rologie i - European Congress of Virology

5 th European Congress of Virologysilencing IE gene expression. This ND10 mediated defense mechanism,however, is counteracted by the immediate early protein (IE1) of HCMV.During infection, IE1 targets ND10 by binding to PML. Subsequently, itinduces PML de SUMOylation and mediates disruption of ND10. However,the exact mode of PML binding and ND10 disruption remains unclear.The aim of our study was to clarify which motifs or regions of IE1 arerequired for binding to the cellular restriction factor PML. Since SUMOinteracting motifs (SIMs) are involved in protein protein interactions, weperformed bioinformatic analyses to identify putative SIMs in IE1. FourSIMs were detected which were subsequently mutated to determine theinfluence on PML binding and ND10 disruption. The mutations had nosignificant effect concerning the ability to disrupt ND10, suggesting thatthe interaction between IE1 and PML is not mediated by SIMs. In addition,we generated C terminal IE1 truncations to better define which region ofIE1 is crucial for PML interaction. Interestingly, the deletion of an alphahelical region of IE1 led to the complete loss of PML binding, therebyhighlighting the role of this domain for PML binding. Taken together,these findings provide new insights into structural requirements for theND10 antagonistic activity of IE1.REF 033Immune recognition of picornavirus RNAs and viral evasion strategiesFrank VAN KUPPEVELD, Qian FENG, Martijn LANGEREISDivision of Virology, Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDSRIG I and MDA5 are cytosolic RNA sensors that play critical roles ininnate antiviral responses. Major advances have been made in identifyingRIG I ligands, but our knowledge of the ligands for MDA5 remains restrictedto data from transfection experiments mostly using poly(I:C). Here, wedetermined the MDA5 stimulatory activity of various viral RNAs producedduring picornavirus infection (i.e., the ss virion RNA, which containsa VPg peptide at the 5 ′ end, the VPg lacking viral ssRNA, and two replicationintermediates – the perfectly duplexed replicative form (RF) andthe partially ds replicative intermediate (RI). Besides transfection studiesusing purified viral RNAs, we also studied RNA recognition during a normalinfection (using mengovirus Zn in which the known IFN antagonistis inactivated) by using inhibitors that interfere with specific steps of viralRNA replication. Our results show that viral ssRNAs (with or without5 ′ VPg) do not activate MDA5, whereas RF is a highly potent MDA5ligand. Moreover, picornaviruses actively suppress IFN a/ response togain replication advantage. While the IFN antagonist of Cardioviruses hasbeen identified, the strategy of Enteroviruses to interfere with IFN a/induction remains unclear. Previous studies demonstrated that the viralproteinase 3C cleaves crucial factors of the IFN a/ pathways. Our preliminarydata show a yet unappreciated role of another viral proteinase, 2A,in antagonizing MDA5 mediated IFN a/ induction.REF 034Study of the effect of CD40 ligand on the lytic cycle of Herpes SimplexVirus type 1 (HSV 1)Virginia Maria VLAHAVA 1 , Eftychia STAVRAKAKI 1 , AristeidisELIOPOULOS 1,2 , Georgios SOURVINOS 11 University of Crete, Medical School, Heraklion, GREECE; 2 Instituteof Molecular Biology and Biotechnology Foundation for Research andTechnology, Heraklion, GREECEPrevious studies have shown that CD40 ligand (CD40L) has the ability todirectly confer antiviral protection in cells expressing the CD40 receptor.On that ground, we sought to investigate how CD40L exerts its protectiveeffect on cells infected by HSV 1. For that reason, we generated a stablytransfected U2OS cell line that expresses the CD40 receptor. After testingthat the CD40 CD40L interaction does not lead to cell death, we wenton to investigate whether there is an effect on the production of progenyvirus following treatment with CD40L. We concluded that indeed thereis a reduction of progeny virus in the presence of CD40L. Growth curvesalso showed that the cells treated with CD40L produced lower viral yieldsduring the course of the infection. The antiviral effect was specific, asthe expression of a mutant CD40 receptor which lacks the ability to bindTRAFs, resulted in similar titers of progeny virus, regardless the presenceof CD40L. In order to examine whether the binding ability of HSV 1virions is compromised by the CD40 CD40L interaction, a binding assaywas performed and confocal microscopy verified the efficient attachmentof the virions. Subsequently, we monitored the distribution of the capsidprotein VP16 after treatment with CD40L and observed that nuclear entryof the virus is delayed. Additionally, replication of the virus was stalledin the presence of CD40L as this was measured by the DNA copies of theEarly ICP8 gene 2, 12 and 24 hpi. Our data, suggest that CD40L poses ablockade on HSV 1 from the very early stages of the infection.REF 035Human Coronavirus EMC/2012 accessory protein 4a functions as atype I interferon antagonistDaniela NIEMEYER 1 , Doreen MUTH 1 , Tasnim SULIMAN 1 , GaborHORVATH 2 , Ron A.M. FOUCHIER 3 , Friedemann WEBER 4 , ChristianDROSTEN 1 , Marcel A. MÜLLER 11 Institute of Virology, University of Bonn Medical Centre, Bonn, GER-MANY; 2 Institute of Innate Immunity, University of Bonn Medical Centre,Bonn, GERMANY; 3 Viroscience Lab, Erasmus MC, Rotterdam, THENETHERLANDS; 4 Institute for Virology, Philipps University Marburg,Marburg, GERMANYA novel human coronavirus, HCoV EMC, emerged recently in the MiddleEast causing severe respiratory tract infections similar to severe acute respiratorysyndrome (SARS). The interferon (IFN) system is a major part ofthe innate immunity controlling viral replication. It can be triggered uponrecognition of double stranded (ds)RNA by RIG I like helicases (RIG I andMDA5). Whereas several SARS CoV accessory proteins were shown toinhibit the IFN response, functions of HCoV EMC accessory proteins areunknown. In the presented study we focused on putative anti IFN mechanismsof the HCoV EMC accessory proteins p3, p4a, p4b and p5. Theaccessory genes were cloned into eukaryotic vectors for overexpressionanalyses in human and primate cells. In an IFN beta promoter reporterassay only p4a inhibited the activation of the IFN response. In confirmatoryassays p4a blocked the nuclear translocation of a co expressed GFPIRF 3 and the secretion of bioactive IFN bioassay. An in silico analysispredicted a dsRNA binding motif for p4a. In HCoV EMC infected cellsp4a co localized with viral dsRNA molecules suggesting direct interaction.An IFN beta promoter assay with targeted IFN stimulation at the levelof the RIG I helicases revealed that p4a inhibited MDA5 but not RIG Iactivation.In conclusion, p4a co localization with dsRNA and its inhibitory effecton MDA5 activation support the idea that p4a is a functional IFN antagonistinhibiting the IFN induction pathway. Direct interaction studiesbetween p4a, dsRNA and MDA5 will be necessary to unravel the detailedmechanism.Virologie, Vol 17, supplément 2, septembre 2013S127