rologie i - European Congress of Virology

5 th European Congress of Virologyof 17.3 per strain. At the protein level, 30 amino acid changes, correspondingto 22 different positions within pUS28, were identified, that is 6.2%of the total codons of the protein. Seven changes were located in putativefunctional motifs and 9 were unpreviously reported. All these polymorphismswere distributed evenly alongside pUS28. Overall, the frequency ofthese changes ranged from 2.4% to 56.1%. Among drug resistant viruses,22 out of the 30 polymorphisms found in drug sensitive viruses were evidencedtogether with 9 additional ones. Three novel polymorphisms werelocated in putative functional motifs. The distribution of US28 genotypeswas similar among drug sensitive and drug resistant viruses. In conclusion,the polymorphism of CMV US28 chemokine receptor is low and does notseem to be affected by CMV susceptibility to current antivirals.REF 134Genotypic characterization of human cytomegalovirus terminaseDavid BOUTOLLEAU 3 , Léa PILORGE 1 , Sonia BURREL 2,3 , ZaïnaAÏT ARKOUB 3 , Henri AGUT 2,31 Service de Virologie, Hôpital Pontchaillou, CHU de Rennes, Rennes,FRANCE; 2 UPMC Univ Paris 06, ER1 DETIV, Paris, FRANCE;3 Service de Virologie, Groupe Hospitalier Universitaire La Pitié Salpêtrière,CharlesFoix, Paris, FRANCEHuman cytomegalovirus (CMV) DNA packaging involves the CMV terminasecomposed of large UL56 and small UL89 subunits. Letermovir(AIC246), a novel anti CMV agent currently tested in phase II clinicaltrial, is able to interfere with CMV terminase. This work aimed to performthe genetic analysis of CMV terminase in genotypically characterized drugsensitive (n=33) and drug resistant (n=30) viruses. Full length CMV UL56and UL89 genes were amplified, sequenced, and compared to that of referencestrain AD169. Among drug sensitive viruses, interstrain identityvaried from 98 to 100% for both genes, with mean numbers of nucleotidemutations of 25.1 and 17.1 per strain for UL56 and UL89, respectively.At the amino acid level, 20 and 8 changes were identified in pUL56 andpUL89, respectively, that is 2.4% and 1.2% of the total codons of the proteins.Overall, the frequency of these changes ranged from 3% to 100%.Seven changes in pUL56, including 1 deletion, and 2 in pUL89 wereunpreviously reported. With regards to the putative functional domainsof CMV terminase subunits, all amino acid changes were located outsideconserved regions, except S345A in pUL89. Conversely to pUL89,polymorphisms in pUL56 clustered mainly within two variable regions.Among drug resistant viruses, 19 out of the 28 polymorphisms foundin drug sensitive viruses were evidenced. Three changes were located inconserved domains (G649D and H698Q in pUL56, A659 V in pUL89).In conclusion, CMV terminase exhibits a low polymorphism and does notseem to be likely impacted by resistance to current antivirals.REF 135Genetic analysis of herpes simplex virus type 1 UL5/UL52 helicaseprimase complexSonia BURREL 2 , Marianne COLLOT 1,2 , Zaïna AIT ARKOUB 2 , HenriAGUT 1,2 , David BOUTOLLEAU 1,21 UPMC Univ Paris 06, ER1 DETIV, Paris, FRANCE; 2 Service de Virologie,Hôpitaux Universitaires La Pitié Salpêtrière Charles Foix, AP HP,Paris, FRANCEAmenamevir (ASP2151, Astellas Pharma) is a non nucleoside drug thatinhibits herpes simplex virus type 1 (HSV 1) UL5/UL56 helicase primase(HP) complex involved in viral DNA replication. Previous data evidencedsome amino acid changes associated with reduced susceptibilities of HSV1 to amenamevir in both subunits of HP complex that might alter viralreplication and pathogenicity. This work aimed to perform the genotypiccharacterization of HSV 1 HP complex among 26 drug sensitive isolates,6 acyclovir resistant isolates and KOS laboratory strain. Full length UL5and UL52 genes were amplified, sequenced, and compared to that of HSV1 reference strain 17. Among drug sensitive isolates, interstrain identityvaried from 99.4% to 99.9% for both genes, with mean numbers of nucleotidemutations of 8.9 and 12.4 per strain for UL5 and UL52, respectively.At the amino acid level, 21 and 26 changes were identified in pUL5 andpUL52, respectively, that is 2.4% and 2.5% of the total codons of theproteins. Of note, 1 isolate harbored a deletion at codons 695 696 betweenpUL52 conserved domains III and IV. For both proteins, all changesidentified lied within nonconserved regions, except V347I in pUL5. Theanalysis HP complex among 6 acyclovir resistant isolates showed polymorphismspreviously reported in drug sensitive isolates together with 6undescribed changes that were located outside functional and conservedregions, except M766 V in pUL52. This work confirms that UL5/UL52HP complex is highly conserved in HSV 1 clinical isolates and constitutesa legitimate antiviral target.REF 136Resistance development in HIV 1 infected individuals failing ARVtherapy: investigation of Gag and Pol polyproteins functional roleIlaria CARLI 1 , Claudia DEL VECCHIO 1 , Saverio Giuseppe PARISI 1 ,Federico DAL BELLO 1 , Arianna CALISTRI 1 , Giorgio PALÙ 1 , CristinaPAROLIN 21 Department of Molecular Medicine, Padua, ITALY; 2 Department of Biology,Padua, ITALYThe introduction of Antiretroviral Therapy (ARV) in the cure of AIDShas decreased the morbidity and mortality rate of HIV 1 positive patients.Nevertheless, since therapy is required lifelong, a significant minority ofpatients are likely to develop resistance to at least one class of drugs. Themechanisms of resistance mainly involve mutations altering the interactionof viral enzymes and inhibitors. Recent studies reveal that, besidesthe enzymes encoding ones, other regions might contribute to the developmentof resistance. In particular, some specific cleavage and non cleavagesite mutations in Gag increase polyprotein processing, thus compensatingfor the catalytic loss of the viral Protease (PR) functional activity inducedby primary resistance mutations. In this context, we are interested instudying the functional role of HIV 1 Gag in the viral life cycle of ARVselected viruses. We analyzed clinical samples of HIV 1 infected patientsfailing PR Inhibitors (PIs) and RT Inhibitors (RTIs) among a cohort offive infectious diseases units located in Veneto in northeastern Italy. Weoptimized PCR amplification and sequencing conditions for gag and polgenes. The relative contribution of Gag, PR and/or RT mutations on virusreplication ability and their interdependence are now examined using HIV1 molecular clones carrying patient derived sequences. Our results wouldcontribute to better characterize the role of Gag and the relations with PRand RT in resistance development, their relevance in viral replication andevolution in the presence or in the absence of drugs.REF 137Design, Synthesis and Biological Evaluation of New 1,3 Diarylpropenonesas Single Site Dual Inhibitors of HIV 1 RTFrancesca ESPOSITO 1 , Rita MELEDDU 1 , Valeria CANNAS 1 , SimonaDISTINTO 1 , Claudia DEL VECCHIO 2 , Giulia BIANCO 1 , AngelaCORONA 1 , Filippo COTTIGLIA 1 , Stefano ALCARO 3 , CristinaPAROLIN 4 , Elias MACCIONI 1 , Enzo TRAMONTANO 11 Department of Life and Environmental Sciences, University of Cagliari,Cagliari, ITALY; 2 Department of Molecular Medicine, University ofPadova, Padova, ITALY; 3 Department “Salvatore Venuta”, Universityof Catanzaro, Catanzaro, ITALY; 4 Department of Biology, University ofPadova, Padova, ITALYA small library of 1,3 diaryl propenones has been designed, and synthesizedas dual inhibitors of both HIV 1 reverse transcriptase DNA polymeraseand ribonuclease H associated functions. Compounds were assayed onS156 Virologie, Vol 17, supplément 2, septembre 2013