rologie i - European Congress of Virology

5 th European Congress of Virologythe functional impact of mutations introduced in N2 expressed alone or incombination with wild type N. Obtained results provided new informationon the cartwheeling model.REF 206Innate immunity activation is required to mediate an effective HumanCytomegalovirus Lytic infection in THP 1 macrophagesDiego GERMINI 1 , Maria Cristina ARCANGELETTI 1 , IsabellaRODIGHIERO 1 , Prisco MIRANDOLA 2 , Flora DE CONTO 1 , MariaCristina MEDICI 1 , Rita GATTI 2 , Carlo CHEZZI 1 , AdrianaCALDERARO 11 Department of Clinical and Experimental Medicine University of Parma,Parma, ITALY; 2 Department of Biomedical, Biotechnological and TranslationalSciences University of Parma, Parma, ITALYToll like receptors (TLR), the main class of immune sensor moleculestriggering the innate immunity pathways, are known to be involved inthe infection of different RNA and DNA viruses, including herpesviruses.Human cytomegalovirus (HCMV) has evolved several strategies to exploitcellular functions connected with the innate immunity activation. Specificviral gene products have an immunomodulatory effect, allowing the virusto evade host defenses and even improve the infection effectiveness. Theaim of the present study was to ascertain whether specific TLRs are exploitedby HCMV to sustain an efficient lytic cycle in THP 1 differentiatedcells, a macrophage like model mimicking one of the most important viraltargets in vivo. We show that TLR 3, 4 and 5 transcripts are stimulatedby HCMV infection of THP 1 macrophages but that only TLR4 expressionincreases in UV inactivated virus infected cells. Using two differentTLR4 antagonists, we analysed the pattern of HCMV immediate earlyviral transcripts and proteins, as well as the viral yield, demonstratingthat the inhibition of the TLR4 pathway induces a significant decreaseof HCMV infection. Furthermore, by confocal microscopy analysis wealso produced data supporting not only HCMV TLR4 interaction at theplasma membrane level but also in the cytoplasmic compartment. Thesedata support a key role for TLR4 in favouring an efficient HCMV productiveinfection in THP 1 macrophages; its involvement seems not onlyconfined to the very early steps but also to later infection stages.REF 207Direct interaction between cyclin T1 and the cyclin dependent kinaseortholog pUL97 of human cytomegalovirusLaura GRAF 1 , Rike WEBEL 1 , Stuart HAMILTON 2 , William D.RAWLINSON 2 , Heinrich STICHT 3 , Manfred MARSCHALL 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Virology Division, MicrobiologySEALS POWH, University of New South Wales, Sydney, AUSTRALIA;3 Division of Bioinformatics, Institute of Biochemistry, University of ErlangenNuremberg, Erlangen, GERMANYThe human cytomegalovirus encoded protein kinase pUL97 regulatesvirus replication at various levels, such as viral DNA replication, geneexpression and nuclear capsid egress, by phosphorylating cellular andviral proteins. Due to structural and functional similarities, pUL97 canbe considered as a cyclin dependent kinase (CDK) ortholog. The primarymechanism of CDK activation in normal human cells is the binding to correspondingcofactors termed cyclins. This study provides first evidence thatpUL97 interacts with cyclin T1 (cycT1), which is the regulatory subunitof CDK9. Indirect immunofluorescence analyses revealed partial colocalizationof pUL97 with cycT1 in subnuclear compartments which wasmost pronounced in viral replication centers. The distribution patterns ofpUL97 and cycT1 were independent of HCMV strain and host cell type.The interaction between pUL97 and cycT1 was demonstrated using yeasttwo hybrid and coimmunoprecipitation analyses. The sequence domain ofpUL97 responsible for the interaction with cycT1 was the region betweenamino acids 231 280, a region which does not contain a known cyclinrecognition motif. The role of the pUL97 cycT1 interaction in phosphorylationdependent regulatory processes and kinase activity of pUL97 iscurrently investigated. This is the first demonstration of interaction betweena herpesviral CDK like protein kinase and a cellular cyclin. Furtherinvestigation of this interaction will contribute to better understanding ofpUL97 function, especially with regard to the role of pUL97 as a CDKortholog.REF 208Upregulation of the transcription elongation factor ELL2 by Tax as anovel mechanism of HTLV 1 gene regulationAndrea K. KRESS, Melanie C. MANN, Sarah STROBEL, BernhardFLECKENSTEINInstitute of Clinical and Molecular Virology, Friedrich Alexander UniversitätErlangen Nürnberg, Erlangen, GERMANYThe viral oncoprotein Tax encoded by Human T cell lymphotropic virustype 1 is a potent modulator of host cell transcription. To test whetherTax could also affect transcriptional elongation, microarray analysis wasperformed. Among all known cellular elongation factors, the eleven nineteenlysine rich elongation factor 2 (ELL2) was the only one selectivelyupregulated in the presence of HTLV 1/Tax. ELL2 is known as the stoichiometricallylimiting factor of the super elongation complex. Furtheranalysis of various HTLV 1 transformed and patient derived cell lines byqPCR and immunoblot revealed ELL2 to be significantly upregulated inthe presence of Tax. Repression of Tax in Tax transformed Tesi cells leadsto reduced amounts of ELL2 mRNA and protein. Moreover, siRNA mediatedknockdown of Tax in MT 2 diminishes ELL2 expression. Transfectionof increasing amounts of Tax in 293T cells leads to an increase of ELL2transcripts, suggesting that ELL2 expression is dependent on Tax. Furthermore,we found that coexpression of ELL2 significantly increases Taxmediated transactivation of the HTLV 1 promotor in a dose dependent manner.The enhanced transactivation is likely due to a promotor independentand Tax specific enhancement of Tax expression by ELL2. Interestingly,siRNA mediated knockdown of ELL2 in MT 2 leads to strong reduction ofTax protein, suggesting a positive feedback loop between ELL2 and Taxin HTLV 1 infected cells. Taken together, we identified ELL2 as a newfactor which may play an important role in HTLV 1 transcription.REF 209Protein kinases determine intracellular localization and transportactivity of the human cytomegalovirus regulatory protein pUL69Manfred MARSCHALL 1 , Laura GRAF 1 , Zin NAING 2 , SabrinaWAGNER 1 , Rike WEBEL 1 , Corina HUTTERER 1 , Gillian M. SCOTT 2 ,William D. RAWLINSON 2 , Thomas STAMMINGER 1 , MarcoTHOMAS 1 , Sabine FEICHTINGER 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Virology Division, MicrobiologySEALS POWH, University of New South Wales, Sydney, AUSTRALIACyclin dependent protein kinases (CDKs) are important regulators of cellularprocesses and are functionally integrated in the replication of humancytomegalovirus (HCMV). We recently demonstrated a regulatory impactof CDK9 on the viral mRNA export factor pUL69. In addition, the HCMVCDK ortholog pUL97 also showed strong pUL69 interaction resulting inpUL69 phosphorylation and regulation of pUL69 nuclear mRNA exportactivity. In vitro kinase analyses demonstrated pUL69 phosphorylation byboth CDK9/cyclin T1 and pUL97 on multiple sites, as single site mutationsdid not prevent phosphorylation. Direct pUL69 cyclin T1 interaction wasdemonstrated using coimmunoprecipitation analyses with proteins fromVirologie, Vol 17, supplément 2, septembre 2013S177