Funktionelle Analyse des KSHV-kodierten vIRF-3: Interferonantagonismus und Onkogenese

Kaposi sarcoma-associated herpesvirus (KSHV) is a human tumor virus and as such associated with at least three proliferative disorders. Originally, it was identified from a Kaposi Sarcoma (KS) biopsy of an HIV infected person. In addition to all forms of KS, it is associated with Primary Effusion Lymphoma (PEL) and Multicentric Castleman Disease (MCD). KSHV codes for more than 80 open reading frames, a number of which display homology to cellular proteins. These include the KSHV-encoded viral Interferon regulatory factors. Of these four vIRFs, so far only KSHV vIRF-3 has been found to be regularly expressed with a latent kinetic in virtually all KSHV-infected PEL cells. In this PhD thesis, cellular IRF-5 was identified and confirmed as a new interaction partner of vIRF-3. IRF-5 is not only part of the innate immune response but also known as a potential tumor suppressor. As such it is involved in cell cycle arrest and apoptosis control. Functional analysis of the IRF 5/vIRF-3 interaction revealed that vIRF-3 inhibits IRF-5-dependent promoter activation. IRF-mediated activation of both a minimal promoter containing Interferon-Stimulated-Response-Elements (ISRE) as well as the Interferon beta promoter and the promoter of cell cycle regulator p21 was inhibited by vIRF-3. As mechanism of action, vIRF-3 reduces binding of IRF-5 to its promoters. In this work the viral IRF-3 was found to be required for proliferation and survival of cultured PEL cells. Knockdown of vIRF-3 by various siRNA approaches unequivocally resulted in a reduced proliferation rate and increased activity of caspase 3 and/or caspase 7. Thus, vIRF-3 can be seen as bona-fide oncogene in these lymphoma cells. Very moderate knockdown of vIRF-3 rendered PEL cells sensitive to interferon, pointing at vIRF-3 as the main viral defense against interferon in latently infected PEL cells. Furthermore, transcription of IRF-5 target genes was strongly enhanced upon knockdown of vIRF-3. As IRF-5 targets are typically involved in anti-proliferative, anti-oncogenic pathways, inhibition of IRF-5 activity by vIRF-3 is likely to contribute to cell proliferation and transformation.Das Kaposi Sarkom-assoziierte Herpesvirus (KSHV) ist ein menschliches Tumorvirus und als solches mit mindestens drei proliferativen Erkrankungen assoziiert. Ursprünglich entdeckt im Gewebe eines Kaposi Sarkoms, gilt es auch als auslösendes Agens des Primären Effusionslymphoms (PEL) und der Multizentrischen Castleman Erkrankung (MCD). Dieses DNA-Virus kodiert für über 80 Proteine, von welchen viele eine ausgeprägte Homologie zu zellulären Proteinen aufweisen, so auch die sogenannten viralen Interferon regulatorischen Faktoren (vIRFs). Von den vier von KSHV kodierten vIRFs konnte bisher nur vIRF-3 regelmäßig in latent infizierten PEL-Zellen nachgewiesen werden. In dieser Arbeit wurde der zelluläre Interferon regulatorische Faktor 5 (IRF-5) als neuer Interaktionspartner von vIRF-3 identifiziert. Diese Interaktion wurde sowohl im Überexpressionssystem als auch in KSHV-infizierten PEL-Zellen auf funktionelle Konsequenzen untersucht. So inhibiert vIRF 3 über diese direkte Interaktion IRF-5-vermittelte Transkription sowohl von einem Minimalpromotor mit Interferon-Stimulated-Response-Elements (ISRE) als auch von den Promotoren der Gene für IFNbeta und des Zellzyklusregulators p21. Durch die IRF-5-vIRF-3-Interaktion ist IRF-5 nicht mehr in der Lage an die Promotoren seiner Zielgene zu binden. Als Konsequenz hieraus werden nach siRNA-vermitteltem temporärem Knockdown von vIRF-3 in PEL-Zellen typische pro-apoptotische und anti-proliferative IRF-5 Zielgene induziert. Eine Reduktion der vIRF-3-Expression in PEL-Zellen führt zudem zu einer stark verminderten Proliferationsrate bei gleichzeitig erhöhter Induktion des programmierten Zelltodes. Dass das kontinuierliche Wachstum dieser Lymphomzellen maßgeblich von der dauerhaften Expression des vIRF-3 abhängig ist, weist auf die essentielle Rolle dieses vIRFs in der Onkogenese des Lymphoms hin. Die in dieser Arbeit präsentierten Daten legen nahe, dass die Blockade der transkriptionellen Aktivität des IRF-5-Proteines eine zentrale Funktion von vIRF-3 ist, über die es zur kontinuierlichen Proliferation der PEL-Zellen beiträgt

Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors▿

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures

Intracellular Localization Map of Human Herpesvirus 8 Proteins▿

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases

A Systems Biology Approach To Identify the Combination Effects of Human Herpesvirus 8 Genes on NF-κB Activation▿

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma. Activation of the cellular transcription factor nuclear factor-kappa B (NF-κB) is essential for latent persistence of HHV-8, survival of HHV-8-infected cells, and disease progression. We used reverse-transfected cell microarrays (RTCM) as an unbiased systems biology approach to systematically analyze the effects of HHV-8 genes on the NF-κB signaling pathway. All HHV-8 genes individually (n = 86) and, additionally, all K and latent genes in pairwise combinations (n = 231) were investigated. Statistical analyses of more than 14,000 transfections identified ORF75 as a novel and confirmed K13 as a known HHV-8 activator of NF-κB. K13 and ORF75 showed cooperative NF-κB activation. Small interfering RNA-mediated knockdown of ORF75 expression demonstrated that this gene contributes significantly to NF-κB activation in HHV-8-infected cells. Furthermore, our approach confirmed K10.5 as an NF-κB inhibitor and newly identified K1 as an inhibitor of both K13- and ORF75-mediated NF-κB activation. All results obtained with RTCM were confirmed with classical transfection experiments. Our work describes the first successful application of RTCM for the systematic analysis of pathofunctions of genes of an infectious agent. With this approach, ORF75 and K1 were identified as novel HHV-8 regulatory molecules on the NF-κB signal transduction pathway. The genes identified may be involved in fine-tuning of the balance between latency and lytic replication, since this depends critically on the state of NF-κB activity

The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5*S⃞

Kaposi's sarcoma-associated herpesvirus encodes four genes with homology to the family of interferon regulatory factors (IRFs). At least one of these viral IRFs, vIRF-3, is expressed in latently Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma (PEL) cells and is essential for the survival of PEL cells. We now report that vIRF-3 interacts with cellular IRF-5, thereby inhibiting binding of IRF-5 to interferon-responsive promoter elements. Consequently, vIRF-3 blocked IRF-5-mediated promoter activation. A central double helix motif present in vIRF-3 was sufficient to abrogate both DNA binding and transcriptional transactivation by IRF-5. Upon DNA damage or activation of the interferon or Toll-like receptor pathways, cytoplasmic IRF-5 has been reported to be translocated to the nucleus, which results in induction of both p53-independent apoptosis and p21-mediated cell cycle arrest. We report here that IRF-5 is present in the nuclei of PEL cells without interferon stimulation. Silencing of vIRF-3 expression in PEL cells was accompanied by increased sensitivity to interferon-mediated apoptosis and up-regulation of IRF-5 target genes. In addition, vIRF-3 antagonized IRF-5-mediated activation of the p21 promoter. The data presented here indicate that vIRF-3 contributes to immune evasion and sustained proliferation of PEL cells by releasing IRF-5 from transcription complexes