rologie i - European Congress of Virology

5 th European Congress of Virologysandwich structure is smaller and more compact when compared with otheradenovirus or reovirus fibre heads, mainly due to it having shorter loops.The DG loop of human adenovirus fibre head is replaced by a short alphahelix.REF 224Host Nucleolin/C23 is an essential partner for vRNPs nucleocytoplasmicexport during Influenza infectionCoralie CARRON 1 , Olivier TERRIER 1 , Aurélien TRAVERSIER 1 ,Gaëlle CARTET 1 , Sabine HACOT 2 , Bruno LINA 1 , Jean JacquesDIAZ 2 , Manuel ROSA CALATRAVA 11 VirCell Team, VirPath Laboratory, EA 4610, Lyon, FRANCE; 2 UMRInserm U1052 CNRS 5286, CRCL, Lyon, FRANCEInfluenza are nuclear replicating viruses that hijack host machineries inorder to replicate and interfere with host antiviral response. Influenzainfection induces an important remodelling of the host cell architecturewith marked modifications of the nucleolar ultrastructure (Terrieret al., 2012). Moreover, several proteomic studies have shown that thenucleolar proteome was strongly impacted during infection, suggestinga “nucleolar experience” for influenza viruses. In this context, we haveshown that subcellular distribution of the three major nucleolar componentswas differentially altered during infection. We then focused onnucleolin, a multifunctional protein implicated in ribosome synthesis andchromatin remodelling processes. We first observed that nucleolin wasdynamically relocated at the nuclear periphery upon infection, followingthe same dynamic pattern as vRNPs during their export towards thecytoplasm. Moreover, we demonstrated a vRNA dependant interactionbetween endogenous nucleolin and the viral nucleoprotein (NP) duringinfection. Finally, nucleolin silencing leads to a delay in vRNPs traffic anda loss of their association with chromatin compartment where they interactwith the host export machinery (Chase et al, 2011), and then reducedyields of viral production. Our results strongly indicate that influenza Aviruses exploit an unusual chromatin targeting strategy to achieve theirvRNP export process. We propose a model in which NP would hijacknucleolin functions for this process and the consecutive snatching of Crm1dependent export machinery.Rhinoviruses (HRVs) are the major cause of virus induced exacerbationsof asthma. Recent findings suggest that HRV pathology may involve disruptionof host cell nucleocytoplasmic trafficking mediated, at least inpart by 3C protease that localises to the nucleus of infected cells as 3CDand 3C. We have investigated nuclear targeting of 3C/3CD using HRVinfected cells and cells transfected to express HRV 3CD. Using 3C specificimmunofluorescence/confocal microscopy and western blotting ofHRV infected HeLa cells, we found that 3CD localises in the nucleusand cytoplasm of infected cells at 6 h post infection concomitant withdegradation of key molecules of the nuclear pore complex; 3C is observedlater, and predominantly in the cytoplasm. When expressed as a GFPfusion protein in transfected cells, 3CD was processed to 3C, localised tothe nucleus and resulted in degradation of specific nucleoporins and mislocalization of an endogenous nuclear protein. Uncleaved 3CD localizedprimarily in the cytoplasm, suggesting that the putative nuclear localizationsignal in 3D is not functional; GFP 3D was also cytoplasmic. GFP 3Cand its proteolyticaly inactive mutant localized in both nucleus and cytoplasmof transfected cells suggesting that 3C does not require cleavage ofnucleoporins to localize to the nucleus.In conclusion, 3C has an inherent ability to localize to the nucleus ofinfected cells; 3CD is unable to facilitate its nuclear localization. Thenuclear accumulation of 3CD in HRV infected cells probably requiresother viral proteins.REF 226Role of the small GTPase Rab27a during Herpes simplex virus infectionof oligodendrocytic cellsJosé Antonio LÓPEZ GUERRERO 1 , Antonio Jesús CRESPILLO 1 ,Alberto FRAILE RAMOS 2 , Enrique TABARÉS 3 , Antonio ALCINA 4 ,Raquel BELLO MORALES 11 Centro de Biología Molecular Severo Ochoa, UAM CSIC, Madrid,SPAIN; 2 Universidad Complutense de Madrid, Facultad de Medicina,Madrid, SPAIN; 3 Universidad Autónoma de Madrid, Facultad de Medicina,Madrid, SPAIN; 4 Instituto de Parasitología y Biomedicina LópezNeyra, CSIC, Granada, SPAINThe morphogenesis of herpes simplex virus type 1 (HSV 1) comprisesseveral events, of which some are not completely understood. It has beenshown that HSV 1 glycoproteins accumulate in the trans Golgi network(TGN) and in TGN derived vesicles. It is also accepted that HSV 1 acquiresits final morphology through a secondary envelopment by budding intoTGN derived vesicles coated with viral glycoproteins and tegument proteins.Nevertheless, several aspects of this process remain elusive. Thesmall GTPase Rab27a has been implicated in regulated exocytosis, and itseems to play a key role in certain membrane trafficking events. Rab27aalso seems to be required for human cytomegalovirus assembly. However,despite the involvement of various Rab GTPases in HSV 1 envelopment,there is, to date, no data reported on the role of Rab27a in HSV 1 infection.Herein, we show that Rab27a colocalized with GHSV UL46, a tegumenttagged green fluorescent protein HSV 1, in the TGN. In fact, thissmall GTPase colocalized with viral glycoproteins gH and gD in thatcompartment. Functional analysis through Rab27a depletion showed asignificant decrease in the number of infected cells and viral production inRab27a silenced cells. Altogether, our results indicate that Rab27a playsan important role in HSV 1 infection of oligodendrocytic cells.REF 225Nuclear Localisation of Rhinovirus 3C/3CDReena GHILDYAL 1 , Erin WALKER 1 , Lora JENSEN 1 , AlexFULCHER 2 , David JANS 21 University of Canberra, Canberra, AUSTRALIA; 2 Monash University,Melbourne, AUSTRALIAREF 227C terminal determinants of the cytomegalovirus GPCR pUS27 forendocytic receptor sortingIna NIEMANN 1 , Anna REICHEL 1 , Heinrich STICHT 2 , ThomasSTAMMINGER 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Emil Fischer Zentrum, Institute ofBiochemistry, University of Erlangen Nuremberg, Erlangen, GERMANYHuman cytomegalovirus (HCMV) encodes several G protein coupledreceptor (vGPCR) homologues, including pUS27, pUS28, pUL33, andpUL78. In order to fully understand vGPCR functions, it is necessaryto precisely define the mechanisms of receptor endocytosis. A rapid andconstitutive internalization of all four vGPCRs was demonstrated by antibodyfeeding experiments. While the well characterized receptors pUS28and pUL78 may play a role for chemokine sequestration, the knowledgeconcerning the sorting abilities of pUS27 is limited. To gain further insightinto the subcellular trafficking of HCMV encoded pUS27, we constructedvarious deletion mutants expressing an N terminal Flag or a C terminalGFP tag. Internalization assays were evaluated by confocal microscopyand FACS analysis. In contrast to recent publication, deletion of 46 aminoacids from the C terminus only partially disrupted endocytosis. Furthermore,we observed pUS27 sorting into different endosomal compartmentsS182 Virologie, Vol 17, supplément 2, septembre 2013