rologie i - European Congress of Virology

5 th European Congress of Virologyprimary fibroblasts infected with HCMV AD169 or Merlin, as well asproteins from transiently transfected cells. Confocal microscopy showedthat selective inhibitors of CDK or pUL97 induced an accumulation ofpUL69 in fine speckled nuclear aggregates, which contained colocalizedCDK9, cyclin T1, RNAP II and to some extent pUL97. Speckled aggregationoccurred at the late phase of viral replication and was independentof viral strain (AD169, Merlin) and host cell type (HFF, MRC 5, TEV1, ARPE 19). Interestingly, speckled aggregation was also observed tooccur spontaneously in ∼1% of cells in the absence of kinase inhibitors.These findings point to a dependence of correct intranuclear localization ofpUL69 on distinct kinase activities. A model describing the phospho regulatedstate of pUL69 responsible for localization and transport function ispresented.REF 210The plant virus NSs protein of the Tomato spotted wilt virus enhancesbaculovirus replication and cell death in Lepidoptera and Diptera celllinesVirgínia Carla OLIVEIRA, Fabricio Da Silva MORGADO, DanielMENDES PEREIRA ARDISSON ARAÚJO, Daniele VITORIANOFREITAS, Bergmann MORAIS RIBEIRO, Renato OLIVEIRARESENDEDepartment of Cell Biology, Brasília, BRAZILWe have shown in a previous work that NSs protein of Tomato spottedwilt virus expressed by a recombinant Autographa californica multiplenucleopolyhedrovirus baculovirus (vAcNSs) can suppress gene silencingin lepidopteran insect cell lines. In this work, replication of the recombinantvirus in this cell line (velvetbean caterpillar derived, UFL AG 286)was shown to be higher than wild type virus (AcMNPV). Interestingly,although with lower absolute values of viral DNA, a difference of 6.75 x(12 h p.i.) and 2.2 x (24 h p.i.) was observed for AcMNPV and vAcNSsinfected nonpermissive C6/36 cells. A cytotoxicity assay resulted in adecrease of 2.65 (UFL AG 286) and 1.77 times (BM 5) in cell viability; ina dipteran cell line (C6/36) a reduction of 1.73 times in cell viability wasobserved; BTI Tn 5B1 4 vAcNSs infected cells showed 2.12 times lessviable cells in relation to AcMNPV infected cells. Furthermore, bioassayswere performed by intrahaemocoelic injection of the baculovirus buddedvirus form and oral infection with purified occluded viruses of the positiveocclusion recombinant virus vAcNSsocc+ and the wild type AcMNPV.For intrahaemocoelic infection, the vAcNSs LT50 values were significantlylower than those for AcMNPV on larvae of S. frugiperda [LT50 of4.82 and 7.52 days with 105 BVs] and A. gemmatalis [LT50 of 3.20 and7.34 days with 105 BVs). In conclusion, NSs is capable of boosting viralreplication in insect cells by probably suppressing gene silence machineryand could efficiently improve baculovirus replication and bioinsecticideaction.of the resulting cell cycle independent IE gene expression for the progressionof the HCMV lytic cycle. Infection of early S phase cells with thecell cycle independent HCMV mutant leads to a stable, p53 independentG2 arrest. Only a small minority of IE positive cells were able to entermitosis. These mitotic cells showed signs of an abortive infection whereasthe G2 arrested cells readily support both early and late viral geneexpression, and accordingly, also viral DNA replication. We hypothesizedthat the recently discovered HCMV pUL21a mediated degradation ofcyclin A2 is required to maintain the virus permissive G2 arrested state. Totest this hypothesis we constructed an HCMV double point mutant whereboth pp150 and pUL21a were disabled in cyclin A2 binding. Intriguingly,infection with this double mutant forced mitotic entry of up to 60% of IEpositive cells, leading to a significant retardation of virus growth. Takentogether, we uncovered the existence of two independent viral mechanismsthat cooperatively assist HCMV to avoid the negative cell cycle effects ofuncontrolled cyclin A2 CDK activity.REF 212PUL21a dependent degradation of Cyclin A2 is essential for the inhibitionof cellular DNA synthesis and mitotic entry during lytic HCMVinfectionLüder WIEBUSCH, Boris BOGDANOW, Martin EIFLER, EllenRICHTER, Ralf UECKER, Barbara VETTER, Christian HAGEMEIERCharité Universitätsmedizin, Berlin, GERMANYLytic human cytomegalovirus (HCMV) infection leads to a pronouncedcell cycle arrest at the G1/S transition. This arrest is characterized by highcyclin E1 but low cyclin A2 levels. Here we show that cyclin A2 downregulation is mediated by the UL21a gene product (pUL21a) of HCMVand represents the crucial step of the viral arrest mechanism. We identifieda short peptide motif within the pUL21a N terminus that is required forspecific pUL21A cyclin A2 binding. Transfection of pUL21a was foundto induce proteasomal degradation of cylin A2 in a cyclin A2 binding(Cy) motif dependent manner. Point mutation of the pUL21a Cy motifwithin the HCMV genome also relieves the block of cyclin A2 expressionduring HCMV infection. The resulting increase of cyclin A2 protein levelsproved to be sufficient to abrogate the viral inhibition of cellular DNA synthesis.Moreover, it triggers the accumulation and nuclear translocation ofcyclin B1 CDK1 complexes and accordingly, the mitotic entry of infectedcells. Deletion of the whole UL21a open reading frame, has only moderateeffects on cyclin A2 expression and cell cycle progression, suggestingthat the pUL21a Cy motif and the previously described pUL21a mediatedinhibition of the APC/C act antagonistically with respect to cyclin A2 proteinstability. Importantly, the cyclin A2 induced mitotic entry of infectedcells results in mitotic catastrophe and, consequently, an abortive infection.Thus, the pUL21a cyclin A2 interaction is essential for the maintenance ofa cell cycle state conducive for the completion of the HCMV replicationcycle.REF 211The cyclin A2 interaction motifs of pp150 and pUL21a cooperateto prevent HCMV from entering a non productive, mitotic state ofinfectionHenry WEISBACH, Christoph SCHABLOWSKY, ChristianHAGEMEIER, Lüder WIEBUSCHLabor für Pädiatrische Molekularbiologie, Charité UniversitätsmedizinBerlin, Berlin, GERMANYThe lytic replication cycle of human cytomegalovirus (HCMV) can onlybe started in the G0/G1 phase of the cell division cycle, due to a cyclinA2 CDK dependent block of immediate early (IE) gene expression inS/G2. This block can be prevented by mutating a cyclin A2 binding sitein the viral tegument protein pp150. Here we analyzed the consequencesREF 213Identification of protein synthesis as a regulatory node in EpizooticHemorrhagic Disease Virus (EHDV) infection and development of anovel imaging technique for its study in single cellsMarcelo EHRLICH 1 , Sima BARHOOM 1 , Ben SHAI 1 , EranSCHMUKLER 1 , Barry COOPERMAN 3 , Zeev SMILANSKY 2 , EranBACHARACH 1 , Ronit PINKAS KRAMARSKI 1 , Orna ELROY STEIN 11 Tel Aviv University, Tel Aviv, ISRAEL; 2 Anima Cell Metrology, Bernardsville,USA; 3 University of Pennsylvania, Philadelphia, USAManipulation of the cellular protein synthesis apparatus is a central regulatorymechanism of the replication cycle of different viruses. Suchmanipulation by orbiviruses, such as the Epizootic Hemorrhagic DiseaseS178 Virologie, Vol 17, supplément 2, septembre 2013