rologie i - European Congress of Virology

5 th European Congress of Virology13. VIRAL REPLICATION STRATEGIESPosters: REF 198 to REF 215characterize the CHIKV RdRp and the molecular details of CHIKV RNAsynthesis in vitro. The in vitro system will be applied in mechanism ofaction studies on inhibitors of CHIKV replication. Determination of theCHIKV RdRp crystal structure should ultimately allow structure baseddrug design.REF 198Herpes Simplex Virus type 1 tegument protein VP11/12 causes disruptionof the Golgi apparatus in neuronsYennyfer ARANCIBIA 1 , Carolina MARTIN 1 , Gonzalo MARDONES 2 ,Carola OTTH 11 Instituto de microbiologia clinica, facultad de medicina, universidadAustral de Chile, Valdivia, CHILE; 2 Instituto de fisiologia, facultad demedicina, universidad Austral de Chile, Valdivia, CHILETegument proteins are layered between the capsid and the envelope ofherpesviruses, and they are delivered into the cytoplasm during viral infectionto manipulate host cell functions. VP11/12 is one of the most abundanttegument proteins of Herpes Simplex Virus type 1 (HSV 1), but its functionduring infection is not well established. During T cell infection VP11/12activates the lymphocyte specific Src family kinase (SFK) Lck, and istyrosine phosphorylated in an Lck dependent manner; however the downstreameffects are poorly understood. Recently, it has been shown that HSV1 has an effect on the dynamics of the Golgi, but it is unknown whethertegument proteins are involved. Considering that VP11/12 has been foundassociated to the trans Golgi network (TGN), we hypothesized that thisprotein plays a role in Golgi fragmentation. The aim of this study was toevaluate both morphological changes and the distribution of Golgi proteinsduring either infection or overexpression of VP11/12 in neurons inprimary culture. We found that infection with HSV 1 induces Golgi fragmentationand redistribution of Golgi proteins. These changes were alsoinduced by overexpression of VP11/12. Our results suggest that duringHSV 1 infection VP11/12 induces changes in the structure and functionof the Golgi apparatus, which may have consequences for neuronal survivaland functionality. Funding: FONDECYT 1120464 and CONICYT24121539.REF 200Functional fitness of ribonucleoprotein complexes reconstituted frominfluenza A H5N1, H1N1pdm09 and H3N2Paul CHAN, Karry NGAI, Martin CHANDepartment of Microbiology, Chinese University of Hong Kong, CHINABackground: Pandemic influenza strains are aroused from reassortmentbetween human and avian viruses. To understand cross species adaptation,we examined the functional fitness of hybrid ribonucleoproteincomplexes reconstituted from avian and human viruses. Methods: ViralRNA polymerase subunits PB1, PB2, PA and NP derived from influenza AH5N1, H1N1pdm09 and H3N2 were co expressed with pPolI vNP Luc inhuman cells, and with its function evaluated by luciferase reporter assay.A quantitative RT PCR was used to measure vRNA, cRNA, and mRNAlevels for assessing the replication and transcription efficiency. Mutationswere created to identify signature of increased adaptability. Results:H5N1 ribonucleoprotein complexes incorporated with PB2 derived fromH1N1pdm09 and H3N2 increased the polymerase activity in human cells.Furthermore, single amino acid substitutions at PB2 of H5N1 could affectpolymerase activity in a temperature dependent manner. Enhancement inreplication and transcription activities of ribonucleoproteins was observedafter introduction of lysine at residue 627 in the H5N1 PB2 subunit.Although less strongly in polymerase activity, E158G mutation appearedto alter the accumulation of H5N1 RNA levels in a temperature dependentmanner.Conclusions: H5N1 viruses can adapt to humans either by acquisition ofPB2 from circulating human adapted viruses through reassortment, or bymutations at critical sites in PB2. This information may help to predict thepandemic potential of newly emerged influenza strains.REF 199Characterization of the Chikungunya virus RNA dependent RNApolymeraseIrina ALBULESCU, Eric SNIJDER, Martijn VAN HEMERTLeiden University Medical Center, Leiden, THE NETHERLANDSChikungunya virus (CHIKV) is a mosquito borne alphavirus that causesfever and an incapacitating arthralgia that may persist for months. A registeredvaccine or specific antiviral therapy are currently lacking to preventor treat CHIKV infection. The objective of this study is to gain more insightinto the structure and function of the CHIKV RNA dependent RNA polymerase(RdRp) by purifying it from bacteria and characterizing it in anin vitro system. The CHIKV RdRp, nsP4, was expressed as a His taggedSUMO fusion protein and was purified from the soluble fraction of a bacteriallysate by immobilized metal affinity chromatography. The tag wasremoved by protease treatment yielding nsP4 with its native N terminaltyrosine residue. Preliminary results suggest that this protein preparationhas terminal adenylyl transferase activity. For the reconstitution of de novoRdRp activity in vitro, a membrane fraction from mammalian cells expressingCHIKV nsP123 is expected to be required. Therefore, nsP123 andvariants with defective nsP1 2 and/or nsP2 3 cleavage sites or an inactivatednsP2 protease domain were expressed in mammalian cells, to analyse theirrole in CHIKV positive and negative strand RNA synthesis. These proteinsin combination with nsP4 and various templates are currently used toREF 201Role of the ubiquitin proteasome system in porcine circovirus type 2replicationShuang CHENG, Qigai HEHuazhong agricultural university, Wuhan, CHINAPorcine circovirus type 2 (PCV2), which is the primary agent of porcinecircovirus associated diseases (PCVAD), resulted in swine immunosuppressivedisease. The primary target cells of PCV2 are mononuclearphagocytes lineages. To characterize the pathogenesis of PCV2 infection,the differential proteomes of porcine alveolar macrophages, with andwithout PCV2 infection, were analyzed at different time points with twodimensional gel electrophoresis (2 DE) followed by MALDI TOF/TOFidentification. Mass spectrometry identified ubiquitin proteasome systemassociated proteins were differential expressed. Then the role of ubiquitinproteasome system in PCV2 infection was further studied. The resultsshow that MG132, a proteasome inhibitor, influence the viral activity asdetermined by decrease in viral genomic copies at the early infectionstage, and decrease in viral protein expression and viral transcription.PCV2 infection led to an accumulation of ubiquitylated proteins, withno apparent change in chymotrypsin like activity. Using small interferingRNA, we demonstrate that gene silencing of ubiquitin reducesthe genomic copies of PCV2. Taken together, our data suggest that theubiquitin proteasome system is associated with effective replication ofPCV2.Virologie, Vol 17, supplément 2, septembre 2013S175