5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>03. INNATE IMMUNITY AGAINSTVIRUSESPosters: REF 016 to REF 036REF 016Characterization <strong>of</strong> human papillomavirus (HPV) integration sitesand somatic mutations in STK11 gene in penile cancerMaria Lina TORNESELLO 1 , Clorinda ANNUNZIATA 1 , LuigiBUONAGURO 1 , Simona LOSITO 2 , Franco BUONAGURO 11 Molecular Biology and Viral Oncology Unit, Istituto Tumori FondazionePascale, Napoli, ITALY; 2 Pathology Unit, Istituto Tumori Fondazione Pascale,Napoli, ITALYBackground: HPV infection accounts for 40 50% <strong>of</strong> all cases <strong>of</strong> penilecarcinoma (PC) suggesting that, besides the virus, host genetic alterationsare relevant for neoplastic transformation. In invasive cervical cancer tw<strong>of</strong>actors have been shown to perturb human genes: 1) HPV integration intoloci relevant for cancer pathogenesis; and 2) mutations/deletions <strong>of</strong> tumoursuppressor genes. In this perspective we have analysed HPV integrationsites and STK11 mutational status in HPV positive and HPV negative PC.Methods: Genomic DNA was extracted from 26 PC cases. Integrationsites <strong>of</strong> HPV were characterized by means <strong>of</strong> Alu HPV based PCR, cloningand sequencing. Genetic alterations in the exons 1 to 9 <strong>of</strong> STK11gene were analysed by PCR and sequencing, and by quantitative real timePCR. Ratios <strong>of</strong> potentially deleted and non deleted exons were indicative<strong>of</strong> specific loss <strong>of</strong> STK11 coding regions. Results: Viral–human DNAjunctions showed that in two cases HPV integration occurred within thechromosome 8q21.3 region, in which the FAM92A1 gene is mapped, andinside the chromosome 16p13.3, within TRAP1 gene. Heterozygous deletions<strong>of</strong> STK11 exon 1and 2 were identified in 14.3% HPV positive andin 8.3%) <strong>of</strong> HPV negative cases (8.3%). Complete nucleotide sequencinganalysis <strong>of</strong> exons 1 9 showed only a single nucleotide change upstreamthe exon 2 coding region in one sample. Conclusions: The present resultssuggest that HPV integration events occur in gene loci relevant for celltransformation and that single nucleotide mutations and/or deletions <strong>of</strong>STK11 gene are rare events in PC.REF 017Breast cancer and virus: Molecular homology between Human MammaryTumor Virus 3 ′ orf and scorpion toxin, a ligand <strong>of</strong> voltage gatedsodium Na+ channel. Omega 3, a Na+ channel modifier, reduces therisk <strong>of</strong> metastatic breast cancerGuy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, Public Health, <strong>European</strong> AIDS Clinical Society (EACS) ScientificCouncil member, Chatenay Malabry, FRANCE; 2 CNRS Jussieu, Paris,FRANCE; 3 Association Positifs, Paris, FRANCE; 4 Association Positifs,Scientific Advisor, Paris, FRANCEHuman Mammary Tumor Virus (HMTV) was found in Breast Cancer(BC), in variable % cases: Tunisia 74%, Australia 42%, Italy 38%, UnitedStates 36%, Argentina 31%, Vietnam 0.8% (Levine PH, 2004). HTMV+(Wang Y, 1995) MCF 7 and MDA MB 231 cell lines injected in nude miceinduce metastatic breast cancer (Shafie SM, 1980; Price JE, 1990). In 123treated BCs, Bougnoux P (1995) found after 48 months 70% metastasisif the breast fat alpha linolenic acid [omega 3 (w 3)] level was low (9% ifit was high): Thus, w 3 protects against metastasis. As w 3 modifies thevoltage gated Na+ channel (NaCh) (Xiao YF, 2001; Banu I, 2006), welooked for a NaCh ligand (scorpion toxin) in HMTV. By amino acid (aa)sequence comparison, we found a molecular homology between HMTV3 ′ orf and scorpion toxin chimera (Possani LD, 2000)/Euscorpius Flavicaudus(AAT76439) (58 71) implicated in development: HMTV 124 AKNYIFTNKT 133 109 KYSSTPQPG 101 (in retro inverso)Toxin chim 1 AKNGYILNKST 11 13 KYSCSTRPG 21HMTV 282 YIYLGTGMHFWGKI 295 265 LTQKEKDDMKQQVH 278Toxin 29 YVAESGYCQ(F,W)GKI 42 58 IVQKEKDQVRQHI H 71The toxin active site (El Ayeb M, 1986, Kharrat R, 1989): N terminus(K2/Y5)/Y14/W38 loop corresponds to HMTV: K125/Y127/Y108/W292.HTMV 3 ′ orf contains a scorpion toxin, explaining the metastasis protectionconferred by w 3, a NaCh blocker; the w 3 rich soya diet in Asia,added to the low HTMV frequency in Asia (Japan/China/Vietnam), mayexplain the low asian BC prevalence. We suggest to use w 3 and otherNaCh ligands (Tran GMK, Allostery EMBO Conf 2013; Djamgoz MBA,2006) with BC chemotherapy.REF 018Toscana virus inhibits the interferon beta response in cell culturesNadege BRISBARRE 1 , Sebastien PLUMET 2 , Philippe DE MICCO 1 ,Isabelle LEPARC GOFFART 2 , Sebastien EMONET 3,21 UMR 7268/Aix Marseille université CNRS/EFS, marseille, FRANCE;2 IRBA Antenne Marseille, marseille, FRANCE; 3 IRBA, lyon, FRANCETOSV is an arthropod borne virus (family Bunyaviridae, genus Phlebovirus)transmitted to humans by sandflies. It is an emerging pathogen inthe Mediterranean basin where it causes outbreaks <strong>of</strong> acute meningitisand meningoencephalitis in native or foreign (tourists) populations duringsummertime when the vector population reaches its maximum density.The innate mammalian immune system can establish a first line <strong>of</strong> defenseagainst invading viruses; cells synthesize and secrete beta interferon (IFN) (type I IFN) which prompt cells to an antiviral state. Previous studiesdemonstrated that some viruses belonging to the genus Phlebovirus maydevelop anti IFN strategies, but information regarding TOSV is widelylacking. We explored the relationships between TOSV and type I interferon(IFN) response. Our main findings were as follows: i) TOSV growthin Vero cells is sensitive to an antiviral state induced by low dose addition<strong>of</strong> exogenous IFN beta; ii) no IFN mRNA or IFN were detectableafter infection <strong>of</strong> HeLa and 293T cells (mammalian cells) by TOSV fromtwo different lineages; iii) TOSV inhibits IFN production induced bySendaï virus, a well known inducer <strong>of</strong> IFN production. These experimentsdemonstrate the ability <strong>of</strong> TOSV to inhibit the IFN production.The existence <strong>of</strong> an active anti IFN system against the mammalian IFNdefense mechanism suggests that a mammalian host exists probably duringthe natural life cycle <strong>of</strong> TOSV; this anti IFN capability is maintained bymeans <strong>of</strong> regular contact with this mammalian host.REF 019Type III interferon (IFN lambda) antagonizes the antiviral activity<strong>of</strong> interferon alpha in vitro against west nile virusDaniele LAPA, Maria Rosaria CAPOBIANCHI, Licia BORDI, EleonoraLALLE, Claudia CAGLIOTI, Serena QUARTU, Antonino DI CARO,Ippolito GIUSEPPE, Concetta CASTILLETTNational Institute for Infectious Disease L. Spallanzani, Rome, ITALYType III interferons (IFN lambda 1 to 4 also known as IL28/29), are themost recently discovered members <strong>of</strong> IFN family. The influence <strong>of</strong> naturalgenetic polymorphisms on spontaneous and therapy driven recoveryfrom HCV infection suggests that this novel IFN type is involved in innateantiviral defence mechanisms. Synergism between different IFN types iswell established, but for type I and type III IFNs no conclusive evidencehas been reported so far. The aim <strong>of</strong> the study was to evaluate the possiblesynergism/antagonism between IFN alpha and IFN lambda1 andVi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S123
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>lambda2 in the inhibition <strong>of</strong> replication <strong>of</strong> WNV (lineages 1 and 2) andin the activation <strong>of</strong> intracellular pathways <strong>of</strong> IFN response in differentcell lines. To this aim, A549 and Vero E6 cells were treated with increasingamounts <strong>of</strong> IFN lambda either IFN lambda1a or lambda2a or IFNalpha, used alone or in combination, then infected with WNV 1 or 2; theextent <strong>of</strong> virus yield inhibition was established; mRNA levels <strong>of</strong> MxA and2 ′ 5 ′ OAS were measured as well. The antiviral potency <strong>of</strong> IFN lambda1and lambda2 was lower than that <strong>of</strong> IFN alpha. When IFN alpha andlambda were used together, the Combination Index (CI) for virus inhibitionwas >1 (although more pronounced in Vero E6 cells infected with WNVlineage 2), indicating antagonistic effect. The same results were obtainedusing other viruses (Chikungunya virus, EMCV and HSV 1). Antagonismbetween IFN alpha and lambda was also observed for the induction<strong>of</strong> mRNA for both MxA and 2 ′ 5 ′ OAS. Elucidating the interplay betweenIFN alpha and lambda may help to better understand innate defencemechanisms against viral infections, including the molecular mechanismsunderlying the possible influence <strong>of</strong> IL-28B polymorphisms on the naturalhistory <strong>of</strong> flavivirus infections. This work was partially supported by the<strong>European</strong> Union Seventh Framework Programme [FP7/2007-2013] underGrant Agreement n ◦ 278433-PREDEMICS.REF 020The measles virus phosphoprotein interacts with the linker domain<strong>of</strong> STAT1Patricia DEVAUX, Lauren PRINISKI, Roberto CATTANEOMayo Clinic, Rochester, MN, USAThe measles virus (MV) phosphoprotein (P) and V proteins block the interferon(IFN) response by impeding phosphorylation <strong>of</strong> the signal transducerand activator <strong>of</strong> transcription 1 (STAT1) by the Janus kinases (JAKs). Wesought to characterize here how STAT1 interacts with MV P. Based onsequence and function conservation between MV and Nipah virus (NiV)P proteins, we postulated that the region <strong>of</strong> STAT1 interacting with NiV P(residues 509 to 712) also interacts with MV P. Within this region, sinceMV P does not interact with STAT2, we mutated segments that have nohomology to it. These segments cover the linker domain, the Src homology2 (SH2) domain and the transactivation domain. We initially producedmutants <strong>of</strong> two or three residues within these segments, and then alsomutants <strong>of</strong> individual residues. The expression and effect <strong>of</strong> these mutantswere tested in STAT1 deficient cells. Certain mutants <strong>of</strong> the linker, theSrc homology 2 domain (SH2), or the transactivation domain had reducedor abolished phosphorylation through JAKs after IFN treatment. Othermutants, mainly localized in the linker, failed to interact with P as documentedby the lack <strong>of</strong> interference with nuclear translocation. Thus, thefunctional footprint <strong>of</strong> P on STAT1 localizes mainly to the linker domain;there is also some overlap with the STAT1 phosphorylation functionalfootprint on the SH2 domain. Based on these observations, we propose amodel <strong>of</strong> how the MV P and V proteins may operate in concert to inhibitthe JAK/STAT pathway.REF 021Mechanism <strong>of</strong> inhibition <strong>of</strong> the innate immune response by alphavirusnsP2 proteinElena FROLOVA 1 , Ivan AKHRYMUK 1 , Sergey KULEMZIN 11 University <strong>of</strong> Alabama at Birmingham, Birmingham, USAOutcome <strong>of</strong> viral infection at the cellular level depends on competitionbetween cellular antiviral response and ability <strong>of</strong> the virus to inhibit itsactivation. The antiviral response induces pathways mediating non cytopathicclearance <strong>of</strong> the virus from infected cells and release <strong>of</strong> interferon andother cytokines, which establish the antiviral state in yet uninfected cells.Numerous viruses promote infection spread and viremia development by atleast partially inhibiting cellular antiviral response. It has been previouslydemonstrated that the Old World alphaviruses, including Sindbis, SemlikiForest and chikungunya viruses, evade the innate immune responseby inducing rapid and global inhibition <strong>of</strong> cellular transcription, whichprevents activation <strong>of</strong> cellular antiviral genes. We have defined the mechanism<strong>of</strong> transcriptional shut<strong>of</strong>f utilized by the Old World alphaviruses.During virus replication, the nonstructural protein nsP2 is transported intothe nucleus <strong>of</strong> the infected cells, where it induces rapid degradation <strong>of</strong>the catalytic subunit <strong>of</strong> the DNA dependent RNA polymerase Rpb1. Wefound that degradation <strong>of</strong> Rpb1 does not depend on the nsP2 associatedprotease activity. Instead, nsP2 imitates activation <strong>of</strong> the transcriptioncoupled repair mechanism, which leads to complete, ubiquitin dependentdegradation <strong>of</strong> Rpb1 within 4 6 h post infection. Interestingly, the NTPaseactivity <strong>of</strong> nsP2 helicase domain is critical for Rpb1 degradation. Thus,the Old World alphaviruses employ a previously unknown and uniquemechanism for robust inhibition <strong>of</strong> cellular antiviral response.REF 022Characterization <strong>of</strong> the effect <strong>of</strong> antiviral enzyme RNase L on differentpathogenic strains <strong>of</strong> influenza A virusWai Yip LAM 1 , Denis Pc CHAN 2 , Paul Ks CHAN 2,3 , Stephen KwTSUI 11 School <strong>of</strong> Biomedical Sciences, Faculty <strong>of</strong> Medicine, The Chinese University<strong>of</strong> Hong Kong, Shatin, HONG KONG; 2 Stanley Ho Centre forEmerging Infectious Diseases, Faculty <strong>of</strong> Medicine, The Chinese University<strong>of</strong> Hong Kong, Shatin, HONG KONG; 3 Department <strong>of</strong> Microbiology,Faculty <strong>of</strong> Medicine, The Chinese University <strong>of</strong> Hong Kong, Shatin, HONGKONGInfluenza A viruses cause a highly contagious respiratory disease inhumans and are responsible for the periodic widespread pandemics, thathave caused high mortality rates. Recently, the World Health Organization(WHO) has recorded a mortality rate <strong>of</strong> greater than 60% in thehighly pathogenic avian influenza A (H5N1) virus infection. The fatal outcome<strong>of</strong> human influenza A H5N1 is found to be associated with reactivehaemophagocytic syndrome and multi organ failure mediated by hypercytokinemia.Ribonuclease L (RNase L) is a latent endoribonuclease in theinterferon regulated double strand RNA (dsRNA) activated antiviral 2 ′ 5 ′oligoadenylate synthetase (OAS)/RNase L pathway. Activated RNase Lwill cleave viral single strand RNA (ssRNA) predominantly at single strandedUA and UU dinucleotides position(s), resulting in small, <strong>of</strong>ten duplexRNA cleavage products. These small RNA cleavage products can furtheractivate the interferon (IFN) as well as other cytokines responses via specificpathogen recognition receptors, such as retinoic acid inducible gene I(RIG I), melanoma differentiation associated gene 5 (MDA5) and toll likereceptors (TLR). We postulate that different viral RNA sequences fromdifferent pathogenic strains <strong>of</strong> influenza A virus will be cleaved by RNaseL in a sequence specific manner and the resulting amount <strong>of</strong> cleavage RNAproducts will thus be different. Bioinformatic data demonstrating a quantitativedifference between the number <strong>of</strong> UA/UU sites between highlypathogenic and low pathogenic viruses. The results were further verifiedusing an in vitro model.REF 023Mononegavirales leader RNA as agonist <strong>of</strong> RIG IJade LOUBER 1 , Louis Marie BLOYET 1 , Eva KOWALINSKI 2,3 ,Stephen CUSACK 2,3 , Denis GERLIER 11 Centre International de Recherche en Infectiologie, CIRI, INSERMU111, CNRS UMR5308, Université Lyon 1, ENS Lyon, Lyon, FRANCE;2 <strong>European</strong> Laboratory <strong>of</strong> Molecular Biology, Grenoble, FRANCE; 3 VirusCell host Interactions Unit, UJF EMBL CNRS, UMI 3265, Grenoble,FRANCES124 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
Change language