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Humane Zytomegalieviren (HCMV) etablieren nach einer Primärinfektion eine lebenslange latente oder persistierende Infektion. Es wird allgemein angenommen, daß hieran die Manipulation der humanen Immunantwort durch das Virus beteiligt ist. Hierzu zählen die Hemmung von zytotoxischen CD8+ T-Zellen durch das Genprodukt US11 und die Beeinträchtigung der Leukozytenwanderung durch die Hemmung des Chemokinsystems durch den Chemokinrezeptor US28. Die Effizienz der US11-vermittelten Hemmung der T-Zell-Aktivierung wurde in einem rekombinanten Modell zur MHC-Klasse-I-vermittelten T-Zell-Aktivierung untersucht. Obwohl die Expression der MHC-Klasse-I-Moleküle durch US11 in dendritische Zellen (DCs) um bis zu 60% vermindert war, konnte keine Hemmung der T-Zell-Proliferation beobachtet werden. US28 ist der einzige funktionelle Rezeptor für die inflammatorischen Chemokine MCP-1, MCP-3, RANTES, MIP-1(, MIP-1( sowie Fraktalkine. Er kann sowohl Liganden-abhängig die Aktivierung von MAPK als auch die konstitutive Aktivierung von NF-(B vermitteln. In der vorliegenden Arbeit konnte mit Hilfe einer Rezeptormutante der Argininrest an Position 129 des DRY-Motivs als Voraussetzung für die Aktivierung der Signalwegen identifiziert werden. Ferner bewirkt die Expression des US28-Rezeptors die Entfernung inflammatorischer Chemokine aus der Umgebung infizierter Zellen. Molekulare Grundlage der Liganden-Depletion stellt die Endozytose des US28-Liganden-Komplexes dar. Es konnte gezeigt werden, daß der US28-Rezeptor eine Umlagerung von (-Arrestin-Molekülen in Vesikel vermittelt, jedoch unabhängig von Arrestin-Molekülen endozytiert wird. Die Endozytose des US28-Rezeptors war abhängig von der GTP-ase Dynamin. Ebenso konnte die Beteiligung des Lipid-Raft-Weges an der US28-Endozytose gezeigt werden. Die Hemmung des Clathrinweges bewirkte jedoch eine zweifach stärkere Verminderung der US28-Endozytose, kann der Clathrin-abhängige Weg als der Hauptweg der US28-Endozytose angesehen werden. / Primary infections of the human cytomegalovirus (HCMV) are followed by a lifelong infection in the state of latency or persistence. It is believed that the virus employs a number of immunomodulatory mechanisms to establish latent infections. Among these are the inhibition of cytotoxic CD8+ T-cells by US11 and the impairment of leukocyte migration by US28. The potency of US11 to mediate the inhibition of T-cell activation was analysed in a model of MHC class I mediated T-cell activation. Surface expression of MHC class I molecules was reduced by 60 % after expression of US11 in murine dendritic cells. In contrast, there was no reduction in the capacity of the dendritic cells to induce T-cell proliferation. The US28 gene product has been characterized as a functional receptor for the inflammatory chemokines RANTES, MCP-1, MCP-3, MIP-1?? MIP-1? and fractalkine.Upon ligand stimulation US28 mediates the activation of MAPK and additionally a constitutive activation of NF-?B. By generating site directed receptor mutant it was shown that the arginine at position 129 represents a structural requirement for both the ligand-induced and the constitutive signaling by US28. Moreover, it was suggested that the US28 dependent sequestration of chemokines from the environment of infected cells hinders leukocytes from the recruitment to sites of viral infection. A molecular mechanism for the ligand depletion is provided by the endocytosis of US28-ligand complexes. Studies revealed that US28 expression induced a redistribution of ?-arrestin molecules into vesicular structures but was dispensable for the endocytosis of the US28 receptor. However, US28 internalization was dependent on the small GTPase dynamin and by impaired receptor endocytosis after inhibition of the lipid raft pathway. Since inhibition of the clathrin dependent pathway resulted in a two-fold stronger reduction of US28 endocytosis, the clathrin-dependent pathway can be considered as the major route of US28 endocytosis.

Human cytomegalovirus (HCMV) is a betaherpesvirus which establishes a lifelong persistent infection, underpinned by its ability to establish latent infection in early myeloid lineage cells, in the infected host. Although well controlled by a healthy immune system, HCMV causes pathological and life threatening disease in individuals with a compromised or immature immune response, which can come from primary HCMV infection or reactivation of latent infection. Although progress is being made in understanding the mechanisms by which HCMV maintains latency and reactivates, a better understanding is essential towards the aim of targeting and killing latently infected cells. In this thesis, I will present evidence that the HCMV-encoded chemokine receptor homologue US28, which is expressed during latent infection of CD14+ monocytes, is necessary for maintaining HCMV latency in these monocytes and, in the absence of US28 protein expression, HCMV undergoes lytic infection. US28 expression was found to attenuate cellular signalling pathways in latently infected cells; in particular, MAP kinase and NFκB. Interestingly, deletion of the US28 gene or inhibition of the US28 protein resulted in the expression of lytic antigens which allowed detection of infected monocytes by the immune system. This observation may lead to a potential new immunotherapeutic strategy against latent HCMV. Having demonstrated that US28 protein is expressed on the surface of latently infected monocytes, I tested whether a new fusion-toxin protein, called F49A-FTP, which binds US28 protein, could be used to target and kill latently infected cells. I developed a protocol for treating latently infected monocytes with F49A-FTP which resulted in a significant reduction in virus reactivation after monocyte differentiation to dendritic cells. I was also able to show that this treatment kills CD34+ progenitor cells, which were experimentally latently infected with HCMV, as well as latently infected monocytes from a healthy, seropositive blood donor. Finally, during my investigations into the role of US28 during HCMV latency, a mass spectrometry screen was performed to measure changes in cellular protein expression when US28 protein is expressed in isolation, in THP-1 monocyte-like cell line. This identified CTCF, a transcription factor which appears to be modified by US28 in THP-1 cells. I showed that CTCF has a repressive effect on the HCMV MIEP, and that CTCF likely plays a role in HCMV latency. In summary, this work provides insights into the role of US28 during HCMV latency, and proposes potential novel therapeutic strategies to kill latently infected cells.

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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