rologie i - European Congress of Virology

5 th European Congress of VirologyHIV 1 reverse transcriptase (RT) performs the viral RNA genome replicationcombining two synthetic and hydrolytic functions: DNA polymeraseand ribonuclease H (RNase H), both validated targets for drug development.HIV 1 RT inhibitors (RTI) are a primary resource for AIDStreatment, but the rapid selection of drug resistance strains moves forwardthe identification of novel RTI classes. Currently, no RNase H functioninhibitor has been approved. Diketoacid derivatives (DKA) have beenshown to inhibit HIV 1 RNase H activity by chelating Mg2+ cofactor in theactive site and to inhibit viral replication in cell culture. The scaffold of theprototype DKA, RDS1643, has been further developed to achieve compoundswith selectivity indexes >100, identifying derivatives RDS1711,RDS2291 and RDS1759. A molecular docking model suggested interactionsbetween DKA and amino acid residues, N474, R448, R557 andY501, located in the RNase H domain. Interestingly, N474 and Y501 arepart of the RNase H primer grip and well preserved for their functionalrole. To test the model, we constructed mutant RTs with single amino acidchanges in these positions and assessed the DKA effects showing that theyare inactive on mutant N474A and Y501A RTs. These results demonstratefor the first time that RNase H inhibition by DKAs is related not only totheir chelating properties, but also to the interaction with conserved residues.Therefore, they provide important insights for a more rational leadoptimization, laying the foundation for non toxic and highly selective HIV1 RNase H inhibitors.REF O111New anti CMV drugs alone or in combination: efficacy in cell cultureand placental explantsDeborah ANDOUARD 1 , Lucie MORERE 1 , Deborah ANDOUARD 1 ,Fadi SAADE 1 , William RAWLINSON 1,2 , Sébastien COTIN 1 , ClaudeCALLISTE 3 , François LABROUSSE 4 , Yves AUBARD 5 , Marie CécilePLOY 1 , Sophie ALAIN 11 Faculty of Medicine/University of Limoges/Inserm U1092 and CNRCytomegalovirus, Limoges, FRANCE; 2 Virology, SEALS MicrobiologyPrince of Wales Hospital, Randwick, AUSTRALIA; 3 Faculty ofPharmacy/University of Limoges/EA4021, Limoges, FRANCE; 4 CHUde Limoges/Pathology department, Limoges, FRANCE; 5 CHU deLimoges/Gynecology obstetrics department, Limoges, FRANCEIntroduction: Cytomegalovirus (CMV) is the first cause of congenitalviral infection. In this process, placenta acts as a gateway and inhibition ofCMV replication in placenta is essential. Current antivirals are toxic for thefetus or the mother. We thus tested the inhibitory effect of foscarnet (PFA)and less toxic new drugs Maribavir (MBV), Baïcalein (BAI), Artesunate(ART) alone or combined.Methods: laboratory strain AD169 and a clinical isolate P*from congenitallyinfected newborn were used. In vitro: Foci reduction assays in humanembryonic lung fibroblasts (MRC 5) were revealed at D5. Ex vivo: placentalvilli from 1st trimester abortion floating on gelfoam sponges wereinfected by capillarity from infected MRC 5 cells in presence of variousconcentrations of drugs. At various times post infection, explants wereremoved for immunohistochemistry and viral load measurement.Results: In vitro: MBV/PFA were synergistic with 66% inhibition at0,25 M MBV+12,5 M PFA (IC50 1 M and 50 M) as were BAI/PFA(42, 72 and 82% inhibition for BAI, PFA, and BAI 0,275 M+PFA50 M)and ART/MBV (26, 34 and 60% inhibition for ART, MBV and ART+MBV0,25 M each). The combination ART/BAI was antagonist. Ex vivo: Viabilityof explants was validated by persistent bHCG secretion and CMVinfection of syncitiotrophoblasts and fibroblasts was maximal at D18. Alldrugs show an inhibitory effect with IC50 close to that observed in vitro.Synergistic and antagonist effects were confirmed.Conclusion: Combination therapy with low toxicity compounds could bean option for prevention of CMV transmission.REF O112Generation and Characterization of defined HCMV UL97 and UL54mutations associated with drug resistanceLena FISCHER 1 , Kerstin LAIB SAMPAIO 2 , Gerhard JAHN 1 , KlausHAMPRECHT 1 , Katharina GÖHRING 1 , Else-Kröner-FreseniusFoundation1 Institute of Medical Virology, Tübingen, GERMANY; 2 Institute of Virology,Ulm, GERMANYIn HSCT recipients, drug resistant human cytomegalovirus (HCMV) infectionremains a serious problem. For characterization of newly emergingmutations marker transfer analysis is performed. Three new HCMV mutations,the UL54 mutations D515E and V715A and the UL97 mutationE596Y as well as a combination of three UL54 mutations (D515E, L516 M,I521T) were characterized.For the generation of HCMV mutants each point mutation as well asthe combination of the three UL54 mutations was introduced into theTB40/E derived bacterial artificial chromosome (BAC) Bac7 UL32 EGFPby en passant mutagenesis (Tischer et al., 2006). After reconstitution ofmutant virus, phenotypic characterization was performed by a modifiedplaque reduction assay using a HFF/ARPE co culture method. Resistancewas defined as a 2 fold decrease in drug sensitivity in comparison to thereference strain HCMV Bac7 UL32 EGFP.The UL97 mutation E596Y showed an IC50 ratio of 6 for ganciclovir(GCV) and was therefore classified as drug resistant. Both UL54 mutationscould also be classified as GCV resistant due to IC50 ratios of >2(D515E: 2,5; V715A: 3,9). Using Foscarnet (PFA), mutation D515E hadan IC50 ratio of 1,7 while mutation V715A had a ratio of 1,8 indicatingPFA sensitivity.The triple UL54 mutant had a IC50 ratio of 9,8 for GCV which is higherthan the ratios described for the single mutations I521T (ratio 3,1 Lurainet al., 2010), L516 M (ratio 1,3 data not published) and D515E (ratio2,5). Therefore the combination of the two resistance associated mutationsI521T and D515E seems to have an additive effect.REF O113Herpes simplex virus infection control with novel small interferingRNA poolsHenrik PAAVILAINEN 1 , Alesia ROMANOVSKAYA 2 , MichaelaNYGÅRDAS 1 , Minna PORANEN 2 , Dennis BAMFORD 2 , VeijoHUKKANEN 11 University of Turku, Turku, FINLAND; 2 University of Helsinki, Helsinki,FINLANDHerpes simplex virus (HSV) is a common pathogen of humans, causinginfections such as cold sores, herpes keratitis, genital herpes and the severeHSV encephalitis. There are antivirals available against HSV, but resistantstrains exist and in some cases current therapy is inadequate. We set outto test a novel RNA interference (RNAi) based approach to control HSVinfection.We elucidated the antiviral effects of pools of small interfering RNAs (siR-NAs), and studied the host responses induced by the RNA molecules, incells of neuronal and epithelial origin. We used swarms of anti HSV dsR-NAs, and commercial single site siRNAs, targeting the essential HSVUL29 gene. The anti HSV dsRNA was synthesized from HSV UL29sequence using the phage phi6 RNA dependent RNA polymerase. Thelong dsRNA was then cleaved into siRNAs with different dicers, creatingsiRNA pools targeting a large portion of the target gene. In neuronal cellsthe inhibition of HSV shedding with siRNA pools reached 99.9% (p