当前位置：文档库 › Expression of the interferon-alpha_beta-inducible bovine Mx1

Expression of the interferon-alpha_beta-inducible bovine Mx1

Expression of the interferon-alpha/beta-inducible bovine Mx1dynamin interferes with replication of rabies virus

M.Leroy,G.Pire,E.Baise,and D.Desmecht *

Department of Pathology,Faculty of Veterinary Medicine,University of Lie `ge,FMV Sart Tilman B43,B-4000Lie `ge,Belgium Received 2March 2005;revised 25August 2005;accepted 28August 2005Available online 3October 2005

Rabies is a fatal anthropozoonotic viral infection of the central nervous system that remains a serious public health problem in many countries.As several animal cases of spontaneous survival to infection were reported and because type 1interferons were shown to protect against the virus,it was suggested that innate resistance mechanisms exist.Among the antiviral proteins that are synthesized in response to interferon-A /B stimulation,Mx proteins from several species are long known to block the replication of vesicular stomatitis virus (VSV).As both VSV and rabies virus belongs to the Rhabdoviridae family,this study was started with the aim to establish whether the anti-VSV activity of a mammalian Mx protein could be extended to rabies virus.This question was addressed by inoculating the virus onto a bovine Mx1or human MxA-expressing Vero cell clone.Plaque formation was unambiguously blocked,and viral yields were reduced 100-to 1000-fold by bovine Mx1expression for both SAG2and SADB19viral strains.In opposition,only SAG2strain could be inhibited by the expression of human MxA protein.The effect of both proteins expression was then evaluated at the viral protein expression level.Again,boMx1was able to repress protein expression in both strain,whereas only SAG2proteins were inhibited in human MxA-expressing cells.These results suggest that protection conferred by interferon-A /B against rabies could be,at least partially,attributable to the Mx pathway.Alternatively,bovine Mx1could be unique in its ability to repress rabies virus which,if confirmed in vivo,would open an avenue for the development of new antirabies therapeutic strategies.D 2005Elsevier Inc.All rights reserved.Keywords:Rabies;Rhabdoviridae;Interferon;Mx

Introduction

Rabies is an anthropozoonotic viral infection in human and animal species that remains a serious public health problem in many developing countries.Being the type member of the Lyssavirus genus in the Rhabdoviridae family,rabies virus is usually trans-

mitted by close contact with the saliva of a rabid animal,then migrates from the inoculation point to the central nervous system through peripheral nerves,which finally results in encephalomye-litis (Hooper et al.,1998;Jackson,2003).The unquestionable general rule is that infection of vertebrates is fatal,but several investigators have reported animal cases where spontaneous resistance and survival occurred (Baer et al.,1977;Hilfenhaus et al.,1977;Murphy,1977;Lodmell et al.,1981;Mendonca and Pereira,1994;Hooper et al.,1998).These atypical cases suggest that some innate resistance mechanisms exist,the understanding of which may open an avenue for the development of a new therapeutic strategy that is crucially needed in the context of the yearly 70,000human death toll worldwide (http://www.who.int/).Although many factors could be involved in antiviral resistance,interferon (IFN)response is a priori presumed to play a crucial role as it is indeed produced in response to rabies virus infection,especially at the site of inoculation (Marcovistz et al.,1986)where the virus stays during most of the incubation period (Jackson,2003).This hypothesis is reinforced by the observation that high levels of IFN occurring in serum early during infection with rabies virus contribute to the resistance of inoculated mice (Mendonca and Pereira,1994).Moreover,as inoculated mice injected with antimouse IFN a /h were rendered more sensitive to the virus,type 1IFNs-dependent mechanisms are likely to be specifically involved (Marcovistz et al.,1986).Until now,three IFN a /h -responsive antiviral proteins have been considered to be involved in antiviral processes:the Mx dynamins (Arnheiter et al.,1996;Haller et al.,1998),the double-stranded RNA-dependent protein kinase R (Katze et al.,1991)and the 2V –5V oligoadenylate synthetases that function through RNase L (Foster,1997).Among them,Mx dynamins belong to a superfamily,the large GTPases,which are present in a variety of cell locations where they perform a wide array of functions including,in the case of murine,rat,chicken,bovine,fish and human Mx proteins,interference with the replication of some viruses the common characteristic of which being that their genome consists of single-stranded negative-sense RNA mole-cules (Haller et al.,1998).Among the viruses strongly repressed by Mx dynamins,vesicular stomatitis virus (VSV)is probably the one that is repressed by the wider spectrum of MX proteins

*Corresponding author.Fax:+3243664565.

E-mail address:daniel.desmecht@ulg.ac.be (D.Desmecht).Available online on ScienceDirect

(https://www.wendangku.net/doc/737994231.html,)https://www.wendangku.net/doc/737994231.html,/locate/ynbdi

Neurobiology of Disease 21(2006)515–521

Fig.1.Expression of bovine Mx1(boMx1,A –C)and human MxA (huMxA,D –F)in stably transfected Vero cell clones.(A –B)Indirect immunofluorescence assay (IFA)in doxycycline-stimulated (1A g/ml)V103cells (V103+,A)and in nonstimulated V103cells (V103à,B).(C)Western blot analysis of boMx1expression in V103+/à.(D –E)IFA in the V A8(D)and V A2(E)Vero cell lines.(F)Western blot analysis of huMxA expression in both cell

lines.

Fig.2.Bovine (boMx1)and human Mx1protein (huMxA)expression confers an antiviral activity against rabies virus.(A)Inhibition of rabies virus plaque formation in stimulated (1A g/ml doxycycline 24h pi,boMx1-producing)compared to nonstimulated V103cells.Confluent monolayers had been infected with successive dilutions of a rabies virus stock (strain SAG2)for 11days.Dark areas represent intact cell sheets.(B)Viral titers retrieved from direct plaque assays.Confluent monolayers of V A2,V A8(huMxA-producing),unstimulated V103and stimulated V103(boMx1-producing)cells had been infected with an identical dilution of the stock virus and the virus allowed to form plaques under soft agar for 11days.Titers (mean T SD)were calculated by direct plaque assay (PFU/ml)and result from 3independent experiments.(C)Rabies virus repression by boMx1(stimulated V103)-and huMxA-producing (V A8)cells,as judged from direct immunofluorescence 48h after infection with the same dilution of the stock virus (SAG2).

M.Leroy et al./Neurobiology of Disease 21(2006)515–521

516

examined so far (Meier et al.,1990;Staeheli and Pavlovic,1991;Schwemmle et al.,1995;Landis et al.,1998;Sandrock et al.,2001;Asano et al.,2002;Baise et al.,2004).As VSV belongs to the Rhabdoviridae family,it is tempting to hypothesize that some Mx proteins may also offer an innate protection against rabies.In this study,we addressed this question by inoculating rabies virus onto a double transgenic Vero cell clone that was engineered to allow tetracycline-subordinated conditional expression of the Bos taurus Mx1protein (Baise et al.,2004)and onto a Vero cell clone constitutively expressing the human MxA protein.

Materials and methods Cell lines and viruses

Vero (ATCC CCL-81and genetically engineered V103)cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10%tetracycline-free fetal calf serum (FCS)(Clontech),1%(v/v)penicillin –streptomycin and 0.5%(v/v)amphotericin (DMEM-10)at 37-C in a 5%CO 2–95%air humidified incubator.Interference of bovine Mx1protein (boMx1)expression with the replication of rabies virus was probed in the induced and noninduced Vero/TetR1/boMx1cell line.Briefly,this double transgenic Vero cell clone (V103)allows tetracycline-subordinated conditional expression of the bovine Mx1CDS under tight transcriptional control of the human

cytomegalovirus enhancer–promoter (Baise et al.,2004).When incubated in doxycycline-free medium,V103cells do not produce detectable levels of boMx1.Conversely,when doxycycline is incorporated (1A g/ml),homogenous expression is observed,with virtually no boMx1-negative remaining cells.The rabies virus strain SAG2(kindly provided by C.Tuffereau)and the rabies virus strain SAD Bern19(SADB19;kindly provided by A.V os)were amplified onto BSR cells and titrated on Vero cells (Lafay et al.,1994).Encephalomyocarditis (EMCV)virus is a clinical isolate prepared from supernatants of infected BHK21cells,aliquoted and stored at à80-C.Its titer was determined by plaque assay on Vero cells.

Generation of cells expressing human MxA protein

The 2.2-kb human MxA (huMxA)cDNA (kindly supplied by O.Haller,Germany)was cloned into the Eco RI and Eco RV restriction sites of the expression vector pcDNA4/TO (Invitrogen,Belgium)which permits constitutive high-level expression of recombinant proteins in various cell types.

Vero cells were transfected by the Lipofectamine 2000procedure with the vector pcDNA4/huMxA.Zeocin (350A g/ml)was added to the culture medium after 48h,and the cells were selected for 2weeks.After two rounds of cloning procedures,cells were probed by immunofluorescence and Western blot analysis for the expression of huMxA with a polyclonal rabbit anti-huMxA antibody (kindly provided by Ilkka Julkunen,

Finland).

Fig.3.Rabies virus repression is not attributable to MX protein-associated cytotoxicity.(A)Encephalomyocarditis virus (EMCV)biological cycle in stimulated/nonstimulated V103,V A2and V A8cell clones is similar,as judged from the titers retrieved by virus yield reduction assay from supernatants sampled 24h pi.Titers (mean T SD)were calculated by the Reed –Muench method and result from 4independent experiments.(B)Viability of nonexpressing cell sheets (nonstimulated V103and V A2)was identical to that of expressing cell sheets (stimulated V103and V A8)for at least 3days.Absorbances (450nm,ref.650nm)refer to the soluble formazan generated by viable cells through bioreduction of an incorporated tetrazolium compound.Values are means T SD from 6independent experiments.

M.Leroy et al./Neurobiology of Disease 21(2006)515–521517

Cytotoxicity assay

The effect of the expression of an exogenous Mx protein on cell viability and metabolism was evaluated by a tetrazolium-based colorimetric method,the[3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay(CellTiter96R Aqueous Assay,Promega).Flat-bottom,96-well plastic microtiter trays were seeded with V103cells in DMEM-10or DMEM-10/doxycycline,Vero-huMxA-expressing cells(clone V A8)or Vero-huMxA-negative cells(V A2).The viability of the cells was quantitated spectrophotometrically at495 nm(reference650nm)using the MTS method according to the instructions of the manufacturer from day0,before stimulation, until day3.

Virus plaque assay

Cell monolayers were infected with several dilutions of the viral stock and incubated for1h at37-C.Unadsorbed viruses were then removed by two washing steps and an overlay agar(DMEM with 2%doxycycline-free FCS,0.4%agar)was added.The cell cultures were incubated until plaques were readily visible(11days),fixed in formaldehyde and stained with1%crystal violet in20%ethanol.

Virus yield reduction assay

Several dilutions of the viral stock were incorporated onto pretreated(doxycycline or vehicle)V103or Vero cells,expressing or not the human MxA protein,monolayers with indicated multiplicities of infection.After1h,unadsorbed viruses were removed by two washings,and fresh medium(DMEM with2% doxycycline-free FCS)was added.After4days of incubation at 37-C,supernatants were sampled and their titers determined onto Vero cell monolayers by the50%tissue culture infective dose method(TCID50).All titers were calculated by the Reed–Muench method.

Immunofluorescence assays

For indirect immunofluorescence,medium was removed24h (SADB19)or48h(SAG2)after infection,cells were washed in PBS and fixed with4%(w/v)paraformaldehyde in PBS(pH7.4) for30min at4-C.Cells were then permeabilized in absolute methanol for6min atà20-C and blocked for1h in washing buffer (1%BSA in PBS)at room temperature.Cells were then probed for 45min with an equine antirabies antiserum FITC-conjugated (VMRD Inc.,Pullman WA).Coverslips were mounted using the Prolong Antifade kit(Molecular Probe),examined with an Olympus BX51fluorescence microscope and photographed using the DP51camera(Olympus,Belgium).

Immunoblotting

Vero cell monolayers were washed three times in PBS and harvested by trypsinization.Cellular proteins were extracted in loading buffer and separated by sodium dodecyl sulphate4–12% polyacrylamide gel electrophoresis.The proteins were then transferred onto a polyvinylidene difluoride membrane(PVDF). For huMxA detection,the membrane was incubated with rabbit anti-huMxA antibodies in Tris-buffered saline(TBS)with0.5?Western blocking reagent(Roche Diagnostics Gmbh,Basel,Switzerland).For rabies virus protein detection,the membrane was incubated with equine antirabies antiserum(1:10).For h-actin,the membrane was stripped(Re-blotTM Plus-Strong, Chemicon International,Temecula,CA)and reprobed with mouse anti-h-actin(1:1000)specific antibodies.Following incubation with the primary antibodies,the membrane was washed in TBS–0.1%(w/v)Tween20and then further incubated with specific HRP-conjugated secondary antibody(1:1000).Immunoreactive bands were revealed on X-ray film(KODAK BioMax MR Film, Amersham Biosciences Europe GmbH,Netherlands)by using chemiluminescent LumiGLO i technology(Cell Signaling Tech-nology,Beverly,USA).

Results and discussion

Several mammalian Mx proteins have been shown to block the replication of VSV,a Rhabdoviridae from the genus Vesiculovirus (Meier et al.,1990;Staeheli and Pavlovic,1991;Schwemmle et al., 1995;Landis et al.,1998;Sandrock et al.,2001;Asano et al.,2002; Baise et al.,2004).Despite the phylogenic proximity of VSV and rabies virus,a Rhabdoviridae from the genus Lyssavirus,

the Fig.4.Rabies virus(SAG2strain)is repressed in Mx-producing cell clones,as judged from the titers retrieved by virus yield reduction assay from supernatants sampled4days pi.Titers(mean T SD)were calculated by the Reed–Muench method and result from3independent experiments.

(A)boMx1-associated repression amounts to2–3log units in stimulated (1A g/ml doxycycline,V103+)compared to nonstimulated(V103à)Vero cells.(B)huMxA-associated repression amounts to¨1.5log unit in expressing(V A8)compared to nonexpressing(V A2)Vero cell clone. Horizontal axis:multiplicity of infection.

M.Leroy et al./Neurobiology of Disease21(2006)515–521 518

repressing capacity of Mx proteins on the latter was not evaluated so far.This question was addressed here by taking advantage of(i) a double transgenic Vero cell clone that allows doxycycline-subordinated conditional expression of boMx1(Figs.1A–C)and (ii)two Vero cell clones stably transfected with pcDNA4/huMxA, one duly expressing the protein(V A8,Figs.1E and F)and another in which expression was extinct(V A2,Figs.1D and F).

Nonstimulated/stimulated V103,V A2and V A8cells were first infected by the SAG2strain of rabies virus.Viral plaque assays unequivocally showed that incorporation of doxycycline24h before infection resulted in a protection of V103monolayers against SAG2-associated cytopathic effect(Fig.2A).Conversely, incorporation of doxycycline onto conventional Vero cells(ATCC/ CCL-81)did not alter the number of plaques(data not shown).To confirm these results,a virus plaque assay was performed and showed a100-fold reduction in viral titer in V103doxycycline-induced monolayers and a10-fold reduction in V A8compared to V A2monolayers.Immunofluorescence analysis was then per-formed48h after infection of the different cell clones.Again, V103stimulated cells showed an important reduction in viral replication(Fig.2C),the reduction being more important than that visible between V A2and V A8cell clones,suggesting that boMx1more strongly repressed rabies virus protein expression than huMxA.In contrast,EMCV-induced cytopathic effect was not different between stimulated and nonstimulated V103or between V A2and V A8cell lines.Moreover,virus yield reduction assay did not show any difference in viral production between the different cell lines(Fig.3A),as observed before for viruses belonging to the Paramyxoviridae family(Leroy et al.,2005).To exclude the hypothesis that the antiviral effect could be due to a cytotoxic effect of Mx expression,an MTS cytotoxicity assay was performed.Clearly,neither boMx1nor huMxA expression altered the global mitochondrial bioreduction potential of the cell monolayers(Fig.3B).

To evaluate the ability of the two Mx proteins to repress the replication of the rabies virus,a virus yield reduction assay was performed after a4-day long infection of stimulated/nonstimulated V103,V A2and V A8cell monolayers at several multiplicities of infection(moi).Again,boMx1expression dramatically repressed viral replication whatever the initial moi had been(Fig.4A),thus confirming the results obtained under agar overlay.Rabies virus yields were reduced100-to1000-fold by boMx1expression, which is quantitatively compatible with the100-to5000-fold reduction observed for VSV in huMxA-or boMx1-expressing

cells Fig.5.Antirabies activity displayed by boMx1-producing cells against SADB19viral strain.(A)Cell clones-specific titers retrieved from direct viral plaque assay after the SADB19inoculum was allowed to form plaques under soft agar for11days.Titers(mean T SD)were calculated by direct plaque assay(PFU/ml) and result from3independent experiments.(B)SADB19strain of rabies virus repression by boMx1but not by huMxA,as judged from direct immunofluorescence24h pi,for successive10-fold dilutions of the inoculum.See preceding figures and text for key.

M.Leroy et al./Neurobiology of Disease21(2006)515–521519

respectively(Schnorr et al.,1993;Landis et al.,1998;Baise et al., 2004).The repression conferred by huMxA was a little weaker, roughly a100-fold reduction(Fig.4B).

To confirm these results,we tested the effect of both Mx proteins against another rabies strain,SADB19.As expected,viral plaque assays unequivocally showed that incorporation of dox-ycycline24h before infection resulted in a100-fold reduction of viral replication in V103cells(Fig.5A).Conversely,expression of huMxA(VA8)did not confer any protection against viral replication in comparison with V A2cell monolayers.These results were further confirmed through a direct immunofluorescence assay in which V103cell monolayers were infected with/without doxycycline conditioning with successive10-fold dilutions and labeled24h after infection.As expected,doxycycline pretreatment resulted in a dramatic reduction in viral replication(Fig.5B,top series).A parallel experiment with clones V A2and V A8failed to highlight a difference in rabies protein expression,thus confirming that huMxA is not able to interfere with the rabies strain SADB19 (Fig.5B,bottom series).

The reduction of viral replication was also sought at the protein level.Immunofluorescence analysis of SAG2virus revealed a drastic inhibition of viral protein accumulation in the cytoplasm of stimulated V103and V A8cell monolayers48h pi,the proportion of positive cells being far more important in negative cells(Fig. 2C).For the SADB19strain,immunofluorescence was performed 24h after infection with several different dilutions of the stock virus.At a dilution of10à3,only stimulated V103revealed a reduction of the proportion of immunopositive cells,the difference being more important at a10à4dilution.

To compare the level of protein synthesis for both virus strains in boMx1-and huMxA-expressing cells,a Western blot analysis was performed.Cells were infected at a moi of1for48(SAG2)or 24h(SADB19).Although both stimulated V103and V A8were able to inhibit the synthesis of SAG2nucleoprotein,the repression was almost complete in stimulated V103cells but only partial in V A8cells(Fig.6A).Western blot analysis of SADB19-infected cell monolayers again revealed an important reduction of virus nucleoprotein expression in stimulated V103cells,whereas it was not affected at all by huMxA,as judged from the nearly identical quantity retrieved in both V A8and V A2cells.

Taken together,the results suggest that the previously reported repression of viral spread and subsequent development of symptoms by type1IFNs(Marcovistz et al.,1986)could at least partially be attributable to the Mx pathway.However,there is substantial evidence that boMx1and huMxA not equally affect the biological cycle of the rabies virus,the former exercising a stronger effect that is strain-independent.It is therefore anticipated that naturally occurring infections with rabies virus could be specifi-cally repressed by specific Mx isoform(s)rather than by Mx proteins in general,and the previously published protection conferred by IFN a/h could then result from yet uncharacterized pathways.

As this is the first observation that an Mx protein represses rabies virus,the molecular mechanism involved remains to be identified.It is nevertheless noteworthy that human MxA has been shown to inhibit VSV primary transcription in cell cultures (Staeheli and Pavlovic,1991)as well as in vitro(Schwemmle et al.,1995),suggesting that Mx proteins alter VSV polymerase functioning.However,as rat intranuclear Mx1blocks the strictly cytoplasmic replication cycle of VSV(Meier et al.,1990),a direct molecular interaction may not be the rule.Further,as a single Mx protein(the human isoform MxA)can display a wide array of antiviral strategies(inhibition of transportation of incoming Thogotovirus nucleocapsids,slowing down of VSV primary transcription,partial inhibition of Morbillivirus mRNA translation or sequestration of La Crosse virus nucleocapsid proteins in cytoplasmic inclusions(Staeheli and Pavlovic,1991;Schnorr et al., 1993;Schwemmle et al.,1995;Kochs and Haller,1999;Kochs

et Fig.6.Repression of rabies virus nucleoprotein expression by boMx1and huMxA.(A)Monolayers of the4cell clones used in the study were infected for48h at a multiplicity of infection of1with the SAG2strain,then cell extracts were immunoblotted and tagged using monoclonal antiactin and polyclonal antirabies antibodies.(B)Same experiment,but after infection with the SADB19strain for24h.See preceding figures and text for key.

M.Leroy et al./Neurobiology of Disease21(2006)515–521

520

al.,2002),it is possible that boMx1could be unique in its way to interfere with,or even ability to significantly repress,rabies virus. Should this specificity be confirmed and the results replicated in vivo,then an avenue would be opened for the development of new antirabies therapeutic strategies.

Acknowledgments

The authors thank Dr.Christine Tuffereau for kindly providing the rabies virus strain SAG2and Dr.Adriaan V os for kindly providing the rabies virus strain SADB19.We thank Ilkka Julkunen for kindly providing the rabbit antiserum directed against human MXA and Otto Haller for the generous gift of HuMxA-containing plasmid.We also wish to thank Kris De Clercq and Nesya Goris(CODA/CERV A,Brussel,Belgium)for their helpful discussion and suggestions.

References

Arnheiter,H.,Frese,M.,Kambadur,R.,Meier,E.,Haller,O.,1996.Mx transgenic mice:animal models of health.Curr.Top.Microbiol.

Immunol.206,119–147.

Asano, A.,Ko,J.H.,Morozumi,T.,Hamashima,N.,Watanabe,T., 2002.Polymorphisms and the antiviral property of porcine Mx1 protein.J.Vet.Med.Sci.64,1085–1089.

Baer,G.M.,Cleary,W.F.,Diaz,A.M.,Perl,D.F.,1977.Characteristics of11 rabies virus isolates in mice:titers and relative invasiveness of virus, inoculation period of infection,and survival of mice with sequelae.

J.Infect.Dis.136,336–345.

Baise,E.,Pire,G.,Leroy,M.,Gerardin,J.,Goris,N.,De Clerck,K., Kerkhofs,P.,Desmecht,D.,2004.Conditional expression of bovine MX1GTPase in a stable transgenic Vero cell line interferes with replication of vesicular stomatitis virus.J.Interferon Cytokine Res.24, 513–521.

Foster,G.R.,1997.Interferons in host defense.Semin.Liver Dis.17, 287–295.

Haller,O.,Frese,M.,Kochs,G.,1998.Mx proteins:mediators of innate resistance to RNA viruses.Rev.Sci.Tech.17,220–230. Hilfenhaus,J.,Weinmann,E.,Majer,M.,Barth,R.,Jaeger,O.,1977.

Administration of human interferon to rabies virus-infected monkeys after exposure.J.Infect.Dis.135,846–849.

Hooper, D.C.,Morimoto,K.,Bette,M.,Weihe, E.,Koprowki,H., Dietzschold,B.,1998.Collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system.J.Virol.72, 3711–3719.Jackson,A.C.,2003.Rabies virus infection:an update.J.NeuroVirol.9, 253–258.

Katze,M.G.,Wambach,M.,Wong,M.L.,Garfinkel,M.,Meurs,E.,Chong, K.,Williams,B.R.,Hovanessian,A.G.,Barber,G.N.,1991.Functional expression and RNA binding analysis of the interferon-induced,double-stranded RNA-activated,68,000-Mr protein kinase in a cell-free system.

Mol.Cell.Biol.11,5497–5505.

Kochs,G.,Haller,O.,1999.Interferon-induced human MxA GTPase blocks nuclear import of Thogotovirus nucleocapsids.Proc.Natl.Acad.

Sci.U.S.A.96,2082–2086.

Kochs,G.,Janzen,C.,Hohenberg,H.,Haller,O.,2002.Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes.Proc.Natl.Acad.Sci.U.S.A.99,3153–3158. Lafay,F.,Benejean,J.,Tuffereau,C.,Flamand,A.,Coulon,P.,1994.

Vaccination against rabies:construction and characterization of SAG2,a double avirulent derivative of SADBern.Vaccine12,317–320. Landis,H.,Simon-Jodicke,A.,Kloti,A.,Di Paolo,C.,Schnorr,J.-J., Schneider-Schaulies,S.,Peter Hefti,H.,Pavlovic,J.,1998.Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins.J.Virol.72,1516–1522.

Leroy,M.,Baise,E.,Pire,G.,Ge′rardin,J.,Desmecht,D.,2005.Resistance of Paramyxoviridae to type I interferon-induced Bos taurus Mx1 dynamin.J.Interferon Cytokine Res.25,192–201.

Lodmell,D.L.,Arai,Y.T.,Ewalt,L.C.,1981.Influence of cell type and virus upon lysis of rabies virus-infected cells by antibody and comple-ment.Arch.Virol.70,147–155.

Marcovistz,R.,Galabru,J.,Tsiang,H.,Hovanessian, A.G.,1986.

Neutralization of interferon produced early during rabies virus infection in mice.J.Gen.Virol.67,387–390.

Meier,E.,Kunz,G.,Haller,O.,Arnheiter,H.,1990.Activity of rat Mx proteins against a rhabdovirus.J.Virol.64,6263–6269. Mendonca,R.Z.,Pereira,C.A.,1994.Relationship of interferon synthesis and the resistance of mice infected with street rabies virus.Braz.J.Med.

Biol.Res.27,691–695.

Murphy,F.A.,1977.Rabies pathogenesis:brief review.Arch.Virol.54, 279–297.

Sandrock,M.,Frese,M.,Haller,O.,Kochs,G.,2001.Interferon-induced rat Mx proteins confer resistance to Rift Valley fever virus and other arthropod-borne viruses.J.Interferon Cytokine Res.21,663–668. Schnorr,J.J.,Schneider-Schaulies,S.,Simon-Jodicke, A.,Pavlovic,J., Horisberger,M.A.,ter Meulen,V.,1993.MxA-dependent inhibition of measles virus glycoprotein synthesis in a stably transfected human monocytic cell line.J.Virol.67,4760–4768.

Schwemmle,M.,Weining,K.C.,Richter,M.F.,Schumacher,B.,Staeheli, P.,1995.Vesicular stomatitis virus transcription inhibited by purified MxA protein.Virology206,545–554.

Staeheli,P.,Pavlovic,J.,1991.Inhibition of vesicular stomatitis virus mRNA synthesis by human MXA protein.J.Virol.65,4498–4501.

M.Leroy et al./Neurobiology of Disease21(2006)515–521521

The way常见用法

The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法）举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法：在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because

The way的用法及其含义(二)

The way的用法及其含义（二）二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中

(完整版)the的用法

定冠词the的用法：定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...

“the way+从句”结构的意义及用法

“tｈｅwａｙ+从句”结构的意义及用法首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法

不对。Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.

way 用法

表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如：我不喜欢他说话的态度。正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。一、in a way的用法表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。三、by the way的用法

The way的用法及其含义(一)

The way的用法及其含义（一）有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析：一、the way作先行词，后接定语从句以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.

way 的用法

way 的用法【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；

the-way-的用法讲解学习

t h e-w a y-的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

way的用法总结大全

way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。

way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句

the_way的用法大全教案资料

t h e_w a y的用法大全

The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的

the way 的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.

The way的用法及其含义(三)

The way的用法及其含义（三）三、the way的语义 1. the way=as（像） Please do it the way I’ve told you.请按照我告诉你的那样做。 I'm talking to you just the way I'd talk to a boy of my own.我和你说话就像和自己孩子说话一样。 Plant need water the way they need sun light. 植物需要水就像它们需要阳光一样。 2. the way=how（怎样，多么） No one can imagine the way he misses her.没人能够想象出他是多么想念她！ I want to find out the way a volcano has formed.我想弄清楚火山是怎样形成的。 He was filled with anger at the way he had been treated.他因遭受如此待遇而怒火满腔。That’s the way she speaks.她就是那样讲话的。 3. the way=according as （根据） The way you answer the questions, you must be an excellent student.从你回答问题来看，你一定是名优秀的学生。 The way most people look at you, you'd think a trash man was a monster.从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物。 The way I look at it, it’s not what you do that matters so much.依我看，重要的并不是你做什么。 I might have been his son the way he talked.根据他说话的样子，好像我是他的儿子一样。One would think these men owned the earth the way they behave.他们这样行动，人家竟会以为他们是地球的主人。

way的用法

一．Way：“方式”、“方法” 1.表示用某种方法或按某种方式 Do it (in) your own way. Please do not talk (in) that way. 2.表示做某事的方式或方法 It’s the best way of studying [to study] English.。 There are different ways to do [of doing] it. 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句正：I don’t like the way he spoke. I don’t like the way that he spoke. I don’t like the way in which he spoke.误：I don’t like the way how he spoke. 4. the way 的从句 That’s the way (=how) he spoke. I know where you are from by the way you pronounce my name. That was the way minority nationalities were treated in old China. Nobody else loves you the way(=as) I do. He did not do it the way his friend did. 二．固定搭配 1. In a/one way：In a way he was right. 2. In the way /get in one’s way I'm afraid your car is in the way， If you are not going to help，at least don't get in the way. You'll have to move－you're in my way. 3. in no way Theory can in no way be separated from practice. 4. On the way (to……) Let’s wait a few moments. He is on the way Spring is on the way. Radio forecasts said a sixth-grade wind was on the way. She has two children with another one on the way. 5. By the way By the way，do you know where Mary lives? 6. By way of Learn English by way of watching US TV series. 8. under way 1. Elbow one’s way He elbowed his way to the front of the queue. 2. shoulder one’s way 3. feel one‘s way 摸索着向前走；We couldn’t see anything in the cave, so we had to feel our way out 4. fight/force one’s way 突破。。。而前进The surrounded soldiers fought their way out. 5.. push/thrust one‘s way（在人群中）挤出一条路He pushed his way through the crowd. 6. wind one’s way 蜿蜒前进 7. lead the way 带路，领路；示范 8. lose one‘s way 迷失方向 9. clear the way 排除障碍，开路迷路 10. make one’s way 前进，行进The team slowly made their way through the jungle.

the way的用法大全

在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms.

“the-way+从句”结构的意义及用法知识讲解

“the way+从句”结构的意义及用法首先让我们来看下面这个句子： Read the following passage and talk about it with your classmates. Try to tell what you think of Tom and of the way the children treated him. 在这个句子中，the way是先行词，后面是省略了关系副词that 或in which的定语从句。下面我们将叙述“the way+从句”结构的用法。 1．the way之后，引导定语从句的关系词是that而不是how,因此，<<现代英语惯用法词典>>中所给出的下面两个句子是错误的：This is the way how it happened. This is the way how he always treats me. 2. 在正式语体中，that可被in which所代替；在非正式语体中，that则往往省略。由此我们得到the way后接定语从句时的三种模式：1) the way +that-从句2) the way +in which-从句3) the way +从句例如：The way(in which ,that) these comrades look at problems is wrong.这些同志看问题的方法不对。

The way(that ,in which)you’re doing it is completely crazy.你这么个干法，简直发疯。 We admired him for the way in which he faces difficulties. Wallace and Darwin greed on the way in which different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way (that) he did it. I liked the way (that) she organized the meeting. 3.the way(that)有时可以与how(作“如何”解)通用。例如： That’s the way (that) she spoke. = That’s how she spoke. I should like to know the way/how you learned to master the fundamental technique within so short a time. 4．the way的其它用法：以上我们讲的都是用作先行词的the way，下面我们将叙述它的一些用法。

定冠词the的12种用法

定冠词the的12种用法定冠词the 的12 种用法，全知道?快来一起学习吧。下面就和大家分享，来欣赏一下吧。定冠词the 的12 种用法，全知道? 定冠词the用在各种名词前面，目的是对这个名词做个记号，表示它的特指属性。所以在词汇表中，定冠词the 的词义是“这个，那个，这些，那些”，可见，the 即可以放在可数名词前，也可以修饰不可数名词，the 后面的名词可以是单数，也可以是复数。定冠词的基本用法： (1) 表示对某人、某物进行特指，所谓的特指就是“不是别的，就是那个!”如： The girl with a red cap is Susan. 戴了个红帽子的女孩是苏珊。 (2) 一旦用到the，表示谈话的俩人都知道说的谁、说的啥。如：

The dog is sick. 狗狗病了。(双方都知道是哪一只狗) (3) 前面提到过的，后文又提到。如： There is a cat in the tree.Thecat is black. 树上有一只猫，猫是黑色的。 (4) 表示世界上唯一的事物。如： The Great Wall is a wonder.万里长城是个奇迹。(5) 方位名词前。如： thenorth of the Yangtze River 长江以北地区 (6) 在序数词和形容词最高级的前面。如： Who is the first?谁第一个? Sam is the tallest.山姆最高。但是不能认为，最高级前必须加the，如： My best friend. 我最好的朋友。 (7) 在乐器前。如： play the flute 吹笛子

Way的用法

Way用法 A:I think you should phone Jenny and say sorry to her. B:_______. It was her fault. A. No way B. Not possible C. No chance D. Not at all 说明：正确答案是A. No way，意思是“别想！没门！决不！” 我认为你应该打电话给珍妮并向她道歉。没门！这是她的错。再看两个关于no way的例句：（1）Give up our tea break? NO way! 让我们放弃喝茶的休息时间？没门儿！（2）No way will I go on working for that boss. 我决不再给那个老板干了。 way一词含义丰富，由它构成的短语用法也很灵活。为了便于同学们掌握和用好它，现结合实例将其用法归纳如下：一、way的含义 1. 路线

He asked me the way to London. 他问我去伦敦的路。 We had to pick our way along the muddy track. 我们不得不在泥泞的小道上择路而行。 2. （沿某）方向 Look this way, please. 请往这边看。 Kindly step this way, ladies and gentlemen. 女士们、先生们，请这边走。 Look both ways before crossing the road. 过马路前向两边看一看。 Make sure that the sign is right way up. 一定要把符号的上下弄对。 3. 道、路、街，常用以构成复合词 a highway（公路），a waterway（水路），a railway（铁路），wayside（路边）

way与time的特殊用法

way/time的特殊用法 1、当先行词是way意思为”方式.方法”的时候,引导定语从句的关系词有下列3种形式： Way在从句中做宾语 The way that / which he explained to us is quite simple. Way在从句中做状语 The way t hat /in which he explained the sentence to us is quite simple. 2、当先行词是time时,若time表示次数时,应用关系代词that引导定语从句,that可以省略; 若time表示”一段时间”讲时,应用关系副词when或介词at/during + which引导定语从句 1.Is this factory _______ we visited last year? 2.Is this the factory-------we visited last year? A. where B in which C the one D which 3. This is the last time _________ I shall give you a lesson. A. when B that C which D in which 4.I don’t like the way ________ you laugh at her. A . that B on which C which D as 5.He didn’t understand the wa y ________ I worked out the problem. A which B in which C where D what 6.I could hardly remember how many times----I’ve failed. A that B which C in which D when 7.This is the second time--------the president has visited the country. A which B where C that D in which 8.This was at a time------there were no televisions, no computers or radios. A what B when C which D that