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Antibody inhibition of the RNA polymerase of a rotavirus: A cryoelectron microscopy and X-ray crystallography study
281
Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-illkirch, France, l-3 July 1998
Infection of a lymphocytic cell line with hepatitis C vlrus. Ultrastructural features of viral morphogenesis and cytopathic effect. Comparison with yellow fever and dengue virus
Antibody inhibition of the RNA polymerase of a rotavirus: A cryoelectron microscopy and X-ray crystallography study
Anne-MarieSteff an, PhilippeMarianneau‘, Vincent Deubel , Catherine Caussin, Cathy Royer, Carine Schmitt, Jean Louis Gendrault, Andre Kirn, Francoise Stall-Keller
Pothier4 et Jean Cohen!f
l
Laboratoire de Virologie de la Facult=.+ de MBdecine, INSERM U74, 3 Rue Koeberk?, 67000 Strasbourg. ‘Unit6des Arbovirus et Virus des Fi.+vres Wnorragiques, lnstitut Pasteur, 25 rue du Dr ROUX, 75724 Paris Cedex 15 Hepatitis C virus (HCV) represents one of the major causes of acute and chmnic hepatitis, cirrhosis, and hcpatoccllular carcinoma around the world. HCV is an RNA virus of the flavivirus family, which includes other human pathogens such as dengue fcvcr and yellow fever. Compared with thcsc two genera. considcrdhly less information is available on the snucturc and the replication of this virus. hi this work we infcctcd Daudi cells, a lymphocytic cell line with scra fmm HCV-positive patients (la and 3d). for different times and looked for HCV gcnomic RNA by RTPCR and in situ hyhridizalion.. Genomic RNA was dclectahlc in the cells for 7 or 19 days. In silu hybridization cxpcrhncms showed an increase of the hllcnshy 01 labclhlg whh the time of infcclion, some cells intcnsivcly lahelcd wcm cnlargcd and vacuolatcd The ultraslructural study of cells 48 hours after infcclion, rcvcaled pictures ol’viral motphogencsis rcscmbling thal of yellow lever virus in a hcpatoma cell: a) HCV particles with a diamctcr of 45 nm, a spherical clcclmndcnsc nuclcocapsid of about 30 nm surrounded by a lipid bilaycr l’ormcd a cristalloid in the cytoplasm of a ccl1 displaying already lypical signs of the early stage of apoptosis, the marginalion of chmmatin to the nuclear mcmbranc, h) numemus mcmbranc-bound organ&s in particular the ER and vacuoles. c) prolifcralion of’ mcmbrancs, d) intracytoplasmic electron-dcnsc arcas correspnding to nuclcocapsids. At lhis time allcr infcaion, some cells displayed a picnolic nucleus, a highly vacuolated cyloplasm and disrupted cytoplasmic organelles inside which panicI= resembling virions wcrc detected. Four days after infeclion, about 6% of the cells displayed striking cytopathic fcaturcs, haloaning with cxlremely enlarged vacuoles. and signs of apoptosis. Aggregates of parliclcs resembling virions were often found inside vacuoles. This cytopathic effect obtained with both vhus genera, which rcscmblcd that induced by dcnguc virus in hcpatoma cells as early as 24 and 48 hours after infection. was ncvcr obscrvcd in uninfected cultures or in infected cultures 56 days after infection when no more HCV RNA was dctcctcd. Our results suggest that HCV vhus replication cycle displays features characlerlsdc of flaviviruses.
Eric Thouveninl,Elizabeth Hewatl, Gu Schoehn2, Felix Rey3, lsabelle Petitpas 3,4, Ma alie Mathleu Evelyne Kohli4, Pierre
T
1 LMES IBS, 41 Avenue des Martyrs, 38027 GRENOBLECedex 1, France ; 2 Birbeck College, London. UK ; 3 LEES, Gif sur Yvette, France ; 4 Laboratoire de Virologie, LIdon, France ; 5 VffQlNRA, Jouy en Jossas, France. The Rotaviruses, members of the Rotaviridae acute gastro-enteritis in children. These viruses million people each year, especially in developing
family, are responsible cause the death of over countries.
They consist of a triple layered icosahedral protein capsid which encloses 11 double strand RNA molecules. On entering the host cell, the virus loses its outer shell (proteins VP4 and VP7) to become a double layered particle (DLP) composed of a core (proteins VP1 VP2 VP3 and the genome), a shell (protein VP6). (Estes M.K. (1996) Fields Virology, 1625. 1655). The virus as well as the DLP possesses its own RNA polymerase. The transcription occurs inside the DLP and the nascent mRNA emerges along the five fold axes (Lawton J.A., Estes M.K., Venkataram Prasad B.V. (1997) Nat Sfruct Biol. 4(Z), 118-121). It has been shown that the trimeric protein VP6 is associated with the polymerase activity. A monoclonal antibody (Mab 238) directed against VP6 inhibits the transcription. (Kohli E., Pothier I’., Tosser G., Cohen J.,Sandino A.M., Spencer E. (1994). Arch. Vim/. 135, 193-200). By combing the stmcture obtained by cryoelectron microscopy of the DLP/MAb 238 complex with that obtained by X-ray crystallography of the VP6/DLP complex, we hope to understand the mechanism by which the polymerase is inhibited. DLP complexed to another monoclonal antibody Mab 133, that is also directed against VP6 but does not inhibit transcription, has also been studied by cryoelectron microscopy. The comparison of both structures DLP/MAb 238 and DLP/MAb 133 shows that the inactivity is not due to a simple covering of the 5 fold axes by MAb 238.
Cyclin A localisation in tobacco BY2 cells during the cell cycle
Visualization of protein trafficking to different vacuoles in plant cells
BrunoCombettes,FabriceSchmit,MartineFlenet andNicoleChaubet
Gian-Pietro
lnstitut de Biologic Mol&ulaire des Plantes du CNRS, 12, rue du G&&al Zimmer, 67084 Strasbourg, France
Laboratoire de Biochimie, lnstitut de Botanique, Universitb de NeuchStel, rue EmileArgand 9, CH2007 Neuchltel, Switzerland
Protein kinases play a central role in the progression of the cell cycle and their activity depends on the association between the catalytic moiety, a cyclin-dependent kinase (CDK), and the regulatory subunit, a cyclin. Otherwise, histone gene expression is cell cycledependent and occurs at the early S-phase. We showed that phosphorylation steps are involved in histone gene induction, probably through a cyclin A/CDK complex specific of the Gl/S transition. Three different cyclins A have been characterised in tobacco BY2 cells (Reichheld, JI’. et al, (1996). Proc. Nat!. Acad. Sci. USA, 93: 13819-13824) and antibodies were obtained by peptide injections. Surprisingly, immunowestern analysis showed a constant level of tobacco cyclins A throughout the cell cycle, whereas animal cyclins A are degraded in mitosis. In order to understand this phenomenon, we immunolocalized cyclins A in BY2 cells at different phases of the cell cycle. By fluorescent microscope observations, a labelling was observed into the nucleus at the S-phase and in cytoplasmic vesicles throughout the cell cycle. By electronic microscope observations, this vesicular label proved to be located in the amyloplasts. This gives an indication that plant cyclins A might be stored in this cellular compartment. Whether this localisation corresponds to an active form of these proteins remains to be determined by kinase activity tests.
for one
Di Sansebastiano,
Nadine Paris , Sophie Marc-Martin
andJean-Marc Neuhaus
In order to visualize protein transport lo the vacuole in living cells, WC adapted a secretory varianl of GFP. mGFPS-ER (Hascloff J. ct al. (1997) Prc~c.Nurf.Acnr[.Sci.USA 94, 2122-2127), replacing its ER retcnlion signal by the C-tcnninnl vacuolar sorting signal (VSS) of tobacco chitinasc A, a mcmbcr of one of lwo well characterized types of VS.% (NcuhauS J.-M. and Rogers J.(1998) Plant Mol. Viol. in press). The resultant SGFPST was transicmly expressed in tobacco mcsophyll protoplasts. Accumulation in vacuoles was observed as expected hut two different paltems were observed after pmlongcd incubation. A large lluoresccnt vacuole was obscrvcd in most transfonncd chlomplast-rich protoplasls, while most transformed chlomplast-poor protoplasts possessed a single small lluorcscenl vacuole ncxl IO their unstained large vacuole. The dil’lcmnt pH of these diffcrcnt vacuoles was visualized by the differential accumulation of Neutral Red, a dye trapped in its prmonatcd form at low pH. The vacuoles accumulaling GFP did noI stain whh Neutral Red and reciprocally. The C-terminal VSS-dcpcndcm soning syslem lhus largcts SGFPST to a neutral vacuolar companmcnt Prcvenling the acidilication of vacuoles by Lrcatment with ammonium chloride or moncnsin did not change the accumulation pattern of GFP, hldicaling thal the spccilic sorling dots no1 dcpcnd on the pH of the companmcnt. On the other hand. wonmannin, which spccilically affects vacuolar targcling mcdialcd by a C-lcrminal VSS prcvcntcd vacuolar accumulation of SGFPST as expcctcd. These results confirmed the shnultaneous exislence in some plant cells of two diffcrcnt vacuolar compartmcnls. a slorage and a lytic vacuolar compartments, charactcrizcd by different complements of proteins (Paris N. ct al. (1996) Cell 85, 563-572). Preliminary results with another GFP, targeted IO the lytic compaflment by a sequence-specific VSS (Ihc other well characterized type of VSS). indicrlcd a dilfcrcnt and novel pattern of accumulation in tobacco prnloplasts. AI early times of expression, the SCFPST could bc seen to accumulate in a paltern similar to the accumulation of the ER-rctaincd isofonn. The same pattcm was also ohscrvcd wilh the GFP targeted to the other vacuolar companmcnt as well as with a secreted GFP which later disappcarcd into the medium. It is thus possible now lo visualize some steps of the transport through the sccrctory pathway in living plam cells.
Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-illkirch, France, l-3 July 1998
Infection of a lymphocytic cell line with hepatitis C vlrus. Ultrastructural features of viral morphogenesis and cytopathic effect. Comparison with yellow fever and dengue virus
Antibody inhibition of the RNA polymerase of a rotavirus: A cryoelectron microscopy and X-ray crystallography study
Anne-MarieSteff an, PhilippeMarianneau‘, Vincent Deubel , Catherine Caussin, Cathy Royer, Carine Schmitt, Jean Louis Gendrault, Andre Kirn, Francoise Stall-Keller
Pothier4 et Jean Cohen!f
l
Laboratoire de Virologie de la Facult=.+ de MBdecine, INSERM U74, 3 Rue Koeberk?, 67000 Strasbourg. ‘Unit6des Arbovirus et Virus des Fi.+vres Wnorragiques, lnstitut Pasteur, 25 rue du Dr ROUX, 75724 Paris Cedex 15 Hepatitis C virus (HCV) represents one of the major causes of acute and chmnic hepatitis, cirrhosis, and hcpatoccllular carcinoma around the world. HCV is an RNA virus of the flavivirus family, which includes other human pathogens such as dengue fcvcr and yellow fever. Compared with thcsc two genera. considcrdhly less information is available on the snucturc and the replication of this virus. hi this work we infcctcd Daudi cells, a lymphocytic cell line with scra fmm HCV-positive patients (la and 3d). for different times and looked for HCV gcnomic RNA by RTPCR and in situ hyhridizalion.. Genomic RNA was dclectahlc in the cells for 7 or 19 days. In silu hybridization cxpcrhncms showed an increase of the hllcnshy 01 labclhlg whh the time of infcclion, some cells intcnsivcly lahelcd wcm cnlargcd and vacuolatcd The ultraslructural study of cells 48 hours after infcclion, rcvcaled pictures ol’viral motphogencsis rcscmbling thal of yellow lever virus in a hcpatoma cell: a) HCV particles with a diamctcr of 45 nm, a spherical clcclmndcnsc nuclcocapsid of about 30 nm surrounded by a lipid bilaycr l’ormcd a cristalloid in the cytoplasm of a ccl1 displaying already lypical signs of the early stage of apoptosis, the marginalion of chmmatin to the nuclear mcmbranc, h) numemus mcmbranc-bound organ&s in particular the ER and vacuoles. c) prolifcralion of’ mcmbrancs, d) intracytoplasmic electron-dcnsc arcas correspnding to nuclcocapsids. At lhis time allcr infcaion, some cells displayed a picnolic nucleus, a highly vacuolated cyloplasm and disrupted cytoplasmic organelles inside which panicI= resembling virions wcrc detected. Four days after infeclion, about 6% of the cells displayed striking cytopathic fcaturcs, haloaning with cxlremely enlarged vacuoles. and signs of apoptosis. Aggregates of parliclcs resembling virions were often found inside vacuoles. This cytopathic effect obtained with both vhus genera, which rcscmblcd that induced by dcnguc virus in hcpatoma cells as early as 24 and 48 hours after infection. was ncvcr obscrvcd in uninfected cultures or in infected cultures 56 days after infection when no more HCV RNA was dctcctcd. Our results suggest that HCV vhus replication cycle displays features characlerlsdc of flaviviruses.
Eric Thouveninl,Elizabeth Hewatl, Gu Schoehn2, Felix Rey3, lsabelle Petitpas 3,4, Ma alie Mathleu Evelyne Kohli4, Pierre
T
1 LMES IBS, 41 Avenue des Martyrs, 38027 GRENOBLECedex 1, France ; 2 Birbeck College, London. UK ; 3 LEES, Gif sur Yvette, France ; 4 Laboratoire de Virologie, LIdon, France ; 5 VffQlNRA, Jouy en Jossas, France. The Rotaviruses, members of the Rotaviridae acute gastro-enteritis in children. These viruses million people each year, especially in developing
family, are responsible cause the death of over countries.
They consist of a triple layered icosahedral protein capsid which encloses 11 double strand RNA molecules. On entering the host cell, the virus loses its outer shell (proteins VP4 and VP7) to become a double layered particle (DLP) composed of a core (proteins VP1 VP2 VP3 and the genome), a shell (protein VP6). (Estes M.K. (1996) Fields Virology, 1625. 1655). The virus as well as the DLP possesses its own RNA polymerase. The transcription occurs inside the DLP and the nascent mRNA emerges along the five fold axes (Lawton J.A., Estes M.K., Venkataram Prasad B.V. (1997) Nat Sfruct Biol. 4(Z), 118-121). It has been shown that the trimeric protein VP6 is associated with the polymerase activity. A monoclonal antibody (Mab 238) directed against VP6 inhibits the transcription. (Kohli E., Pothier I’., Tosser G., Cohen J.,Sandino A.M., Spencer E. (1994). Arch. Vim/. 135, 193-200). By combing the stmcture obtained by cryoelectron microscopy of the DLP/MAb 238 complex with that obtained by X-ray crystallography of the VP6/DLP complex, we hope to understand the mechanism by which the polymerase is inhibited. DLP complexed to another monoclonal antibody Mab 133, that is also directed against VP6 but does not inhibit transcription, has also been studied by cryoelectron microscopy. The comparison of both structures DLP/MAb 238 and DLP/MAb 133 shows that the inactivity is not due to a simple covering of the 5 fold axes by MAb 238.
Cyclin A localisation in tobacco BY2 cells during the cell cycle
Visualization of protein trafficking to different vacuoles in plant cells
BrunoCombettes,FabriceSchmit,MartineFlenet andNicoleChaubet
Gian-Pietro
lnstitut de Biologic Mol&ulaire des Plantes du CNRS, 12, rue du G&&al Zimmer, 67084 Strasbourg, France
Laboratoire de Biochimie, lnstitut de Botanique, Universitb de NeuchStel, rue EmileArgand 9, CH2007 Neuchltel, Switzerland
Protein kinases play a central role in the progression of the cell cycle and their activity depends on the association between the catalytic moiety, a cyclin-dependent kinase (CDK), and the regulatory subunit, a cyclin. Otherwise, histone gene expression is cell cycledependent and occurs at the early S-phase. We showed that phosphorylation steps are involved in histone gene induction, probably through a cyclin A/CDK complex specific of the Gl/S transition. Three different cyclins A have been characterised in tobacco BY2 cells (Reichheld, JI’. et al, (1996). Proc. Nat!. Acad. Sci. USA, 93: 13819-13824) and antibodies were obtained by peptide injections. Surprisingly, immunowestern analysis showed a constant level of tobacco cyclins A throughout the cell cycle, whereas animal cyclins A are degraded in mitosis. In order to understand this phenomenon, we immunolocalized cyclins A in BY2 cells at different phases of the cell cycle. By fluorescent microscope observations, a labelling was observed into the nucleus at the S-phase and in cytoplasmic vesicles throughout the cell cycle. By electronic microscope observations, this vesicular label proved to be located in the amyloplasts. This gives an indication that plant cyclins A might be stored in this cellular compartment. Whether this localisation corresponds to an active form of these proteins remains to be determined by kinase activity tests.
for one
Di Sansebastiano,
Nadine Paris , Sophie Marc-Martin
andJean-Marc Neuhaus
In order to visualize protein transport lo the vacuole in living cells, WC adapted a secretory varianl of GFP. mGFPS-ER (Hascloff J. ct al. (1997) Prc~c.Nurf.Acnr[.Sci.USA 94, 2122-2127), replacing its ER retcnlion signal by the C-tcnninnl vacuolar sorting signal (VSS) of tobacco chitinasc A, a mcmbcr of one of lwo well characterized types of VS.% (NcuhauS J.-M. and Rogers J.(1998) Plant Mol. Viol. in press). The resultant SGFPST was transicmly expressed in tobacco mcsophyll protoplasts. Accumulation in vacuoles was observed as expected hut two different paltems were observed after pmlongcd incubation. A large lluoresccnt vacuole was obscrvcd in most transfonncd chlomplast-rich protoplasls, while most transformed chlomplast-poor protoplasts possessed a single small lluorcscenl vacuole ncxl IO their unstained large vacuole. The dil’lcmnt pH of these diffcrcnt vacuoles was visualized by the differential accumulation of Neutral Red, a dye trapped in its prmonatcd form at low pH. The vacuoles accumulaling GFP did noI stain whh Neutral Red and reciprocally. The C-terminal VSS-dcpcndcm soning syslem lhus largcts SGFPST to a neutral vacuolar companmcnt Prcvenling the acidilication of vacuoles by Lrcatment with ammonium chloride or moncnsin did not change the accumulation pattern of GFP, hldicaling thal the spccilic sorling dots no1 dcpcnd on the pH of the companmcnt. On the other hand. wonmannin, which spccilically affects vacuolar targcling mcdialcd by a C-lcrminal VSS prcvcntcd vacuolar accumulation of SGFPST as expcctcd. These results confirmed the shnultaneous exislence in some plant cells of two diffcrcnt vacuolar compartmcnls. a slorage and a lytic vacuolar compartments, charactcrizcd by different complements of proteins (Paris N. ct al. (1996) Cell 85, 563-572). Preliminary results with another GFP, targeted IO the lytic compaflment by a sequence-specific VSS (Ihc other well characterized type of VSS). indicrlcd a dilfcrcnt and novel pattern of accumulation in tobacco prnloplasts. AI early times of expression, the SCFPST could bc seen to accumulate in a paltern similar to the accumulation of the ER-rctaincd isofonn. The same pattcm was also ohscrvcd wilh the GFP targeted to the other vacuolar companmcnt as well as with a secreted GFP which later disappcarcd into the medium. It is thus possible now lo visualize some steps of the transport through the sccrctory pathway in living plam cells.