receptors lectina de tipus C NKG2 en cèl·lules Natural Killer i T CD4+. Demostrem que les cèl·lules T CD4+ específiques pel Cytomegalovirus Humà poden expressar diferents receptors NK, i que el receptor lectina tipus C NKG2D s'expressa en cèl·lules citotòxiques i de memòria, potenciant la proliferació i secreció de citocines depenent del TCR. La segona part d'aquesta tesi es centra en l'estudi de l'expressió dels receptors CD94/NKG2 en cèl·lules NK. Mostrem com l'expressió de CD94/NKG2A s'indueix en cèl·lules CD94/NKG2C+ estimulades amb IL-12 o cultivades amb cèl·lules dendrítiques infectades pel Cytomegalovirus Humà, i que l'expressió de CD94/NKG2A inhibeix la resposta de clons NK CD94/NKG2C+ envers dianes HLA-E+, constituint un possible mecanisme de feedback negatiu per controlar l'activació cel·lular. En resum, els nostres resultats demostren que l'expressió dels receptors lectina tipus C NKG2 pot ser modificada durant les infeccions víriques consitutint un possible mecanisme per regular la resposta tant de cèl·lules NK com T CD4+.

S'ha establert un sistema experimental autòleg per a poder estudiar la resposta de les cèl.lules Natural Killer (NK) contra les cèl.lules dendrítiques derivades de monòcits (moDC), infectades pel Cytomegalovirus humà (HCMV). Els nostres resultats mostren que les cèl.lules NK responen contra les moDC infectades per HCMV, que presenten una expressió de les molècules MHC de classe I a superficie reduïda. Específicament, demostrem que la infecció per HCMV disminueix l'expressió en superficie d'HLA-E en les moDC, alliberant així la inhibició de les cèl.lules NK NKG2A+. Mostrem que els NKR anomenats NKp46 i DNAM-1 tenen un paper dominant en el reconeixement de les moDC infectades per HCMV i evidenciem la importància de la dinàmica dels mecanismes d'immunoevassió en la susceptibilitat a la resposta NK. Finalment, trobem que els interferons de tipus I i la IL-12 secretats en resposta a la infecció per HCMV, a més de participar en l'activació de la cèl.lula NK i en la secreció d'IFN-, inhibeixen l'expressió i la funció de NKG2D en les cèl.lules NK, com un mecanisme de regulació potencial per prevenir la reactivitat NK contra cèl.lules veïnes sanes. / Suitable experimental conditions have been established to dissect the role of NK cell receptors (NKR) and cytokines in the NK cell response against autologous human cytomegalovirus (HCMV) infected monocyte derived dendritic cells (moDC). Our results reveal that NK cells are capable of responding to HCMV infected moDC that have down-regulated surface MHC class I molecules. In particular, we prove that HCMV infection decreases surface HLA-E expression on moDC, thus releasing NKG2A+ NK cells from inhibition. We show that NKp46 and DNAM-1 NKR play a dominant role in the recognition of HCMV infected moDC and we provide evidences stressing the importance of the dynamics of viral immune evasion mechanisms in NK cell susceptibility. Finally, we find that type I interferons and IL-12 secreted in response to HCMV infection, beyond their participation in NK cell activation and IFN- secretion, transiently inhibit the expression and function of NKG2D in NK cells, thus providing a potential regulatory feedback mechanism to prevent NK cell reactivity against bystander healthy cells.

In the present study, we have approached the molecular characterization of the HCMV specific UL1. To this end a HCMV (AD169-derived HB5 background) recombinant with an HA-epitope tagged UL1 and a mutant with a full UL1 deletion in the endotheliotropic HCMV TB40/E strain were generated. Our data reveal that the UL1 is transcribed with late kinetics. pUL1 is glycosylated and localizes at the site of virus assembly and secondary envelopment in infected cells forming part of the envelope of HCMV virions. A HCMV mutant with a targeted deletion of UL1 exhibits a growth defect phenotype in retinal pigment epithelium cells but not in fibroblasts, indicating that this ORF encodes a cell-type specific tropism factor. / En aquest treball hem investigat la pauta oberta de lectura de UL1 del Cytomegalovirus humà (HCMV), el gen UL1 es específic del HCMV. Hem caracteritzat la proteïna UL1 modificada amb un epítop HA en la soca HB5, derivada de AD169. L'UL1 s’expressa com una glicoproteïna que es pot detectar a les 48 i 72h post-infecció. En fibroblasts humans infectats, UL1 co-localitza al citoplasma, al lloc d’assemblatge del virió, amb proteïnes estructurals del virus. A més a més, els anàlisis de virions AD169 purificats que contenen UL1-HA mostren que UL1 és un nou constituent de l’envolta del HCMV. La delecció de UL1 en el context de la soca TB40/E del HCMV disminueix el creixement viral de manera selectiva en determinats tipus cel•lulars, suggerint que UL1 podria estar involucrat en la regulació del tropisme cel•lular del HCMV.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T09:16:08Z (GMT). No. of bitstreams: 1 Andrade_PaulaDurante_M.pdf: 3994483 bytes, checksum: eca47ce9f7df65d68a6da3ad72ad056b (MD5) Previous issue date: 2009 / Resumo: O Citomegalovírus Humano (HCMV) é o principal causador de complicações pós-transplante. Métodos específicos que permitam identificar, precocemente, os pacientes com risco de desenvolvimento de doença, para os quais tratamento é indicado, têm sido requeridos, a fim de que poucos pacientes sejam desnecessariamente tratados e para a efetiva instituição terapêutica. A "Nested-PCR", dupla Reação em Cadeia da Polimerase, é um teste comumente utilizado no diagnóstico da infecção ativa por HCMV, contudo, quando realizada em leucócitos do sangue periférico, devido à sua alta sensibilidade, não apresenta boa correlação com o desenvolvimento de doença por HCMV. A detecção do DNA do HCMV no soro, pela PCR, tem sido associada com o desenvolvimento de doença por HCMV. Neste estudo, nós aplicamos a "Nested-PCR" em leucócitos do sangue periférico (denominada "L-PCR"), o método Convencional do Laboratório, e em soro ("sPCR"), para o diagnóstico da infecção ativa por HCMV, a fim de estabelecermos a correlação dos resultados obtidos, de ambos os métodos, com o desenvolvimento de infecção sintomática. Com este propósito, nós avaliamos, prospectivamente, amostras de 37 pacientes, 20 submetidos a transplante renal, e 17 submetidos a transplante de células tronco hematopoiéticas. Para excluir resultados falso negativos, na reação de amplificação pela "sPCR", um controle interno foi construído e todas as reações foram realizadas utilizando-o. De acordo com os critérios estabelecidos neste estudo, 21 pacientes (21/37 - 57%) desenvolveram infecção ativa por HCMV. Todos os pacientes com infecção ativa por HCMV foram positivos para a "L-PCR" (p= 0,0003). A "sPCR" foi positiva para somente 10 pacientes (10/37 - 27%) (p= 0,01). Resultados discordantes foram observados em 11 pacientes que foram positivos para infecção ativa para a "L-PCR", mas negativos para a "sPCR", 5 dos quais desenvolveram sintomas clínicos para o HCMV. O coeficiente Kappa de concordância observado para ambos os métodos foi de 0,44 (acordo moderado). Em dezesseis pacientes (43%), dos 37 estudados, não foram observados resultados positivos por nenhum dos métodos empregados - não desenvolveram infecção ativa -, contudo, 2 destes desenvolveram sintomas clínicos de provável doença por HCMV e um apresentou, posteriormente, biópsia confirmativa para doença por HCMV. Sintomas clínicos foram observados em 14 pacientes, em 12 deles infecção ativa foi diagnosticada através da "L-PCR" (p= 0,007) e, em 7, através da "sPCR" (p= 0,02). Ausência de sintomas clínicos foram observados em 9 pacientes nos quais a "L-PCR" detectou infecção ativa e em 3 pacientes nos quais a "sPCR" detectou (coeficiente Kappa 0,57, acordo moderado). De 14 pacientes sintomáticos, 2 pacientes soronegativos para o HCMV que receberam rins soropositivos desenvolveram infecção primária. Analizando os dois métodos para o diagnóstico da infecção ativa, nós observamos maior sensibilidade e valor preditivo negativo da "L-PCR" ("L-PCR" 100% vs. "sPCR" 62%), e maior especificidade e valor preditivo positivo da "sPCR" ("sPCR" 81% vs. "L-PCR" 50% e 72%). O valor dos testes positivos, "L-PCR" e "sPCR", para predizer doença por HCMV foram, respectivamente, 57% e 70%, e o valor dos testes negativos para predizer que doença não desenvolveria foi 88% para "L-PCR" e 74% para a "sPCR". A análise comparativa entre os primeiros resultados positivos para a presença de infecção ativa por HCMV e o aparecimento de sintomas mostrou que a "L-PCR" precedeu o inicio dos sintomas clínicos 19 dias (mediana) em 8 pacientes. Em 2 pacientes, a "sPCR" precedeu 7 dias (mediana) a "L-PCR" na detecção de infecção sintomática por HCMV. A "L-PCR" e a "sPCR" foram considerados métodos complementares para o diagnóstico e monitoramento da infecção sintomática por HCMV. / Abstract: The Human Cytomegalovirus (HCMV) is the main cause of post-transplant complications. Specific methods that allow to identify early patients with risk of developing disease for which treatment is indicated, have been required so that few patients are treated unnecessarily and for the effective therapeutic institution. The "Nested-PCR" has been commonly used in the diagnosis of HCMV infection, however, when performed in peripheral blood leukocytes, due to its high sensitivity does not present good correlation with the development of HCMV disease. Detection of HCMV DNA in serum by PCR has been associated with the development of HCMV disease. In this study, we apply the "Nested-PCR" in peripheral blood leukocytes (termed "L-PCR"), the conventional method of Laboratory, and in serum ("sPCR"), for the diagnosis of active HCMV infection, in order to establish the correlation of results obtained of both methods with the development of symptomatic infection. With this purpose, we evaluated prospectively samples of 37 patients, 20 undergoing kidney transplantation, and 17 submitted to haematopoietic stem cells transplantation. To exclude false negative results in the amplification reaction by "sPCR", an internal control was constructed and all reactions were carried out by using it. According to the criteria established in this study, 21 patients (21/37 - 57%) developed active HCMV infection. All patients with active HCMV infection were positive by LPCR (p= 0,0003). The serum PCR were active HCMV infection positive for only 10 patients (10/37 - 27%) (p= 0,01). Discordant results were observed in 11 patients who had active HCMV infection positive for "L-PCR" but negative for "sPCR", of which 5 developed clinical symptoms for HCMV. The observed Kappa coefficient of agreement for both assays was 0,44 (moderate agreement). Sixteen of 37 patients (43%) did not develop active HCMV infection by neither of the methods employed, however 2 of these developed clinical symptoms of probable HCMV disease and one presented later confirmative biopsy for HCMV disease. Clinical symptoms were observed in 14 patients, in 12 of whom active infection was diagnosed by "LPCR" (p= 0,007) and in 7 by "sPCR" (p= 0,02). Absence of clinical symptoms were observed in 9 patients in which the "L-PCR" detected active HCMV infection and in 3 patients in which the "sPCR" detect it (kappa coefficient 0,57, moderate agreement). Of the fourteen symptomatic patients, 2 HCMV-seronegative patients who received seropositive Kidneys developed primary infection. Analyzing the two methods for the diagnosis of active infection, we observed higher sensitivity and negative predictive value of the "L-PCR" ("L-PCR" 100% vs. "sPCR" 62%), and higher specificity and positive predictive value of "sPCR" ("sPCR" 81% vs. "L-PCR" 50% e 72%). The value of positive "L-PCR" or "sPCR" tests to predict HCMV disease were respectively 57% and 70% and, the value of the negative tests to predict that HCMV disease would not develop was 88% to "L-PCR" and 74% to "sPCR". The comparative analysis between the first positive results for the presence of active HCMV infection and the onset of symptoms, showed that the "L-PCR" test preceded the onset of clinical symptoms 19 days (median) in 8 patients. In two patients the "sPCR" preceded 7 days (median) the "L-PCR" in the detection of symptomatic HCMV infection. Therefore the "L-PCR" and the "sPCR" were considered complementary methods for the diagnosis and manegement of HCMV symptomatic infection. / Mestrado / Mestre em Farmacologia

Human cytomegalovirus (HCMV) is a ubiquitous virus of the herpes type that infects a high percentage of some populations. One of the most researched genes expressed by HCMV with close homology to human chemokine receptors is the US28 G protein-coupled receptor. Study design: This study was initiated to elucidate the intracellular signalling pathways of an inflammatory factor (IL-6) and an angiogenic factor (STAT3) triggered by the viral US28 oncogene and the presence of US28 in the HCMV viral particle. These pathways were observed by introducing the US28 gene into two human cell lines by infection with a HCMV strain that expresses the US28 gene (wild type), and two HCMV strains where the US28 gene was deleted (ÄUS28 and ÄUS28/UL33). Special attention was directed at the expression of IL-6 after promotion of the US28 gene and subsequent phosphorolation of STAT3. A new US28 antibody was validated and a method developed in an attempt to determine US28 on the viral particle. The following techniques were applied: Cell culture work, two mammalian cell lines were used, HFF’s and U373 MG. Virus stock titre determination to determine the multiplicity of infection. Protein quantitation to determine very small quantities of protein for Western blot analysis. ELISA for the quantitative determination of IL-6. Western blotting for phospho- STAT3 determination and validation of the US28 antibody. Immunocytochemistry was used for back titrations of virally infected cells. Immunofluorescence assay and use of confocal microscopic techniques was used for the location of the US28 gene in the virion and for tSTAT3 translocation to the nucleus. Conclusion: A clear increase in IL-6 secretion (495% ± 1%) was seen, and this was after only an hour in HCMV WT infected cells. From the increase in IL-6 secretion a subsequent increase in STAT3 phosphorylation was detected in the same samples. A clear link has been established between IL-6 and STAT3. A method to determine whether US28 was present in the HCMV viral particle was designed and preliminary results obtained. The results were inclusive. / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

Der DNA-Verpackungsmechanismus des humanen Cytomegalievirus (HCMV) ist charakteristisch für große DNA-Viren wie Herpesviren und ds-Bakteriophagen. Er beruht auf der Spaltung der konkatemeren DNA durch einen viralen, hetero-oligomeren Proteinkomplex, der Terminase. In der vorliegenden Arbeit konnten die funktionellen Domänen der Terminase-Untereinheit pUL89 in vitro identifiziert und charakterisiert werden. Neben einer Nuklease-Aktivität besitzt pUL89 auch die Fähigkeit dsDNA sequenz-unabhängig zu binden. Durch Nuklease-Untersuchungen konnte gezeigt werden, dass pUL89 sowohl dsDNA, als auch lineare DNA spaltet. PUL89 weist dabei eine größere Spezifität zu dsDNA auf. Des Weiteren konnte nachgewiesen werden, dass die Aminosäure D463 eine zentrale Funktion innerhalb der Nuklease-Aktivität besitzt. Durch kolorimetrische DNA-Bindungsuntersuchungen konnte die Aminosäure R544 als essenziell für die dsDNA-Bindungsfähigkeit von pUL89 identifiziert werden. Basierend auf den in vitro Ergebnissen wurden rekombinanten TB40/E-Virusmutanten mit Mutationen im ORF UL89 durch die En Passant Mutagenese generiert. Mit Hilfe dieser Viren sollte der Einfluss der Mutationen auf die Replikation des Virus charakterisiert werden. Es war möglich nachzuweisen, dass die Aminosäuren E534 und R544 eine essenzielle Aufgabe innerhalb von HCMV erfüllen, da die Mutation einer dieser Aminosäure zu nicht wachstums-fähigen BAC-Mutanten führte. Zur Charakterisierung dieser Konstrukte wurden die Zelllinien HELF Fi301-UL89 und HELF Fi301-vProm-UL89 verwendet. Durch Untersuchungen hinsichtlich der Wachstumseigenschaften, Proteinexpression, DNA-Spaltung, DNA-Bindung sowie elektronenmikroskopischen Aufnahmen, konnte gezeigt werden, dass die wachstums-kompetenten BAC-Mutanten keinen signifikanten Unterschied zum Wildtyp-Virus TB40/E zeigten. Sodass nachgewiesen werden konnte, dass die basischen Aminosäuren H565 und H571 keine essenzielle Funktion in pUL89 erfüllen. / The human cytomegalovirus DNA packaging mechanism is characteristic for large DNA viruses like Herpes viruses and ds bacteriophages. This mechanism is based on the cleavage of concatemeric DNA by the viral heterooligomeric protein complex terminase. This dissertation includes the identification and characterization of functional domains of the HCMV terminase subunit pUL89. PUL89 contain a nuclease activity and the ability to bind dsDNA. This protein shows the property to cut as well dsDNA as linear DNA. The amino acid D463 shows a significant role in this cleavage event. Colorimetric DNA binding experiments show the central role of R544 in DNA binding by pUL89. Based of the in vitro results recombinant TB40/E viruses with mutations in the ORF UL89 were generated. These viruses allow a characterization of the impact of virus replication. It was possible to show that the amino acids E534 and R544 have a functional role in HCMV. The mutation of one of these amino acids was enough to generate a growth deficient mutant. The stable cell lines HELF Fi301-UL89 and HELF Fi301-vProm UL89 were used for the characterization of the growth deficient mutants. The growth competent mutants H656A and H571A show no significant differences in comparison with the wild type TB40/E virus. This was verified by growth kinetics, protein expression characterizations, pulse field gel electrophoresis, DNA binding assays and electron microscopy.

Computationally driven drug design/discovery efforts generally rely on accurate assessment of the forces that guide the molecular recognition process. HINT (Hydropathic INTeraction) is a natural force field, derived from experimentally determined partition coefficients that quantifies all non-bonded interactions in the biological environment, including hydrogen bonding, electrostatic and hydrophobic interactions, and the energy of desolvation. The overall goal of this work is to apply the HINT-based atomic level description of molecular systems to biologically important proteins, to better understand their biochemistry – a key step in exploiting them for therapeutic purposes. This dissertation discusses the results of three diverse projects: i) structural modeling of human sphingosine kinase 2 (SphK2, a novel anticancer target) and binding mode determination of an isoform selective thiazolidine-2,4-dione (TZD) analog; ii) structural modeling of human cytomegalorvirus (HCMV) alkaline nuclease (AN) UL98 (a novel antiviral target) and subsequent virtual screening of its active site; and iii) explicit treatment of interfacial waters during protein-protein docking process using HINT-based computational tools. SphK2 is a key regulator of the sphingosine-rheostat, and its upregulation /overexpression has been associated with cancer development. We report structural modeling studies of a novel TZD-analog that selectively inhibits SphK2, in a HINT analysis that identifies the key structural features of ligand and protein binding site responsible for isoform selectivity. The second aim was to build a three-dimensional structure of a novel HCMV target – AN UL98, to identify its catalytically important residues. HINT analysis of the interaction of 5’ DNA end at its active site is reported. A parallel aim to perform in silico screening with a site-based pharmacophore model, identified several novel hits with potentially desirable chemical features for interaction with UL98 AN. The majority of current protein-protein docking algorithms fail to account for water molecules involved in bridging interactions between partners, mediating and stabilizing their association. HINT is capable of reproducing the physical and chemical properties of such waters, while accounting for their energetic stabilizing contributions. We have designed a solvated protein-protein docking protocol that explicitly models the Relevant bridging waters, and demonstrate that more accurate results are obtained when water is not ignored.

This work is part of a project focused on the study of the variability of human cytomegalovirus (HCMV) among clinical isolates with the aim to map the geographical distribution of HCMV genotypes, reveal the relationships between genotypes and the severity of HCMV-associated diseases, and identify regions in the HCMV genome with a potential for use as diagnostic and therapeutic targets. Attention was paid to the development of the methodology for the preparation of the material for next-generation sequencing (NGS) from HCMV clinical isolates and evaluation of the obtained sequencing data. Blood and urine samples collected from hematopoietic stem cell transplantat recipients and congenitally infected children were analyzed. Samples suitable for NGS were sequenced by the Illumina platform and sequences were created by de novo assembly followed by mapping assembly. Urine samples in comparison to blood samples had higher yield of material for NGS. Of the samples positive for HCMV DNA (7 of 50) after amplification in the cell cultures, only one sample had high purity of the viral DNA (98%) while six samples had purity of less than 7%. The sample containing 98% of the viral DNA was fully sequenced and the sequence was compared to the sequences of other clinical isolates from Belgium in 11 polymorphic...

Das Humane Cytomegalievirus (HCMV) ist ein ubiquitäres Pathogen, welches den Metabolismus der Wirtszelle auf vielfältige Weise manipuliert, um seine eigene Vermehrung zu begünstigen. In der vorliegenden Arbeit konnte nachgewiesen werden, dass auch das Ubiquitin-Proteasom-System in die HCMV-Replikation involviert ist. So konnte zunächst gezeigt werden, dass die Chymotrypsin-ähnliche (CT-L) Aktivität des konstitutiven Proteasoms in HCMV-infizierten Zellen signifikant erhöht ist. Wurde die CT-L Proteasomaktivität durch Proteasominhibitoren (PI) blockiert, so hatte dies die Hemmung der HCMV-Replikation zur Folge. Die Charakterisierung des Einflusses von PI auf die virale Proteinexpression ergab, dass bei niedriger MOI (MOI 0.1) deutlich verringerte Mengen der sehr frühen Proteine vorlagen, dieser Effekt jedoch bei hoher MOI (ab MOI 1) aufgehoben war. Die Expression früher Proteine war MOI-unabhängig reduziert. Hingegen war die Expression der späten Proteine MOI-unabhängig vollständig unterdrückt. Studien mit dem Nukleosidanalogon BrdU ergaben zudem, dass die de novo Synthese viraler DNA blockiert war. Um erste Hinweise auf den Wirkungsmechanismus von PI zu erhalten, wurde untersucht, ob der Transkriptionsfaktor NF-kappaB oder zelluläre Transkriptionsrepressoren wie z.B. hDaxx am anti-HCMV-Effekt beteiligt sind. Durch die Charakterisierung einer Virusmutante mit Deletion der NF-kappaB-Bindestellen im MIE-Enhancer/Promotor konnte gezeigt werden, dass der antivirale Effekt von PI nicht auf der Hemmung der Aktivierung von NF-kappaB beruht. Experimente mit hDaxx-knockdown Zellen ergaben hingegen, dass die Stabilisierung des Transkriptionsrepressors hDaxx partiell zum anti-HCMV-Effekt von PI beiträgt. Darüber hinaus müssen jedoch weitere virale oder zelluläre Zielproteine existieren, deren Beeinflussung durch PI kritisch für die Virusreplikation ist. Zusammenfassend stellt das Proteasom somit einen neu identifizierten potentiellen Angriffspunkt für die anti-HCMV-Therapie dar. / The Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that manipulates many aspects of the host cell metabolism to enhance viral replication. This work demonstrates that the ubiquitin-proteasome system is also involved in HCMV replication. First of all, the chymotrypsin-like (CT-L) activity of the constitutive proteasome was significantly increased in HCMV infected cells. In the presence of proteasome inhibitors (PI) viral replication was efficiently blocked. Characterisation of the influence of PI on viral protein expression showed that immediate early protein expression was clearly reduced at low MOI (MOI 0.1); however, this effect was abolished at high MOI (starting from MOI 1). Expression of early proteins was significantly decreased independently of the MOI used for infection. In contrast, late protein expression was completely suppressed at both low and high MOI. Additionally, studies using the nucleoside analogue BrdU showed that PI block the de novo synthesis of viral DNA. In order to gain insight into the working mechanism of PI the involvement of the transcription factor NF-kappaB and cellular repressors of transcription (e.g. hDaxx) in the antiviral effect of PI was examined. Studies using a mutant virus carrying deletions of the NF-kappaB binding sites in the MIE-enhancer/promoter revealed that the anti-HCMV effect of PI is not due to inhibition of NF-kappaB activation. Analyses using hDaxx-knockdown cells showed that stabilisation of the transcriptional repressor hDaxx partially contributes to the antiviral effect of PI. However, the existence of additional viral or cellular target proteins of PI is very likely. In summary, the proteasome thus represents a newly identified and promising target for anti-HCMV therapy.