J OURNAL OF V IROLOGY,Aug.2009,p.7948–7958Vol.83,No.16 0022-538X/09/$08.00?0doi:10.1128/JVI.00554-09
Copyright?2009,American Society for Microbiology.All Rights Reserved.
Functional Characterization of Negri Bodies(NBs)in Rabies
Virus-Infected Cells:Evidence that NBs Are Sites of Viral
Transcription and Replication?
Xavier Lahaye,1Aurore Vidy,1?Carole Pomier,1Linda Obiang,1Francis Harper,2
Yves Gaudin,1and Danielle Blondel1*
CNRS,UMR2472,INRA,UMR1157,IFR115,Virologie Mole′culaire et Structurale,91198,Gif sur Yvette,France,1and CCNRS, FRE2937,IFR89,Laboratoire de Replication de l’ADN et Ultrastructure du Noyau,94801Villejuif,France2
Received18March2009/Accepted26May2009
Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies
found in the cytoplasm of some infected nerve cells.We have studied the morphogenesis and the role of these
Negri body-like structures(NBLs)during viral infection.The results indicate that these spherical structures
(one or two per cell in the initial stage of infection),composed of the viral N and P proteins,grow during the
virus cycle before appearing as smaller structures at late stages of infection.We have shown that the
microtubule network is not necessary for the formation of these inclusion bodies but is involved in their
dynamics.In contrast,the actin network does not play any detectable role in these processes.These inclusion
bodies contain Hsp70and ubiquitinylated proteins,but they are not misfolded protein aggregates.NBLs,in
fact,appear to be functional structures involved in the viral life cycle.Speci?cally,using in situ?uorescent
hybridization techniques,we show that all viral RNAs(genome,antigenome,and every mRNA)are located
inside the inclusion bodies.Signi?cantly,short-term RNA labeling in the presence of BrUTP strongly suggests
that the NBLs are the sites where viral transcription and replication take place.
Viral infection can cause extensive cellular rearrangements leading to the formation of cytoplasmic inclusions that concen-trate viral components.These inclusions are often considered as side-products of the infectious process due to the passive accumulation of large quantities of proteins produced in excess during infection.Nevertheless,in some cases,it has been dem-onstrated that these inclusions are in fact viral factories in which essential events of the viral cycle take place(30,38). Indeed,in these compartments,the high concentration of the viral components increases the ef?ciency of many steps of the viral infection(including transcription and/or replication of the genome and assembly of subviral particles)such that they represent“viral factories.”Such factories have been identi?ed for a variety of unrelated viruses,including large DNA viruses (such as Poxviridae,Iridoviridae,and the closely related African swine fever virus),small DNA viruses(such as adenovirus and poliomavirus),double-stranded RNA viruses(such as rotavi-ruses),and positive-stranded RNA viruses(such as togavirus) (39).
Similar inclusions called aggresomes are formed in response to protein misfolding(7,20).Proteins aggregates of misfolded proteins are toxic to cells and are driven along microtubules to aggresomes for immobilization and subsequent degradation by proteasomes.The similarity between aggresomes and viral in-clusions raises the possibility that viruses hijack the aggresome pathway to facilitate their own replication and assembly(14, 39).Alternatively,aggresomes may be part of the innate cel-lular response that recognizes viral components as foreign or misfolded and targets them for storage and degradation. Rabies virus,the prototype of the lyssavirus genus that be-longs to the Rhabdoviridae family(Mononegavirales order), causes a fatal disease associated with intense viral replication in the central nervous system.Its single-stranded negative-sense RNA genome(?12kb),which encodes?ve viral pro-teins,is encapsidated by the nucleoprotein N(50kDa)to form the nucleocapsid that is associated with the RNA-dependent RNA polymerase L(220kDa)and its cofactor the phospho-protein P(33kDa).Inside the viral particle,this nucleocapsid has a tightly coiled helical structure that is associated with the matrix protein M(22kDa)and surrounded by a membrane containing a unique glycoprotein G(62kDa).The virus enters the host cell through the endosomal transport pathway via a low-pH-induced membrane fusion process catalyzed by glyco-protein G(9).The nucleocapsid released into the cytoplasm serves as a template for transcription and replication processes that are catalyzed by the L-P polymerase complex.During transcription,a positive-stranded leader RNA and?ve capped and polyadenylated mRNAs are synthesized.The replication process yields nucleocapsids containing full-length antigenome-sense RNA,which in turn serve as templates for the synthesis of genome-sense RNA.During their synthesis,both the nas-cent antigenome and the genome are encapsidated by N pro-teins.The neosynthesized genome either serves as a template for secondary transcription or is assembled with M proteins to allow budding of the neosynthesized virion at a cellular mem-brane.Some electron microscopy studies suggest that the bud-
*Corresponding author.Mailing address:CNRS,UMR2472,INRA, UMR1157,IFR115,Virologie Mole′culaire et Structurale,91198,Gif
sur Yvette,France.Phone:33169823837.Fax:33169824308.
E-mail:blondel@https://www.doczj.com/doc/f816764745.html,rs-gif.fr.
?Present address:Max von Pettenkofer Institute fu¨r Virologie,Lud-
wig Maximilians University,Pettenkoferstrasse9a,80336Munich, Germany.
?Published ahead of print on3June2009.
7948
ding step occurs at the level of internal cellular compartments (27,28).
Rabies virus infection induces the formation of cytoplasmic inclusion bodies(IBs)that are called Negri bodies(29).Since these structures are typical of rabies infection of the brain,they have a diagnostic value and have been used as de?nite histo-logical proof of such infection.These spherical structures hav-ing a diameter of a few micrometers(2to10?m)are found in the cytoplasm of some infected nerve cells.By electron micros-copy observations,the Negri bodies were found to be com-posed by a matrix of granular or?lamentous material consist-ing of viral nucleoprotein.Viral particles were also seen budding from this structure(21,22,24).Cytological stainings have shown that they contain nucleic acids,suggesting that they may be replication complexes.However,RNA labeling(with [3H]thymidine or[3H]uridine)failed to detect the IBs,arguing against this conclusion(23).Rabies virus RNA was detected by in situ hybridization in human brain,but no colocalization of viral RNA and Negri bodies have been reported(17,18).
In cultured cells from a neuronal or nonneuronal origin,IBs are also formed during viral infection.Based on their size and shape,it has been considered that these inclusions might be Negri body-like structures(NBLs).
In the present study,we have shown that the IBs formed in infected cells do indeed have similar characteristics to those of Negri https://www.doczj.com/doc/f816764745.html,ing this model,we have been able to study the mechanisms involved in the formation of NBL structures in infected cells and to investigate the role of these IBs during viral infection,revealing that they may be factories where viral RNAs synthesis occurs.
MATERIALS AND METHODS
Cells and viruses.Human neuroblastoma SK cells,human glioblastoma as-trocytoma U373-MG cells and BSR cells cloned from BHK21(baby hamster kidney)were grown in Dulbecco modi?ed Eagle medium supplemented with 10%fetal bovine serum.
The CVS strain of rabies virus was grown in BSR cells.Virus titers were determined by standard plaque assays into BSR cells.
Antibodies and drugs.The mouse polyclonal anti-P antibody has been de-scribed previously and the anti-P monoclonal antibodies(MAbs)25C2and30F2 have been previously described(35).The rabbit polyclonal anti-N antibody conjugated to?uorescein isothiocyanate(FITC)was kindly provided by Pierre Perrin(Institute Pasteur);anti-N MAb62B5was produced in mice immunized with the virus.Rabbit polyclonal anti-P antibody and mouse polyclonal anti-M were obtained by repeated injection of puri?ed recombinant protein produced in Escherichia coli.Anti-G MAb30AA5has been previously described(8).Anti-HSp70MAb(SPA-810)and anti-ubiquitin(SPA-203)were obtained from Stressgen.Anti-bromodeoxyuridine(anti-BrdU)MAb(clone B1C9318)from Roche and anti-?tubulin MAb(N356)from Amersham were used.Fluorescent secondary antibodies were purchased from Molecular Probes,and anti-vimentin (V6630)and phalloidin(P1951)from Sigma.MitoTracker Red was obtained from Invitrogen(M7513).Nocodazole(M1404),paclitaxel(T1402),cytochalasin D(catalog no.30385),latrunculin A(L5163),and actinomycin D(ActD;A9415) were obtained from Sigma;jasplakinolide(420107)was purchased from Calbio-chem.
Plasmids.The Plasmids pCDM8-P encoding the P protein(CVS strain)and pCDM8-N encoding the N protein(CVS strain)have been described previously (3).The plasmid pP-RFP has been generated by cloning?rst the P gene in fusion with the red?uorescent protein(RFP)gene in the plasmid pDsRedM(Clontech; gift of David Pasdeloup,MRC Virology Unit,Glasgow,United Kingdom),and then the fusion product P-RFP was inserted into pcDNA-3vector between NheI and XbaI restriction sites.The plasmid pP?143AQ147A-RFP was obtained from the previous construct pCDM8encoding pP?143AQ147A(33)by using the strategy described above.The dynamitin-green?uorescent protein(GFP)-ex-pressing construct(p50-GFP)was a generous gift from Richard Vallee(Colum-bia University,New York,NY)(5).
Cell infection and transfection.Monolayers of U373-MG cells or BSR cells were grown on sterile glass coverslip in six-well plates(from50to80%con?u-FIG.1.Rabies virus RNA species(genome,antigenome,and mRNAs encoding N,P,M,G,and L proteins)and location of sense and antisense primers(indicated by arrows)used in the FISH exper-iments.Abbreviations:Le,leader;Tr,trailer;An,poly(A).
TABLE1.Sequences of the probes used in the FISH experiments a
FISH probe Sequence(5?-3?biotinylated)First nucleotide position b Leader RNA GCTTTGCAATTGACGCTGTCTTTTTCTTCTTTGATCTGGTTG9
N mRNA CACCTGATTATTGACTTTGAACACAATCTTGTCGGCATCCAT71
P mRNA TATTTGCACTCCAACATTATCCAGGTACGATTGGAACAGGAG1784
M1mRNA AGGAGAGGGCTTTTGAGTGTCCTCATCCCTACATTTTTTCAC2517
M2mRNA CATAGTTGACATGCCCTCGAGTTCGAGTTGATACACCAGATC2996
G1mRNA GCATCCTTCGTCCTCCACTACCAAATTGTTTGGGCAGCTGAG3438
G2mRNA GGATCTCATTCCAAGTTCTGACTGACTTGTAGTGAGCATCGG4348
L1mRNA TGGGTCAATAGGGTCATCATAGACCTCTCCAGGATCGAGCAT5417
L2mRNA GTACTGATACAGAAACTCTAGGATTGCCTCCTCCTGTTGCTC8820
L3mRNA CTGTTATACAGGTCGTGAAGTCTTGCGAAGCTAGGGACGTCA11470
N genome ATGGATGCCGACAAGATTGTGTTCAAAGTCAATAATCAGGTG71
P genome CTCCTGTTCCAATCGTACCTGGATAATGTTGGAGTCCAAATA1784 Antigenome GAGAAAAACAATCTCACACCAGAGGTTCGGATTCAAGATCTT11862
Poly-A d(T)x42
M mRNA VSV CCTAATTTCTTAGATTTCTTAACCTTTCCCCTTCAGACCGAG2271 GADPH mRNA TGGCCAAGGTCATCCATGACAACTTTGGCATTGTGGAAGG
a The name of the probe corresponds to the viral RNA detected(the probe N mRNA is used to detect the N mRNA).
b Nucleotides are numbered according to their distance from the5?end of rabies virus and VSV virus antigenome(positive strand).
V OL.83,2009FUNCTIONAL CHARACTERIZATION OF RABIES VIRUS NEGRI BODIES7949
ence)and infected at different multiplicity of infection(MOI)of rabies virus (CVS strain)and at different times postinfection(p.i.).
U373-MG cells or BSR cells were grown on sterile glass coverslip in six-well plates(from50to80%con?uence)and were transfected with2.5?g or5?g of plasmid DNA by the calcium phosphate coprecipitation procedure.For N and P coexpression,the T7vaccinia virus system was used as described previously(3). Treatments of cells with drugs.Cells were kept in Dulbecco modi?ed Eagle medium containing2?M nocodazole,1.25nM paclitaxel,2.5?M cytochalasin D,1?M latrunculin A,or0.5?M jasplakinolide for1h before virus inoculation and during infection.Since all drugs were reconstituted in dimethyl sulfoxide (DMSO),untreated cells were maintained in medium containing the same con-centration of DMSO.Cells were?xed and processed for microscopy. Immuno?uorescence staining and confocal microscopy.Cells monolayers grown on glass coverslip were?xed20min with4%(wt/vol)paraformaldehyde in phosphate-buffered saline(PBS)and permeabilized for5min in0.1%Triton X-100 in PBS.Cells were then prepared for double-immuno?uorescence staining and analyzed by confocal microscopy.The intracellular distribution of viral antigen P,N, M,and G were analyzed by using the speci?c antibody at the dilution of1/500to 1/1,000,followed by incubation with the corresponding immunoglobulin G(IgG) antibody conjugated to Alexa568or Alexa488at the dilution of1/1,000.The cells were mounted onto glass slides by using Immu-Mount(Shandon)containing DAPI (4?,6?-diamidino-2-phenylindole)to stain nuclei.
Confocal laser microscopy was performed on a Leica SP2microscope(?63 oil-immersion objective lens)using UV excitation at351nm(DAPI)and blue laser excitation at488nm(Alexa488)and green laser excitation at545nm (Alexa568)in sequential recording mode.
Sellers’staining.Infected cells grown on coverslips were stained with a mix-ture of saturated solution of basic fuschin and methylene blue dissolved in methanol as described by Sellers in1927(40).Cells were?xed and permeabilized with methanol at?20°C,dipped into the staining solution for several seconds, and then washed with water.Cells were observed under a light microscope. Quanti?cation of Negri body-like structures.BSR cells were infected with CVS at different MOIs(0.5,1,3,5,and10)in the presence or not of nocodazole (2?M).Cells were?xed and permeabilized at different times p.i.(4to16h).The viral N protein was stained by a rabbit polyclonal anti-N conjugated to FITC. Quanti?cation of IBs was performed by counting the viral inclusions(containing the N protein)present in infected cells.An average of400cells from three independent experiments were counted at each time p.i.and for each MOI.The results are expressed as a percentage of uninfected cells and cell containing one, two,three,four,and more IBs.
Poisson distribution predicts that the relative frequency(p)of one cell to be infected by n virus at an MOI of?is p(n)?(e????n)/n!.
FISH.Fluorescence in situ hybridization(FISH)was performed by using3?biotinylated oligonucleotides(biotin569.61,obtained from Eurogentec)(42 nucleotides)complementary to speci?c sequences of viral RNA(Table1and Fig.1).According to the amount of viral RNA expected to be present during rabies virus infection,the number of probes mixed during hybridization was different:the NmRNA and PmRNA were detected by using one probe,the MmRNA and GmRNA were detected by using a mixture of two probes(M1 and M2or G1and G2),the LmRNA was detected by using three probes(L1, L2,and L3),and the genome was detected by using a mixture of two probes. The probe used to detect the antigenome was designed in order to avoid the detection of the mRNA,whereas the probe used to detect the mRNA hy-bridizes also to the antigenome.
Infected cells grown on coverslips were?xed with paraformaldehyde and permeabilized as described above.After treatment with200U of DNase I
FIG.2.Characterization of the viral IBs.(A)BSR cells were in-fected with rabies virus(CVS strain)at an MOI of3for16h and stained by the Sellers’staining protocol as described in Materials and Methods.Viral IBs are indicated pointed by arrows(left panel).A higher magni?cation of an area is also presented(right panel).The scale bar corresponds to12?m.(B)BSR cells were infected as de-scribed above.After?xation and permeabilization,cells were pro-cessed for double-immuno?uorescence staining with a rabbit poly-clonal anti-P antibody(P)and the MAb62B5anti-N antibody(N),a mouse polyclonal anti-M antibody(M),or the MAb30AA5anti-G antibody(G).In all cases,DAPI(blue)was used to stain the nuclei (merge).Colocalization is apparent as yellow coloration in the merged panel.The scale bars correspond to12?m.(C)Infected BSR cells as described above were treated for immunogold labeling.P protein was detected by using two MAbs,25C2and30F2;N protein was detected by using MAb64B6;and M and G proteins were detected by using mouse polyclonal anti-M antibody and by anti-G MAb30AA5,respec-tively.The scale bars correspond to0.5?m.
7950LAHAYE ET AL.J.V IROL.
(RNase-free D7201;Sigma)/ml,cells were prehybridized in hybridization buffer (50%formamide,10%dextran sulfate,4?SSC [1?SSC is 0.15M NaCl plus 0.015M sodium citrate])for 30min at 37°C before being incubated with 10?l of hybridization mix containing 10ng of biotinylated probe,10?g of herring testis DNA,and 8.5?l of hybridization buffer for 2min at 95°C in presence or not of 15U of RNase A (catalog no.10109142001;Roche)/ml.Coverslips were then put in a humidi?ed hybridization chamber at 37°C overnight and washed three times at 42°C for 5min with 50%formamide–2?SSC and then three times at 42°C with 0.5?SSC.Cells were then processed for immuno?uorescence staining.The N protein was stained with a mouse MAb anti-N (62B5)antibody,followed by incubation with Alexa 488-goat anti-mouse IgG.Viral biotinylated RNA were detected by incubation with streptavidin conjugated to cyanin 3(Cy3;catalog no.016160081;Jackson Immunotech).
BrUTP labeling of viral RNA.BrUTP incorporation of newly synthezised viral RNA was performed as described by Georgi et al.(11).Brie?y,the infected cells (16h p.i.)were treated with 15?g of ActD/ml for 1h and then transfected with BrUTP (B 7166;Sigma)at a ?nal concentration of 10mM by using Lipo-fectamine 2000(Invitrogen)and maintained in the presence of ActD for 20min.The cells were ?xed and permeabilized as described above and processed for immuno?uorescence.P protein was detected by using the rabbit polyclonal anti-P antibody,followed by incubation with Alexa 568-conjugated goat anti-rabbit IgG.The BrUTP-labeled RNAs were stained by using mouse MAb to BrdU at a dilution of 1/50,followed by the addition of goat anti-mouse Alexa 488-conjugated antibody.
Electron microscopy.For structural analysis,infected BSR cells were ?xed for 1h at room temperature with 2%glutaraldehyde (Sigma)in 0.1M cacodylate buffer (pH 7.4;Sigma).Cells were washed with 0.1M sucrose (Sigma)in 0.1M cacodylate buffer (pH 7.4).Cells were then post?xed for 1h at room temperature with 1%aqueous osmium tetroxide (OsO 4)and 1.5%potassium ferrocyanide (Sigma)in 0.1M cacodylate buffer (pH 7.4)and stained with 2%uranyl acetate in water.The cells were then dehydrated in increasing concentrations of ethanol and embedded by Epon with 2,4,6-tris(dimethylaminomethyl)phenol (DMP30;Delta Microscopies).Polymerization was carried out for 48h at 60°C.Ultrathin
sections of Epon-embedded material were collected on copper palladium grids (200mesh)and stored until use.These sections were stained with lead citrate (Delta Microscopies).Sections were examined with a Zeiss EM902electron microscope operated at 80kV,and images were acquired with a charge-coupled device camera (Megaview III)and analyzed with ITEM software (Elo?se,France;MIMA2platform [INRA-CRJ]).
For immunogold detection of rabies virus protein,cells were ?xed for 1h at 4°C with 4%formaldehyde (Merck)in 0.1M phosphate buffer (pH 7.3).During ?xation,cells were scraped and centrifuged.Pellets were dehydrated in increas-ing concentrations of methanol and embedded in Lowicryl K4M (Chemische Werke Lowi)at a low temperature according to the method of Roth et al.(36).Polymerization was carried out for 5days at ?30°C under long-wavelength UV light (Phillips ?uorescent tubes [TL 6W]).Ultrathin sections of Lowicryl-embed-ded material were collected on carbon-coated gold grids (200mesh)and stored until use.For immunogold labeling,grids bearing Lowcryl thin sections of in-fected cells were ?rst placed for 2min over drops of bovine serum albumin (5%PBS).The grids were then ?oated for 2h,at room temperature,on drops of af?nity puri?ed mouse monoclonal or polyclonal anti-rabies virus proteins anti-bodies (diluted 1/20in PBS)or over normal mouse serum as control (diluted 1/20in PBS).After 15-min washes with 3drops of PBS,the grids were incubated for 30min over drops of a 1/30dilution in PBS of goat anti-mouse IgG conjugated to gold particles (10nm in diameter;Biocell Research Laboratories).After 5-min passages on 3drops of PBS,the grids were rapidly rinsed in jet-distilled water,air dried,and stained for 10min with 5%aqueous uranyl acetate.Grids were observed with a Philips 400transmission electron microscope,at 80kV at magni?cations of ?8,000to 22,000.
RESULTS
Characterization of the IBs found in rabies virus-infected cells.Rabies virus infection induces the formation of round IBs in the cytoplasm of neuronal and nonneuronal cells.We
ob-
FIG.3.Evolution of the viral IBs during the viral cycle.(A)BSR cells were infected by CVS at an MOI of 3for different times p.i.as indicated and stained with rabbit polyclonal anti-N conjugated to FITC antibody and DAPI.The scale bars correspond to 12?m.(B and C)The numbers of IBs per cell were quanti?ed at different times p.i.for an MOI of 3(B)or an MOI of 10(C).A histogram represents the average values from three independent experiments by counting 400cells per experiment.The results are indicated as the percentages of cells that were uninfected or that contained one,two,three,four,and more IBs and are depicted by six variously shaded columns from left to right,respectively,for each time point.Error bars indicate the standard deviations.
V OL .83,2009FUNCTIONAL CHARACTERIZATION OF RABIES VIRUS NEGRI BODIES 7951
served IBs in infected BSR cells (Fig.2).Consistent with the known properties of Negri bodies,these inclusions could be stained with the Seller’s solution containing the dye,basic fuschin (40)(Fig.2A).
Unsurprisingly,confocal and electron microscopy analyses revealed that the IBs are full of N and P proteins (Fig.2B and C).It has also been shown that the polymerase L is present in the IBs (6).In contrast and as expected,the M and G proteins were excluded from these structures,as indicated by the fact that IBs were neither labeled by a polyclonal serum against the M protein nor by an MAb directed against the G protein (Fig.2B and C).Thus,the staining characteristics,the size (a few micrometers),and the shape,as well as the viral protein com-position of the IBs formed in infected BSR cells,indicated that these structures share the de?ned features as Negri bodies and can thus be considered NBLs.
Morphogenesis of the NBLs during viral infection.Little is known about the formation and evolution of Negri bodies during viral infection.Our model system permits us to perform such characterization.This is the ?rst point that we have ad-dressed.
BSR cells or human neuroblastoma SK cells were infected at different MOIs (0.5to 10)and analyzed at different times p.i.by immuno?uorescence using anti-N or anti-P antibodies.At different times p.i.,IBs in the cytoplasm of cells infected at different MOIs were counted.At an early stage of infection (4h p.i.),infected cells presented one or two viral inclusions that then grew in size (10to 12h p.i.)(Fig.3A).These values were observed regardless of the MOI used (between 3and 10).Smaller IB structures appeared at later stage of infection (16to 20h p.i.)(Fig.3A).
The number of NBLs per cell was independent of the MOI and consequently was independent of the number of incoming virus per cell (Fig.3B and C)that was expected to follow a Poisson distribution as mentioned in the Materials and Meth-ods.These results suggest that there are a limited number of sites per cell that allow the formation of IBs.
The morphogenesis and structural evolution of NBLs were also analyzed in infected BSR cells by electron microscopy (Fig.4).NBLs are spherical structures (a few micrometers in size)that often contain a cavity.Although initially devoid of membranes (4to 12h p.i.,Fig.4A),NBLs progressively be-came associated with a double membrane (16h p.i.,Fig.4B and C).The cytoplasmic granular surface of the membrane suggests that it might be derived from rough endoplasmic re-ticulum (Fig.4C).Finally,NBLs were completely surrounded by these double membranes (16to 24h p.i.).This membrane organization is reminiscent of the organization of the
double
FIG.4.Morphology and evolution of viral IBs.Ultrastructural aspect of BSR cells infected for 12h (A),16h (B and C),and 24h (D and E)p.i.as indicated.Double ?xation was performed with glutaraldehyde and osmium tetraoxide .Epon embedding and uranium-lead staining.IBs display a granular dense structure with a cavity (indicated by arrows in panels A,B,C,and E).Note the recruitment of membranes around IBs at 16h p.i.(B and C)and the presence of virus particles close to the viral inclusion at 24h p.i.(indicated by arrow in panel D).Nu,nucleus;Cy,cytoplasm.Bars correspond to 0.5?m.Higher magni?cation (?13)of two membrane areas reveals a double-membrane with a granular surface are shown for panel C.
7952LAHAYE ET AL.J.V IROL .
membrane around the cell nucleus.After24h p.i.,the shape of the bodies appeared to be altered,and viral particles were also seen budding from some of these structures(Fig.4D and E), most probably inside the lumen of the compartments that surround the bodies.
Comparison of the NBLs and aggresomes.Aggresomes are misfolded protein aggregates that appear when the protea-some and chaperones can no longer deal with the aggregated proteins.They are driven on microtubules to the microtubule organizing center(MTOC),they are surrounded by vimentin cages to isolate and reduce the toxicity of misfolded proteins, and they recruit mitochondria that may provide the ATP re-quired by chaperones and proteasomes for protein folding and degradation(7,20).For many viral families,it has been pro-posed that the aggresome pathway is hijacked in order to concentrate viral components and to facilitate viral replication and assembly(14,39).Therefore,we investigated whether NBLs share the characteristics of aggresomes described above. We observed that NBLs do not colocalize with the MTOC,as revealed by anti-?-tubulin staining(Fig.5A).Costaining with anti-vimentin antibodies did not show any rearrangement of vimentin around the cytoplasmic inclusions at any stage of infection(Fig.5A).Furthermore,we did not observe recruit-ment of mitochondria in close proximity to the NBLs(Fig.5A). However,Hsp70chaperone and ubiquitinylated proteins accu-mulate inside the IBs,as revealed by an anti-Hsp70and anti-poly-ubiquitin staining,respectively(Fig.5B).These results indicate that the NBLs do not share all of the features of aggresomes,although they contain Hsp70and ubiquitinylated proteins.
Role of the cytoskeleton in the morphogenesis of NBLs.We next investigated the mechanism by which the different pro-teins gather and are recruited in NBL structures by analyzing the role of the cytoskeleton in the morphogenesis and evolu-tion of the IBs.
We studied the role of the microtubule network by using two different speci?c inhibitors of MT dynamics(nocodazole and paclitaxel).BSR or U373-MG infected cells(MOI?3and MOI?10)were treated with nocodazole,which results in the disruption of microtubules,for1h before virus inoculation and during infection.In the presence of nocodazole,the“early”(large,one or two per cell)NBLs formed ef?ciently without any apparent delay(Fig.6).These results indicate that micro-tubules are not needed for the IB formation.
In striking contrast,the formation of“late”(small)IBs was affected by nocodazole treatment since at later stages of infec-tion(16to20h p.i.),we observed NBLs that were much larger than those in the absence of the drug.Furthermore,at these late stages,no smaller structures were observed in nocodazole-treated cells(Fig.6).This was in contrast to untreated cells where small IBs(more than15per cell)were detected.Thus, the drug inhibited the formation of additional IBs and/or the appearance of smaller structures.
We did not observe any effect of paclitaxel,a drug that stabilizes microtubules,on the formation and evolution of IBs (data not shown).This suggests that the mechanisms involved in these processes do not require dynamic microtubules.
As P protein has been shown to interact with LC8,the light chain of the molecular motor dynein(19,34),we checked whether this interaction plays a role in the targeting of the major P component to the viral inclusions.When cells were infected and transfected with the plasmid encoding the P pro-tein mutant unable to interact with LC8(P?LC8-RFP)(33), RFP?uorescent viral inclusions were formed as in cells in-fected and transfected with the plasmid encoding the wild-type P-RFP protein(Fig.7A).This indicates that LC8is not re-quired in the recruitment of P inside the IBs.This is consistent
FIG.5.Viral IBs exhibit some features of aggresomes but are dis-
tinct structures.(A)BSR cells were infected for16h and costained
with a rabbit polyclonal anti-P antibody(P)and either mouse anti-?
tubulin MAb(Tub),mouse anti-vimentin MAb(Vim),or the Mito-
Tracker Red(Mito),followed by incubation with Alexa488-goat anti-
rabbit IgG and Alexa568-goat anti-mouse IgG.Note that IBs are not
localized at the MTOC(indicated by arrows)and that no vimentin
cage or no recruitment of mitochondria(as would be expected for
aggresomes)are observed around the IBs.(B)IBs contain Hsp70and
ubiquitinylated proteins.Infected BSR cells were immunostained with
a rabbit polyclonal FITC-conjugated anti-N antibody(N)and a mouse
MAb anti-ubiquitin antibody(Ub)or a mouse MAb anti-Hsp70anti-
body(Hsp70),followed by incubation with Alexa568-goat anti-mouse
IgG.The scale bars correspond to12?m.
V OL.83,2009FUNCTIONAL CHARACTERIZATION OF RABIES VIRUS NEGRI BODIES7953
with the fact that overexpression of the inhibitor of dynein-mediated transport,dynamitin (dynamitin-GFP)during infec-tion did not result in the inhibition of the formation of viral inclusions (Fig.7B).
The role of actin was also investigated in BSR cells or in U373-MG cells by using inhibitors resulting in depolymeriza-tion of actin (cytochalasin D and latrunculin A)or resulting in stabilization of actin (jasplakinolide).None of the inhibitors signi?cantly affected the formation and evolution of NBLs,indicating that actin ?laments are not required in these pro-cesses.
NBLs are potential sites of viral transcription and replica-tion.We then analyzed whether NBLs are functional struc-tures where some essential steps of the virus cycle take place.Since the viral N,P,and L proteins involved in viral transcrip-tion and replication accumulate in IBs,we investigated the presence of viral RNA in these structures by using FISH.We therefore designed biotinylated oligonucleotide probes to de-tect speci?cally viral mRNAs,antigenomic RNA,and genomic RNA (Table 1and Fig.1).
After 16h of infection,BSR cells were simultaneously pre-pared for FISH to detect the viral RNA and immunostained with anti-N antibody to visualize the Negri bodies.
All viral mRNAs encoding the ?ve viral proteins (N,P,M,G,and L)were detected inside the IBs with no viral mRNA readily detected elsewhere (i.e.,nucleus/cytoplasm)outside of IBs (Fig.8A).The relative quantities of the viral RNAs were determined (Fig.8D).Although the FISH method does not allow a precise quanti?cation,it clearly indicated that the N mRNA was more abundant than the L mRNA according to the known gradient of decreasing mRNAs concentration that re-?ects the gene order (N,P,M,G,and L)(1).These hybrid-izations were speci?c since no positive signal was observed in mock-infected cells (data not shown)or in infected cells treated with a probe detecting the mRNA of the M protein of vesicular stomatitis virus (VSV)(Fig.8C).A positive control carried out in infected cells with a poly(dT)probe or a probe against the housekeeping GADPH mRNA gave rise to a signal diffusely distributed in the nucleus and the cytoplasm as ex-pected (Fig.8C).RNase A treatment of infected cells prior to FISH with poly(dT)probe prevented the hybridization signal in the nucleus and the cytoplasm but left intact the signal in the IBs (Fig.8C).These results indicate that the viral mRNA accumulate inside the IBs and are protected against RNase digestion,most probably by the cage formed by the N and P proteins.Indeed,the N protein formed an apparent ringlike structure (cage)around the RNA (Fig.2B and Fig.8A).
By using speci?c probes,the viral genomic RNA and
anti-
FIG.6.Effect of nocodazole on formation and evolution of IBs during infection.(A)BSR cells were infected and treated with nocodazole (2?M;NCZ)or mock treated (DMSO).At different times p.i.as indicated,the cells were costained with rabbit polyclonal anti-N conjugated to FITC antibody and with mouse MAb anti-?tubulin antibody,followed by incubation with Alexa 568-goat anti-mouse IgG.The scale bars correspond to 12?m.(B and C)The numbers of IBs per cell were quanti?ed in nocodazole-treated cells at different times p.i.as indicated for an MOI of 3(B)or an MOI of 10(C).A histogram represents the average values from three independent experiments by counting 400cells per experiment.The results are expressed as the percentages of uninfected cells or cells containing one,two,three,four,and more IBs and are depicted by six variously shaded columns from left to right,respectively,for each time point.Error bars indicate the standard deviation.
7954LAHAYE ET AL.J.V IROL .
genomic RNA were also detected exclusively in the IBs(Fig. 8B).The relative quanti?cation of the viral RNAs indicated that the amount of genomic RNA is higher than the amount of antigenomic RNA(Fig.8D).
The experiments described above demonstrated that NBLs are structures where viral transcription and replication prod-ucts accumulate.In order to establish whether the viral RNAs are synthesized inside IBs or synthesized outside IBs and then translocated into these structures,we performed short-term RNA labeling in the presence of BrUTP and ActD to inhibit cellular transcription.Cells were infected for16h before treat-ment without or with ActD for1h and transfected with BrUTP for20min.Cells were then examined by double-immuno?uo-rescence staining using anti-BrdU and anti-P antibodies(Fig.
9).Without ActD,cellular RNA was mainly detected in the nucleus,as expected(Fig.9A,upper panel).In the presence of the drug,newly synthesized RNA were detected inside the viral inclusions(Fig.9A,lower panel).Control experiments were performed in which rabies virus N and P proteins were coex-pressed(with the vaccinia virus T7system)to mimic NBLs,as previously shown(3),and provide nonviral IBs.The fact that no BrUTP signal was detected in these nonviral IBs in the presence of the drug indicated that the BrUTP signals were dependent on the incorporation of BrUTP by the rabies virus RNA polymerase(Fig.9B).These results provide evidence that NBs are functional structures where viral transcription and replication take place.
DISCUSSION
The present study characterizes the morphogenesis and functions of the IBs that are found in the cytoplasm of cells infected by rabies virus.We show that these IBs have staining characteristics that are similar to those of the Negri bodies found in certain nerve cells infected by rabies virus.These structures are not canonical aggresomes containing misfolded proteins aggregates en route to degradation by the proteasome (consistent with the observations of Menager et al.[26])but are functional structures with a central role in viral infection. The detection of viral RNA in IBs indicates either that viral RNAs are synthesized within IBs or are synthesized elsewhere before import and accumulation within IBs.Our?ndings indi-cate that the former is the case and that Negri bodies are most probably specialized sites of viral transcription and replication. Signi?cantly,the labeling period used in the BrUTP experiments is short compared to the duration of viral mRNA synthesis.In-deed,it has been reported that due to a quite slow elongation rate of transcription(3.1to4.3nucleotides),it takes about10min for the VSV N mRNA and1to2h for the LmRNA to be synthesized (16).This is also consistent with the elongation rates recently reported for another Mononegavirales,the measles virus(three nucleotides)(32).Thus,the newly BrUTP-labeled RNAs are al-most certainly synthesized within NBLs(during the20-min label-ing period),as indicated by the BrUTP labeling experiment pre-viously used for VSV(4).
This would be consistent with the fact that proteins L,P, and N involved in viral RNA synthesis are accumulated in these structures.Interestingly,viral genomes,antigenomes, and mRNAs synthesized inside the IBs appear to be sur-rounded by a cage formed by N and P proteins that may protect them from degradation.We did not investigate whether NBLs are also sites of viral protein synthesis.Whether the viral proteins L,N,and P are translated inside or at the periphery of the viral inclusions or anywhere else,and in this latter case how these proteins are recruited in such IBs,re-mains an open question.Cellular proteins are also detected in these structures.Although the exact composition of the NBLs remains unknown,the Toll-like receptor3has been shown to be an important compound of the NBLs(26).In addition,the heat shock protein Hsp70and ubiquitinylated proteins,which are also found associated with aggresomes,were accumulated inside these structures.This enrichment might be correlated with an intense production of viral proteins(particularly N and P)in these viral factories.However,in addition to continuously surveying the folding status of proteins as part of its quality control function,Hsp70is also involved in many cellular pro-cesses such as cell cycle,apoptosis,or innate immunity(25). Similarly,ubiquitinylation of proteins is also involved in many cellular functions other than degradation,such as signalization events.Since viruses need to interfere with cellular processes to create a favorable environment for their multiplication, Hsp70and ubiquitin ligases are frequently diverted and re-cruited by viruses(2,25).In the case of rabies virus infection,
FIG.7.Formation of IBs does not require dynein-mediated trans-
port.(A)BSR cells were transfected with a plasmid encoding the
P-RFP or a P-RFP mutant that does not interact with LC8(P?LC8-
RFP)and then infected for24h.Cells were stained with the MAb
62B5anti-N antibody(N),followed by incubation with Alexa488-goat
anti-mouse IgG and DAPI(merge).The scale bars correspond to12
?m.(B)BSR cells were transfected with a plasmid encoding dyna-
mitin-GFP and then infected for24h.Cells were stained with a rabbit
polyclonal anti-P antibody(P),followed by incubation with Alexa
568-goat anti-rabbit IgG and DAPI(merge).Colocalization is appar-
ent as yellow coloration in the merged panel.The scale bars corre-
spond to12?m.
V OL.83,2009FUNCTIONAL CHARACTERIZATION OF RABIES VIRUS NEGRI BODIES7955
7956LAHAYE ET AL.J.V IROL.
Hsp70might play a proviral role,as previously indicated by the
presence of Hsp70in puri?ed rabies virus(37).In addition,
NEDD4,an E3ubiquitin ligase,is recruited by rabies virus
matrix protein via a PPXY motif and is involved in the budding
process(13,15).
Electron-microscopy studies indicate that viral inclusions
initially devoid of membranes recruit cellular membranes,sug-
gesting that the budding step could occur inside intracellular
compartment derived from the rough endoplasmic reticulum
and wrapped around IBs as previously shown(24).
The fact that IBs may support viral transcription and repli-
cation leads us to consider that such structures may form
around incoming genomes.Our results indicate that early after
infection,infected cells contain one or two viral factories.This
limited initial number of formation sites is independent of the
MOI and consequently is independent of the number of in-
coming viruses per cell.The mechanism involved in this re-
striction is not known,but it might occur at the cell entry to
limit the number of incoming viral particles and/or inside the
cytoplasm to reduce the number of functional transcription
and replication sites.It is also possible that a viral factory arises
from many incoming genomes.The restriction would then be
due to the requirement of speci?c cellular structures or or-
ganelles(present in a limited number in the cell)for the IBs to
form.
We did not observe any preferential intracellular localiza-
tion of the viral inclusions in the cells such as the MTOC,
where aggresomes are typically located,as well as some other
viral factories(31).The lack of a link between MTOC and
rabies viral factories is consistent with the fact that microtubule
networks are not involved in the morphogenesis of NBLs that
are formed ef?ciently and without any delay in the presence
of an inhibitor of microtubule polymerization.Furthermore,
FIG.8.Viral RNAs accumulate in viral IBs.BSR cells were in-
fected(MOI?3)for16h p.i.(A and B)FISH analyses were per-
formed with biotinylated oligonucleotides(described in Materials and
Methods),followed by incubation with streptavidin conjugated to Cy3
to detect NmRNA,PmRNA,MmRNA,GmRNA,and LmRNA as
indicated(A)and to detect viral genomic and antigenomic RNA as
indicated(B).Note that the probe used to detect the mRNA also
allows the detection of the antigenome.In contrast,the probe used to
detect the antigenome was designed in order to avoid the detection of
the mRNA.Note that for the antigenomes,the photomultiplier tube
(PMT)has been increased in order to clearly visualize the?uorescence
signals resulting in an increase of the background signal.No positive
signal was observed in mock-infected cells(indicated by an arrow).
(C)FISH was also performed to detect the housekeeping GADPH
mRNA or cellular mRNA(Poly-A)after treatment with RNase
(?RNase)or without RNase(–RNase).No signal was observed with a
control probe complementary to the MmRNA of VSV.In all cases,the
N protein(N)was stained with the mouse MAb anti-N(62B5)anti-
body,followed by incubation with Alexa488-goat anti-mouse IgG(A,
B,and C).The scale bars correspond to12?m.(D)Relative quanti-
?cation of the viral RNA accumulated inside the Negri bodies.FISH
was performed as described above.Quanti?cation of Cy3?uorescent
intensity(arbitrary units[ua])was performed with the same PMT(the
PMT adjusted with the N mRNA probe)on a Leica SP2confocal
microscope.The surface area and the Cy3?uorescence intensity of
each Negri body was calculated by using Leica confocal software.Two
independent experiments were performed by counting an average of
300IBs.The results were expressed as the log10of Cy3’s intensity per
?m2of Negri body and per probe(ua/?m2).
overexpression of dynamitin that blocks dynein/dynactin-me-diated retrograde transport along microtubules has no effect on the morphogenesis of viral inclusions.All of these data indicate that the mechanism to gather the different viral and cellular components in viral inclusions does not require an active cellular transport along the microtubules.
Although microtubule depolymerization does not inhibit the formation of Negri bodies,it prevents the appearance of smaller IBs that otherwise form late in infection.Further ex-periments are required to establish whether these small inclu-sions are derived from the initial IBs or may be newly formed by infection at a later stage by new viral progeny or just by delayed formation.In contrast,the actin network does not play a prominent role in these processes.
It is possible that NBLs might be the result of a cytoplasmic compartmentalization due to a cellular defense mechanism involving components of the aggresome pathway,such as host factors involved in stress response,the storage or degradation of proteins to target virus components for storage,and degra-dation,as previously suggested for other viruses.These struc-tures may be hijacked by the rabies virus to concentrate host and viral components in one place to facilitate viral replication, to immobilize and inactivate proteins that would otherwise inhibit infection,and also to mask rabies virus presence from the cellular components of the innate immune system.This is consistent with the?ndings of Menager et al.(26),who sug-gested that the sequestration of Toll-like receptor3in the Negri body could be a strategy whereby the virus prevents the antiviral or apoptotic effect of this cellular protein.Cytoplas-mic inclusions have been reported for?loviruses(10)and some paramyxoviruses(12),suggesting that virus-induced formation of intracellular compartments might be general among viruses of the Mononegavirales order that contains numerous human and animal pathogens.It is probable that the mechanisms and the machineries hijacked by rabies virus are similarly diverted by these other viruses.
ACKNOWLEDGMENTS
We are grateful to Greg Moseley(from Monash University,Clayton, Victoria,Australia)for helpful discussions and careful reading of the manuscript.We thank Manos Mavrakis for helpful discussions.We thank David Pasdeloup for providing the plasmid pDsRedM and for technical advice for the FISH.We acknowledge Richard Vallee for the gift of the plasmid encoding dynamitin-GFP.We acknowledge Christine Longin for helpful advice and assistance with the electron microscopy(Unite′UR1196Ge′nomique et Physiologie de la Lac-tation,INRA,Plateau de Microscopie Electronique MIMA2,Jouy-en-Josas Cedex,France).Confocal microscopy was performed on the Plate-forme Imagerie et Biologie Cellulaire of the CNRS cam-pus supported by the Institut Fe′de′ratif de Recherche87,La Plante et Son Environnement,and the program ASTRE of the Conseil Ge′ne′ral de l’Essonne.
We acknowledge support from the CNRS and INRA.
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How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
THE PEOPLE’S REPUBLIC OF CHINA MARRIAGE CERTIFICATE Ministry of Civil Affairs of the People’s Republic of China (The Special Seal of Management of Marriage Certificate) This application conforms to the Marriage Law of the People’s Republic of China. We hereby give them the permission to register and issue this marriage certificate. Authorized by: Special Seal for Marriage Register of XX District, Civil Affairs Bureau of Beijing Undertaker (signature): XX XXX Certificate Holder: X XX Registration Date: xx xxx 2009 Marriage Certificate No.: Jing Chao Jie Zi 050920381 Note: Name: X XX Sex: Female Nationality: Chinese Date of Birth: xx xxx 1980 ID No.: 110105xxxxxxxxxxxx Name: X X Sex: Male Nationality: Chinese Date of Birth: xx xxx 1978 ID No.: 120104xxxxxxxxxxxx
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i
The People’s Republic of China Marriage Certificate Marriage Certificate of Ministry of Civil Affairs of the People’s Republic of China (seal) Under the supervision of Ministry of Civil Affairs of the People’s Republic of China Your Marriage application is in accordance with the regulations of <
Dear teacher and colleagues: my topic is on “spare time”. It is a huge blessing that we can work 996. Jack Ma said at an Ali's internal communication activity, That means we should work at 9am to 9pm, 6 days a week .I question the entire premise of this piece. but I'm always interested in hearing what successful and especially rich people come up with time .So I finally found out Jack Ma also had said :”i f you don’t put out more time and energy than others ,how can you achieve the success you want? If you do not do 996 when you are young ,when will you ?”I quite agree with the idea that young people should fight for success .But there are a lot of survival activities to do in a day ,I want to focus on how much time they take from us and what can we do with the rest of the time. As all we known ,There are 168 hours in a week .We sleep roughly seven-and-a-half and eight hours a day .so around 56 hours a week . maybe it is slightly different for someone . We do our personal things like eating and bathing and maybe looking after kids -about three hours a day .so around 21 hours a week .And if you are working a full time job ,so 40 hours a week , Oh! Maybe it is impossible for us at
关于人英语演讲稿(精选多篇) 关于人的优美句子 1、“黑皮小子”是我对在公交车上偶遇两次的一个男孩的称呼代号。一听这个外号，你也定会知道他极黑了。他的脸总是黑黑的；裸露在短袖外的胳膊也是黑黑的；就连两只有厚厚耳垂的耳朵也那么黑黑的，时不时像黑色的猎犬竖起来倾听着什么；黑黑的扁鼻子时不时地深呼吸着，像是在警觉地嗅着什么异样的味道。 2、我不知道，如何诠释我的母亲，因为母亲淡淡的生活中却常常跳动着不一样的间最无私、最伟大、最崇高的爱，莫过于母爱。无私，因为她的爱只有付出，无需回报；伟大，因为她的爱寓于
普通、平凡和简单之中；崇高，是因为她的爱是用生命化作乳汁，哺育着我，使我的生命得以延续，得以蓬勃，得以灿烂。 3、我的左撇子伙伴是用左手写字的，就像我们的右手一样挥洒自如。在日常生活中，曾见过用左手拿筷子的，也有像超级林丹用左手打羽毛球的，但很少碰见用左手写字的。中国汉字笔画笔顺是左起右收，适合用右手写字。但我的左撇子伙伴写字是右起左收的，像鸡爪一样迈出田字格，左看右看，上看下看，每一个字都很难看。平时考试时间终了，他总是做不完试卷。于是老师就跟家长商量，决定让他左改右写。经过老师引导，家长配合，他自己刻苦练字，考试能够提前完成了。现在他的字像他本人一样阳光、帅气。 4、老师，他们是辛勤的园丁，帮助着那些幼苗茁壮成长。他们不怕辛苦地给我们改厚厚一叠的作业，给我们上课。一步一步一点一点地给我们知识。虽然
他们有时也会批评一些人，但是他们的批评是对我们有帮助的，我们也要理解他们。那些学习差的同学，老师会逐一地耐心教导，使他们的学习突飞猛进。使他们的耐心教导培养出了一批批优秀的人才。他们不怕辛苦、不怕劳累地教育着我们这些幼苗，难道不是美吗？ 5、我有一个表妹，还不到十岁，她那圆圆的小脸蛋儿，粉白中透着粉红，她的头发很浓密，而且好像马鬓毛一样的粗硬，但还是保留着孩子一样的蓬乱的美，卷曲的环绕着她那小小的耳朵。说起她，她可是一个古灵精怪的小女孩。 6、黑皮小子是一个善良的人，他要跟所有见过的人成为最好的朋友！这样人人都是他的好朋友，那么人人都是好友一样坦诚、关爱相交，这样人与人自然会和谐起来，少了许多争执了。 7、有人说，老师是土壤，把知识化作养分，传授给祖国的花朵，让他们茁壮成长。亦有人说，老师是一座知识的桥梁，把我们带进奇妙的科学世界，让
精品word. 1 / 5 Ministry of Civil Affairs of the People ’s Republic of China (seal) Supervised by the Ministry of Civil Affairs of the People ’s Republic of China The application for marriage is in accordance with the Marriage Law of the People ’s Republic of China and allowed to register. Hereby this certificate is issued. Registration Authority: Department of Civil Affairs of XXX City XXXXXXX Province (special seal for marriage registration) Marriage Registrar: (signature) (unclear )
精品word. 2 / 5 Certificate Holder XXXXXXXX Registration Date xxx. xxth, xxxx Marriage Certificate Zi No. XXXXXXXXX Remark No Name Xxx xxxxxx Sex Male Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000 Name Xxx xxxxxx Sex Female Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000
关于时间的英语演讲稿范文_演讲稿 and organizing people to learn from advanced areas to broaden their horizons in order to understand the team of cadres working conditions in schools, the ministry of education has traveled a number of primary and secondary schools to conduct research, listen to the views of the school party and government leaders to make school leadership cadres receive attention and guidance of the ministry of education to carry out a variety of practical activities to actively lead the majority of young teachers work hard to become qualified personnel and for them to put up the cast talent stage, a single sail swaying, after numerous twists and turns arrived in port, if there is wind, a hand, and naturally smooth arrival and guide students to strive to e xcel, need to nazhen “wind” - teacher. teachers should be ideological and moral education, culture education and the needs of students organically combining various activities for the students or students to carry out their own. for example: school quiz competitions, essay contests, ke benju performances and other activities to enable students to give full play to their talents. teachers rush toil, in order to that will enable students to continue to draw nutrients, to help them grow up healthily and become pillars of the country before. for all students in general education, the government departments have also not forget those who cared about the
good afternoon everybody! I’m very glad to stand here and give you a short speech about challenge and opportunity,双手交握微笑 So, what’s an opportunity? It’s a great chance to achieve dreams, it’s a platform to show ourselves.Just as now,I’m standing here showing myself,it's one of my dreams,also my opportunity. Then, what about challenge? Challenge is a new or difficult task which test our ability and skill. it is a fighter, a sense of courage, the fearless spirit towards all difficulties, Many people are afraid of being challenged, worrying about change and failure.I used to be a member of them，but now,I find that sth we haven’t done isn’t as difficult as it seems, fear always hindered our courage. Said so much,此刻我最想与你们分享的一句话就是： we should welcome being challenged,and try to enjoy it.它很可能是你进步的机会 Cruel competition isn 't the excuse for failure. No one is regularly on a winner or a loser. To the strong willed, competition is the spring of hope; To the weak minded, it means the winter of despair. So turning into opportunities means taking advantage of these challenges and benefiting from them.In this way,you can be the boss of your life.这句语法有没有错误？指着他，手势 That’s all, thank you!