Laboratoire de Microbiologie Fondamentale et Pathogénicité

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Intracellular dynamic of subviral structures.

 

 

Responsible :

Harald WODRICH.

Team members :

 

Research project :

Our group is working on the intracytoplasmic and the nuclear transport of different viral components that are involved in nuclear delivery of viral genomes. The main focus is the human hepatitis B virus as this virus exhibits an extreme efficiency. Being at the interphase between virology and cell biology our research targets the molecular interactions with cellular partners. The obtained data will result in a better understanding of basic cell biology and may help developing efficient vectors for gene therapy as well as identifying essential targets for antiviral therapy.

 

Introduction :

Fig1

 

For replication viruses have to transport their genome to the place where multiplication of the genomic information occurs. Viruses that make use of the nuclear replication processes and infect non-dividing cells have to deliver their genome through cytoplasm towards the nuclear envelope followed by entry into the karyoplasm by passing the nuclear pore complex (NPC) (for overview see figure 1). As nucleic acids are not karyophilic per se attached viral proteins have to mediate distinct and well coordinated interactions with the different cellular transport machineries. Evidently the location of the virus (or its subviral structure) has to be related with genome release in order to prevent premature abortion of the viral life cycle.

 

 

 

 

 

 

 

 

 

 

 

Hepatitis B virus :

Amongst the depicted viruses human hepatitis B virus (HBV) is particular (figure 2). HBV is an enveloped pararetrovirus that multiplies via RNA synthesis. It enters the cell by a receptor(s) that have not been unequivocally identified yet. Independent upon acidification it releases the viral capsid into the cytoplasm. Subsequently the capsid surrounding the partially double stranded viral DNA is transported to the nucleus in which the genome becomes released.Fig2Cellular enzymes convert the viral genome to the covalently closed circular DNA (cccDNA) that is the template for mRNA synthesis. One mRNA of supergenomic length, the pregenomic RNA (PG) is bicistronic and encodes for the capsid protein and the viral polymerase (pol).Pol binds speci-fically to PG thereby facilitatin - ing encapsidation into the assembling capsid. Being only active in the capsid environment Pol converts the encapsidated RNA into the partially double stranded viral DNA. In contrast to all other viruses (exception: caulimoviruses of plants) the HBV capsids derived from cellular entry are identical to the mature progeny capsids.Hence not only incoming capsids but also the progeny ones are targeted to the nucleus (fig.1, red arrow) leading to amplification of the nuclear cccDNA. Only after sufficient amounts of surface proteins are synthesized the mature capsids become enveloped by the surface proteins leading to secretion of progeny HBV. becomes released. Cellular enzymes convert the viral genome to the covalently closed circular DNA (cccDNA) that is the template for mRNA synthesis. One mRNA of supergenomic length, the pregenomic RNA (PG) is bicistronic and encodes for the capsid protein and the viral polymerase (pol).Pol binds speci-fically to PG thereby facilitatin - ing encapsidation into the assembling capsid. Being only active in the capsid environment Pol converts the encapsidated RNA into the partially double stranded viral DNA. In contrast to all other viruses (exception: caulimoviruses of plants) the HBV capsids derived from cellular entry are identical to the mature progeny capsids.Hence not only incoming capsids but also the progeny ones are targeted to the nucleus (fig.1, red arrow) leading to amplification of the nuclear cccDNA. Only after sufficient amounts of surface proteins are synthesized the mature capsids become enveloped by the surface proteins leading to secretion of progeny HBV.

 

Our lab interests are focused on the intracytoplasmic and nuclear transport of the HBV by three reasons: (i) HBV is a major human pathogen causing one million deaths per year (ii) HBV is extremely efficient; practically all viruses represent an infectious unit. Learning the molecular interactions causing this performance can help to develop efficient vectors in gene therapy. (iii) The analysis of the cellular interaction partners will result in a better understanding of basic transport-related cell biology.

 

Intracytoplasmic transport. The main obstacle in HBV research is the lack of a commonly available cell line that is susceptible. Even primary human hepatocytes loose their susceptibility a few days after taken in culture.kann3 How ever after transfection of viral DNA hepatoma cell lines produce in vivo-likeamounts of virus indicating restrictions in viral entry. We there-fore replaced the viral envelope by lipids allowing the analysis of HBV travel towards the nucleus. Figure 3 depicts the time course of capsid transport and genome release. This system allowed determining the intracytoplasmic transport system and participating motor complexes. Detailed analysis shall identify the domains on capsid and motor proteins.

 

 

 

 

 

 

 

 

 

 

Nuclear transport. As depicted, the capsids release the genome exclusively into the nucleus implying a tight regulation of capsid disintegration. We analyzed the interactions at the nucleus in more detail using Digitonin-permeabilized cells. Digitonin permeabilizes the plasma membrane leaving the nuclear membrane integer.

We showed that the capsids interact with the NPC via the cellular nuclear import receptors importin a and ß. However RNA-containing capsids did not interact with the NPC (figure 4A), capsid with an immature DNA genome interact with the NPC without becoming imported (figure 4B) into the karyoplasm, while mature capsids containing the partially double stranded DNA caused nuclear capsid stain (figure 4C) kann4_5and released genomes. Consistently only those capsids capable to interact with the nucleus exposed a nuclear localization signal (NLS) on their surface, while RNA-containing capsids hide the NLS in their interior. NLS are stretches of basic amino acids that interact with importin a. Nuclear transport mechanisms are phylogenetically well conserved. Consequently electron microscopy after microinjection of capsids into the cytoplasm of Xenopus laevis oocytes confirmed the interaction with the NPCs (Figure 5; collaboration with Nelly Panté, UBC, Canada). Further immature and mature capsids were depicted to enter the nuclear basket integer leading to a redefinition of the maximal nuclear pore size to be 39 nm (Panté & Kann, MBC, 2002). The basket is a cage-like structure on the karyoplasmic side of the NPC where cargo and import receptors dissociate, allowing that the cargo (in this case the capsid) diffuse deeper into the karyoplasm. So why do immature capsids arrest in the basket? And why do mature capsids enter the karyoplasm? Cross-linked mature capsids, unable to dissociate, behave like immature ones. They become arrested, implying that both capsid types interact with a protein of the nuclear basket. In collaboration with my lab at Giessen University, Germany, this protein could be identified to be nucleoporin 153 (Nup153), which is essential for cell viability.

 

Having these assays allows us (i) to evaluate Nup153 on import and export processes, (ii) to identify its function in capsid disassembly and (iii) evaluate the further fate of the genome.

 

 

 

 

 

 

 

HIV integrase and paroviruses

The studies are complemented by studying other viruses or subviral structures. In order to learn more about cargo-motor protein interactions we are currently evaluating the intracytoplasmic transport of the HIV integrase (HIV IN) in collaboration with Michel Fournier and Sébastien Desfarges in our department. HIV IN is not only of interest as it is an essential protein of the HIV preintegration complex but also as its small size allows a rapid identification of interaction domains.

Further nuclear interactions are investigated using parvoviruses. Like HBV capsids parvoviruses replicate in the nucleus, are small enough to pass the nuclear pore and contain a potential NLS hidden on one capsid protein. Nonetheless the nuclear entry of the parvoviral genome is controversial. Our analyses showed that despite of the potential NLS different parvoviruses cause local nuclear envelope break-down (NEBD). Physiologically NEBD occurs in mitosis, meiosis and apoptosis and our cell-free assays may help to unravel the signalling and nuclear degradation in these essential processes.

 

Important message

Important message for students and scientists who would like to perform training in our group: if your working program requires experiments with intact adeno-, parvo- and hepatitis B viruses or analysis by time lapse microscopy you must be successfully vaccinated against hepatitis B. A certificate on vaccination will be asked the first day of your training. When you are not vaccinated or if you do not know your vaccination titre (anti HBs antibodies) please consider that the vaccination takes some time and that you should contact your general practitioner at least 2 month ahead.

Publications :

Rayne F, Wittkop L, Bader C, Kassab S, Tumiotto C, Berciaud S, Wodrich H, Lafon ME. Rapid Adenovirus typing method for species identification.J Virol Methods. 2017 Nov;249:156-160

Montespan C, Wiethoff CM, Wodrich H. A Small Viral PPxY Peptide Motif To Control Antiviral Autophagy.J Virol. 2017 Sep 15;91(18)

Saltel F, Giese A, Azzi L, Elatmani H, Costet P, Ezzoukhry Z, Dugot-Senant N, Miquerol L, Boussadia O, Wodrich H, Dubus P, Jacquemin-Sablon H. Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation.J Cell Sci. 2017 May 15;130(10):1796-1808

Montespan C, Marvin SA, Austin S, Burrage AM, Roger B, Rayne F, Faure M, Campell EM, Schneider C, Reimer R, Grünewald K, Wiethoff CM, Wodrich H. Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.PLoS Pathog. 2017 Feb;13(2):e1006217

Gallucci L, Kann M. Nuclear Import of Hepatitis B Virus Capsids and Genome.Viruses. 2017 Jan 21;9(1)

Osseman Q, Kann M. Intracytoplasmic Transport of Hepatitis B Virus Capsids.Methods Mol Biol. 2017;1540:37-51

Quemener C, Baud J, Boyé K, Dubrac A, Billottet C, Soulet F, Darlot F, Dumartin L, Sire M, Grepin R, Daubon T, Rayne F, Wodrich H, Couvelard A, Pineau R, Schilling M, Castronovo V, Sue SC, Clarke K, Lomri A, Khatib AM, Hagedorn M, Prats H, Bikfalvi A. Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.Cancer Res. 2016 Nov 15;76(22):6507-6519

Austin S, Taouji S, Chevet E, Wodrich H, Rayne F. Using AlphaScreen(®) to Identify Small-Molecule Inhibitors Targeting a Conserved Host-Pathogen Interaction.Methods Mol Biol. 2016;1449:453-67

Chen C, Wang JC, Pierson EE, Keifer DZ, Delaleau M, Gallucci L, Cazenave C, Kann M, Jarrold MF, Zlotnick A. Importin β Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.PLoS Pathog. 2016 Aug;12(8):e1005802

Komatsu T, Robinson DR, Hisaoka M, Ueshima S, Okuwaki M, Nagata K, Wodrich H. Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments.Traffic. 2016 Nov;17(11):1168-1180

Alarcon V, Hernández S, Rubio L, Alvarez F, Flores Y, Varas-Godoy M, De Ferrari GV, Kann M, Villanueva RA, Loyola A. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.Sci Rep. 2016 May 13;6:25901

Blondot ML, Bruss V, Kann M. Intracellular transport and egress of hepatitis B virus.J Hepatol. 2016 Apr;64(1 Suppl):S49-S59

Komatsu T, Will H, Nagata K, Wodrich H. Imaging analysis of nuclear antiviral factors through direct detection of incoming adenovirus genome complexes.Biochem Biophys Res Commun. 2016 Apr 22;473(1):200-205

Boyer A, Couallier V, Clouzeau B, Lasheras A, M'zali F, Kann M, Rogues AM, Gruson D. Control of extended-spectrum β-lactamase-producing Enterobacteriaceae nosocomial acquisition in an intensive care unit: A time series regression analysis.Am J Infect Control. 2015 Dec 1;43(12):1296-301

Komatsu T, Dacheux D, Kreppel F, Nagata K, Wodrich H. A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells.PLoS One. 2015;10(9):e0137102

Gil-Ranedo J, Hernando E, Riolobos L, Domínguez C, Kann M, Almendral JM. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly.PLoS Pathog. 2015 Jun;11(6):e1004920

Zeisel MB, Lucifora J, Mason WS, Sureau C, Beck J, Levrero M, Kann M, Knolle PA, Benkirane M, Durantel D, Michel ML, Autran B, Cosset FL, Strick-Marchand H, Trépo C, Kao JH, Carrat F, Lacombe K, Schinazi RF, Barré-Sinoussi F, Delfraissy JF, Zoulim F. Towards an HBV cure: state-of-the-art and unresolved questions--report of the ANRS workshop on HBV cure.Gut. 2015 Aug;64(8):1314-26

Martinez R, Schellenberger P, Vasishtan D, Aknin C, Austin S, Dacheux D, Rayne F, Siebert A, Ruzsics Z, Gruenewald K, Wodrich H. The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.J Virol. 2015 Feb;89(4):2121-35

Cassany A, Ragues J, Guan T, Bégu D, Wodrich H, Kann M, Nemerow GR, Gerace L. Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214.J Virol. 2015 Feb;89(3):1719-30

Deroubaix A, Osseman Q, Cassany A, Bégu D, Ragues J, Kassab S, Lainé S, Kann M. Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus.J Gen Virol. 2015 Jan;96(Pt 1):183-95

Couzi L, Pitard V, Sicard X, Garrigue I, Hawchar O, Merville P, Moreau JF, Déchanet-Merville J. Antibody-dependent anti-cytomegalovirus activity of human γδ T cells expressing CD16 (FcγRIIIa).Blood. 2012 Feb 9;119(6):1418-27

Couzi L, Helou S, Bachelet T, Moreau K, Martin S, Morel D, Lafon ME, Boyer B, Alain S, Garrigue I, Merville P. High incidence of anticytomegalovirus drug resistance among D+R- kidney transplant recipients receiving preemptive therapy.Am J Transplant. 2012 Jan;12(1):202-9

Debaisieux S, Rayne F, Yezid H, Beaumelle B. The ins and outs of HIV-1 Tat.Traffic. 2012 Mar;13(3):355-63

Gasnault J, Costagliola D, Hendel-Chavez H, Dulioust A, Pakianather S, Mazet AA, de Goer de Herve MG, Lancar R, Lascaux AS, Porte L, Delfraissy JF, Taoufik Y. Improved survival of HIV-1-infected patients with progressive multifocal leukoencephalopathy receiving early 5-drug combination antiretroviral therapy.PLoS One. 2011;6(6):e20967

Schmitz A, Schwarz A, Foss M, Zhou L, Rabe B, Hoellenriegel J, Stoeber M, Panté N, Kann M. Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.PLoS Pathog. 2010 Jan 29;6(1):e1000741

Riolobos L, Valle N, Hernando E, Maroto B, Kann M, Almendral JM. Viral oncolysis that targets Raf-1 signaling control of nuclear transport.J Virol. 2010 Feb;84(4):2090-9

Rabe B, Delaleau M, Bischof A, Foss M, Sominskaya I, Pumpens P, Cazenave C, Castroviejo M, Kann M. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids.PLoS Pathog. 2009 Aug;5(8):e1000563

Kantelhardt VC, Schwarz A, Wend U, Schüttler CG, Willems WR, Trimoulet P, Fleury H, Gerlich WH, Kann M. Re-evaluation of anti-HBc non-reactive serum samples from patients with persistent hepatitis B infection by immune precipitation with labelled HBV core antigen.J Clin Virol. 2009 Oct;46(2):124-8

Seiradake E, Henaff D, Wodrich H, Billet O, Perreau M, Hippert C, Mennechet F, Schoehn G, Lortat-Jacob H, Dreja H, Ibanes S, Kalatzis V, Wang JP, Finberg RW, Cusack S, Kremer EJ. The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.PLoS Pathog. 2009 Jan;5(1):e1000277

Lang M, Kann M, Zahner H, Taubert A, Hermosilla C. Inhibition of host cell apoptosis by Eimeria bovis sporozoites.Vet Parasitol. 2009 Mar 9;160(1-2):25-33

Cassany A, Gerace L. Reconstitution of nuclear import in permeabilized cells.Methods Mol Biol. 2009;464:181-205

Smith JG, Cassany A, Gerace L, Ralston R, Nemerow GR. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.J Virol. 2008 Jul;82(13):6492-500

Bellecave P, Cazenave C, Rumi J, Staedel C, Cosnefroy O, Andreola ML, Ventura M, Tarrago-Litvak L, Astier-Gin T. Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells.Antimicrob Agents Chemother. 2008 Jun;52(6):2097-110

Lainé S, Thouard A, Komar AA, Rossignol JM. Ribosome can resume the translation in both +1 or -1 frames after encountering an AGA cluster in Escherichia coli.Gene. 2008 Apr 15;412(1-2):95-101

Kann M, Schmitz A, Rabe B. Intracellular transport of hepatitis B virus.World J Gastroenterol. 2007 Jan 7;13(1):39-47

Damianov A, Kann M, Lane WS, Bindereif A. Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs.Biol Chem. 2006 Oct-Nov;387(10-11):1455-60

Wodrich H, Cassany A, D'Angelo MA, Guan T, Nemerow G, Gerace L. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.J Virol. 2006 Oct;80(19):9608-18

Cazenave C, Bathany K, Rayner B. Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis.Oligonucleotides. 2006 Summer;16(2):181-5

Bannwarth S, Lainé S, Daher A, Grandvaux N, Clerzius G, Leblanc AC, Hiscott J, Gatignol A. Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes.J Mol Biol. 2006 Feb 3;355(5):898-910

 

 

 

 

 

 

 

 

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