Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis

Regulation zellulärer Signalwege durch die Onkoproteine T-lymphotroper Herpesviren

The T-lymphotropic tumorviruses, Herpesvirus saimiri (HVS) and the closely related Herpesvirus ateles (HVA), persistently infect their natural hosts, the squirrel and the spider monkeys, respectively, without causing any apparent disease. In other New World primate species like marmosets they induce T-cell lymphomas. Induction of proliferation of marmoset T cells in vitro is also known. Distinct pathogenic properties and a divergent genomic sequence region coding for the viral oncoproteins classifies HVS isolates into three subgroups: HVS-A, -B, -C. Only HVS-C is able to transform human T cells to permanent proliferation in vitro. In general, growth transformation by HVS and HVA depends on the expression of virus-specific oncoproteins StpA, -B, -C, Tip und Tio. The first part of this thesis presents a comprehensive in vitro study revealing that all HVS subgroups and HVA transform T cells of two different marmoset species, whereas human T-cell transformation is restricted to HVS-C as well as HVA. In transiently transfected human Jurkat T cells, comparative analyses demonstrate that NF-κB is activated by oncoproteins of all virus strains. Furthermore, NF-κB activity is essential for survival of HVS and HVA transformed cell lines. Likewise, Src-family kinase (SFK) activity is also required for survival. However, SFK-related signaling in human Jurkat T cells is only induced by oncoproteins of HVS-C and HVA, Tip and Tio. They are thereby distinguished from the other viral oncoproteins. These results suggest that the unique potential of HVS-C and HVA to transform human T cells correlates with the particular SFK targeting by their oncoproteins. The second part of this thesis focusses on serum response factor (SRF) activation in Tip expressing human T cells. Induction of an SRF luciferase reporter depends on the cofactor MAL, which is known to be regulated by cytoskeletal reorganization. Accordingly, Tip-mediated SRF induction requires RhoGTPase activity and actin polymerization. This pathway is not only triggered by the viral oncoprotein Tip, but also by T-cell stimulation and constitutively active RhoGTPases. Thus, Tip induces MAL:SRF activity and thereby may link cytoskeletal modifications with target gene activation responsible for viral oncogenesis.Die T-lymphotropen Tumorviren Herpesvirus saimiri (HVS) und das nahe verwandte Herpesvirus ateles (HVA) infizieren persistent ihre natürlichen Wirtspezies, die Totenkopfäffchen bzw. Spinnenaffen, ohne ersichtliche Erkrankungen zu verursachen. In anderen Neuweltprimaten wie Krallenaffen rufen sie T-Zell-Lymphome hervor. Die Induktion anhaltender, Proliferation von T-Zellen aus Krallenaffen in vitro ist ebenfalls bekannt. Aufgrund verschiedener pathogener Eigenschaften und divergierender genomischer Sequenzen, welche die transformations-relevanten viralen Onkoproteine kodieren, werden HVS-Isolate in drei Untergruppen eingeteilt: HVS-A, -B, -C. Nur HVS-C ist in der Lage humane T-Zellen zu permanentem Wachstum in vitro zu transformieren. Generell ist die Wachstumstransformation durch HVS und HVA abhängig von der Expression der Virus-spezifischen Onkoproteine StpA, -B, -C, Tip und Tio. Im ersten Teil dieser Arbeit zeigt eine umfassende in vitro Untersuchung, dass T-Zellen von zwei verschieden Krallenaffen-Arten durch alle HVS-Untergruppen und HVA transformiert werden, wohingegen humane T-Zell-Transformation auf HVS-C und HVA beschränkt ist. In transient transfizierten humanen Jurkat T-Zellen zeigen vergleichende Analysen der Onkoproteine, dass NF-κB von Onkoproteinen aller Virusstämme aktiviert wird. Weiterhin ist NF-κB-Aktivität essentiell für das Überleben von HVS- und HVA-transformierten Zell-Linien. Src-Familie-Kinase (SFK)-Aktivität wird gleichermaßen für deren Fortbestand benötigt. SFK-assoziierte Signalweiterleitung wird jedoch nur beeinflußt durch die Onkoproteine von HVS-C und HVA, Tip und Tio. Diese unterscheiden sich dadurch von den anderen Onkoproteinen. Diese Daten sprechen für eine Korrelation zwischen der besonderen Fähigkeit von HVS-C und HVA, humane T-Zellen zu transformieren, und der speziellen Ansteuerung von SFK durch ihre Onkoproteine. Der zweite Teil dieser Arbeit befaßt sich mit der Aktivierung des Serum Response Faktors (SRF) in Tip-exprimierenden T-Zellen. Die Induktion eines SRF-Luziferase-Reportergens ist abhängig von dem Kofaktor MAL, welcher durch Umstrukturierung des Zytoskeletts reguliert wird. Entsprechend benötigt die Tip-vermittelte SRF-Induktion RhoGTPase-Aktivität und Aktin Polymerisierung. Dieser Weg wird nicht nur durch das virale Onkoprotein Tip aktiviert, sondern auch durch T-Zell-Stimulation und konstitutiv aktive RhoGTPasen. Somit induziert Tip die Aktivität von MAL:SRF und könnte dadurch Zytoskelett-Veränderungen mit der Aktivierung von Ziel-Genen verbinden, welche verantwortlich sind für die virale Onkogenese

QuaNCAT:Quantitating proteome dynamics in primary cells

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli. © 2013 Nature America, Inc. All rights reserved