rologie i - European Congress of Virology

5 th European Congress of VirologyFriday 13 th September 2013, 15h00 – 17h15WORKSHOP 13: “VIRAL REPLICATION STRATEGIES”Chairpersons: Luis ENJUANES (Madrid, SPAIN)& Juan ORTIN (Madrid, SPAIN)Room Gratte-Ciel 1, 2, 3for virus viability was also demonstrated by reverse genetics, showed thatD99A, P116A and R190A mutations severely impaired or even abolishednsp12/nsp8 interaction and consequently polymerase activity. Moreover,conserved residues in nsp8 (K58) and nsp7 (K7 and N37) modulate RNAtemplate recognition by nsp7/nsp8/nsp12. Nsp14 is the first description ofa mismatch excision enzyme bound to a viral RNA polymerase, illustratingthe integration of RNA polymerization, repair and capping activitiesinto a single multi active complex.KEYNOTE:Influenza polymerase host adaptationAnna CAULDWELL, Jason LONG, Hongbo ZHOU, OlivierMONCORGE, Wendy BARCLAYSection of Virology, Imperial College London, UNITED KINGDOMTypical avian influenza A viruses do not replicate efficiently in mammals.This host range block is recapitulated in the laboratory in a cellbased polymerase activity assay in which viral polymerases reconstitutedfrom an avian virus do not amplify minigenomes inside human cells. Wepreviously showed that avian/human heterokaryons could support avianvirus polymerase activity suggesting there is an avian host factor lackingin human cells that could complement avian virus polymerase. Several distincthost adaptive mutations map to the polymerase complex (PB1, PB2and PA), and in addition it was recently reported that the nuclear exportprotein NEP encoded on RNA segment 8 can also alleviate the host rangeblock in polymerase activity. Some of these viral mutations increase polymerasefunction in both human and avian cells whereas others are humancell specific, suggesting the mechanisms by which they act and the hostfactors involved may be different. Some mutations which enhance activityin the reconstituted polymerase assay are not selected for in nature. Thismay be because they lead to attenuated virus replication, for example byincreasing interferon activation. Taken together these analyses can tell usmore about how influenza polymerase functions within the infected celland by which mechanisms one of the several host range barriers that limitavian influenza viruses in humans might be breached.ORAL COMMUNICATIONSREF O115Processive RNA synthesis, repair and capping activities embedded ina SARS Coronavirus multi protein complexLorenzo SUBISSI 1 , Clara C. POSTHUMA 2 , Jessika ZEVENHOVEN 2 ,Axelle COLLET 1 , Alexander E. GORBALENYA 2 , Eric J. SNIJDER 2 ,Etienne DECROLY 1 , Bruno CANARD 1 , Isabelle IMBERT 11 AFMB AMU, Marseille, France; 2 LUMC, Leiden, THE NETHERLANDSCoronaviruses include lethal human pathogens like the recently emergedHuman Coronavirus EMC (HCoV EMC) and Severe Acute RespiratorySyndrome Coronavirus (SARS CoV). They carry largest known viral RNAgenomes encoding replication/transcription machineries of unusual complexityand composed of, at least, 16 viral non structural proteins (nsps).The RNA dependent RNA polymerase (RdRp) nsp12 is poorly active invitro, but supposedly plays a central role in the efficient replication of thelarge (>30 kb) coronavirus genome. Here, we report that SARS CoV nsp7and nsp8, previously co crystallized as a hexadecamer reminding a slidingclamp, confer highly efficient and processive RNA synthesis properties tonsp12. The nsp7/nsp8/nsp12 complex further interacts in a stable fashionwith nsp14, a bi functional enzyme bearing 3 ′ 5 ′ exoribonuclease (ExoN)and RNA cap N7 guanine methyltransferase activities. The processiveRNA synthesis of nsp7/nsp8/nsp12 is not affected by nsp14 ExoN activity.Mutational analysis of conserved nsp8 residues, whose importanceREF O116Crystal structure of the N0 P complex of Nipah virus and of VSVprovide new insights into the encapsidation mechanism of the Paramyxoviridaeand RhabdoviridaeFilip YABUKARSKI 1 , Cédric LEYRAT 1 , Nicolas TARBOURIECH 1 ,Malene RINGKJØBING JENSEN 2 , Martin BLACKLEDGE 2 , RobRUIGROK 1 , Marc JAMIN 11 UMI3265 UJF EMBL CNRS UVHCI, Grenoble, FRANCE; 2 UMR5075CEA CNRS UJF Institut de Biologie Structurale, Grenoble, FRANCEEncapsidation of newly synthesized viral RNA genomes and antigenomesin a helical homopolymer of nucleoprotein (N) is an essential step in thereplication of non segmented negative strand RNA viruses. For Rhabdoviridaeand Paramyxoviridae, encapsidation relies on the continuousproduction of soluble RNA free nucleoprotein (N0) in the form of acomplex with the phosphoprotein (P), named the N0 P complex. In bothfamilies, this complex is formed by the interaction between the N terminalregion of P and the core of the N protein.We developed a strategy to reconstruct the N0 P complex from purifiedcomponents using an N mutant deleted of its N terminal arm (∼20 aa) anda peptide of P containing the N0 binding site. For vesicular stomatitis virus(VSV), a rhabdovirus, and for Nipah virus (NiV), a paramyxovirus, wecharacterized the structure of the N terminal region of P alone in solutionand we solved the crystal structure of the reconstructed N0 P complex. Thiswork demonstrates that the N terminal region of P is globally disorderedbut contains transiently populated a helices, and that it folds upon bindingto the N0. The structure of the reconstituted N0 P complexes revealedhow P prevents N0 from polymerizing and thereby from binding RNA.These structures also suggest mechanisms by which P stimulates initiationof RNA synthesis and controls encapsidation of the RNA genome. Thestructure of the NiV complex provides the first glimpse at the structure ofthe nucleoprotein of a Paramyxovirinae.REF O117Role of host factors in enterovirus RNA replicationFrank VAN KUPPEVELD, Hilde VAN TONGEREN, CristinaDOROBANTU, Lonneke VAN DER LINDEN, Lucian ALBULESCU,Jeroen STRATINGDivision of Virology, Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDSEnteroviruses (poliovirus, coxsackievirus, rhinovirus, enterovirus 71)extensively modify host cell membranes to create a scaffold for genomicRNA replication. As yet little is about the structure and biogenesis ofthese viral replication organelles and the identity of the host factors thatare involved in their formation. Previously, we showed that the enterovirus3A protein of enteroviruses recruits GBF1, a GEF of Arf1, to membranesand demonstrated a critical role of this protein in RNA replication(Wessels et al, 2006, Dev Cell). Recently, Golgi localized PI4KIII, whichis an effector of Arf1, was also recognized to be recruited to membranes by3A and to be important for viral RNA replication (Hsu et al., 2012, Cell).Here, evidence will be presented that PI4KIII is recruited to membranesVirologie, Vol 17, supplément 2, septembre 2013S89