Inhibitor NS-2009-Novel influenza virus NS1 antagonists

J OURNAL OF V IROLOGY,Feb.2009,p.1881–1891Vol.83,No.4 0022-538X/09/$08.00?0doi:10.1128/JVI.01805-08

Copyright?2009,American Society for Microbiology.All Rights Reserved.

Novel In?uenza Virus NS1Antagonists Block Replication and Restore

Innate Immune Function??

Dipanwita Basu,1Marcin P.Walkiewicz,1Matthew Frieman,2Ralph S.Baric,2

David T.Auble,3and Daniel A.Engel1*

Department of Microbiology,University of Virginia School of Medicine,Charlottesville,Virginia229081;Department of Epidemiology, School of Public Health,University of North Carolina at Chapel Hill,Chapel Hill,North Carolina275992;and Department of

Biochemistry and Molecular Genetics,University of Virginia School of Medicine,Charlottesville,Virginia229083

Received27August2008/Accepted24November2008

The innate immune system guards against virus infection through a variety of mechanisms including

mobilization of the host interferon system,which attacks viral products mainly at a posttranscriptional level.

The in?uenza virus NS1protein is a multifunctional facilitator of virus replication,one of whose actions is to

antagonize the interferon response.Since NS1is required for ef?cient virus replication,it was reasoned that

chemical inhibitors of this protein could be used to further understand virus-host interactions and also serve

as potential new antiviral agents.A yeast-based assay was developed to identify compounds that phenotypically

suppress NS1function.Several such compounds exhibited signi?cant activity speci?cally against in?uenza A

virus in cell culture but had no effect on the replication of another RNA virus,respiratory syncytial virus.

Interestingly,cells lacking an interferon response were drug resistant,suggesting that the compounds block

interactions between NS1and the interferon system.Accordingly,the compounds reversed the inhibition of

beta interferon mRNA induction during infection,which is known to be caused by NS1.In addition,the

compounds blocked the ability of NS1protein to inhibit double-stranded RNA-dependent activation of a

transfected beta interferon promoter construct.The effects of the compounds were speci?c to NS1,because they

had no effect on the ability of the severe acute respiratory syndrome coronavirus papainlike protease protein

to block beta interferon promoter activation.These data demonstrate that the function of NS1can be

modulated by chemical inhibitors and that such inhibitors will be useful as probes of biological function and

as starting points for clinical drug development.

In?uenza is associated with signi?cant morbidity and mor-tality and is a continuing worldwide public health problem. Seasonal in?uenza epidemics affect ca.5to15%of the world’s population,and estimates of annual mortality range from 250,000to500,000(75),including approximately30,000deaths and200,000hospitalizations in the United States(68).Groups at high risk include the elderly,the very young,and those suffering from chronic illness.Medical complications include pneumonia and exacerbation of symptoms associated with chronic illness(60).

In the20th century,three in?uenza pandemics were recorded—in 1918,1957,and1968.The1918pandemic was the most severe and was responsible for an estimated20to40million deaths, including a signi?cant percentage of young adults(58,67).The epidemiology of transmission and the genetics of the in?uenza viruses make it likely that additional pandemics will occur due to emergence of new strains,for which the world’s healthcare network is not yet prepared(16,50,64).In this regard the spread of H5N1among avian species and sporadic spillage into humans has attracted much attention(48,51).Whereas this virus has not yet acquired the ability to transmit from person to person,the small number of humans infected by H5N1due to direct contact with birds has revealed a dangerously high rate of mortality,ca.60%(1,16).

Control of seasonal in?uenza is an ongoing challenge(73). Due to antigenic drift the widely used seasonal vaccine is unevenly effective from year to year,and its use is lower than optimal even in developed countries such as the United States (7,46).There are currently two classes of anti-in?uenza virus drugs that have been used effectively in prevention and treat-ment.These drugs target the viral M2ion channel(e.g.,aman-tadine)and neuraminidase proteins(e.g.,oseltamivir),respec-tively(25,44).Despite these successes there remain concerns regarding drug ef?cacy,resistance,and cost(26).

In light of the continuing threat to public health,the current state of prevention and treatment options,and the likelihood of emergence of a pandemic strain for which the human pop-ulation is immunologically unprepared,it makes sense to at-tempt to develop novel antiviral agents that could be used alone or in combination with existing modalities of treatment. Such agents could take advantage of steps in the virus repli-cative cycle that have not yet been exploited pharmacologi-cally.These agents could also be designed to attack cellular functions that are required to support virus replication or to enhance the host innate or adaptive immune responses.Novel agents that block virus replication could also be used as mo-lecular probes of the biology of the virus,as well as virus-host interactions.

We have explored the use of a novel target for the develop-ment of anti-in?uenza virus compounds,the NS1protein.NS1

*Corresponding author.Mailing address:Department of Microbi-ology,University of Virginia School of Medicine,1300Jefferson Park Ave.,Charlottesville,VA22908.Phone:(434)924-8633.Fax:(434) 982-1071.E-mail:dae2s@https://www.wendangku.net/doc/1f2249640.html,.

?Supplemental material for this article may be found at http://jvi

https://www.wendangku.net/doc/1f2249640.html,/.

?Published ahead of print on3December2008.

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is a nonstructural protein encoded by segment8of in?uenza virus A.Genetic analyses of NS1have shown that viral repli-cation,spread,and pathogenesis are very dependent on the function of this protein(3,4,6,10,11,13,17,18,22,27,29,30, 36,63,66,74).This satis?es an important criterion for an anti-in?uenza virus target,since drugs that inhibit the action of the target must be able to slow virus production and/or patho-genesis as a consequence.Several interesting functions for NS1 have been described.NS1is an RNA-binding protein that can interact with a variety of RNA species,including double-stranded RNA(dsRNA)(10,21,23,24,54–56,70).Binding of NS1to dsRNA inhibits the2-5A oligoadenylase/RNase L path-way for degradation of viral RNAs,blocks the activity of tran-scription factor pathways that depend on dsRNA,and inhibits activation of cellular PKR,to which NS1also binds directly (14,20,32,35,37,41–43,52,65,71).NS1also inhibits the induction of RNA interference through its ability to sequester small interfering RNAs(5,33).NS1modi?es cellular pre-mRNA processing,including3?-end formation,by binding to the30-kDa subunit of CPSF(for cleavage and polyadenylation speci?city factor)and binding to poly(A)-binding protein II (31).It also inhibits cellular pre-mRNA splicing(69)and blocks nuclear RNA export by associating with several cellular proteins that mediate RNA export(15,55,61).Some of the functions of NS1serve to inhibit the host antiviral response that is mediated by interferon(IFN)(18;reviewed in refer-ences14and31).For instance,NS1blocks induction of IFN gene transcription and mRNA maturation,thereby preventing the cell from mounting an ef?cient innate defense(47,65,71). Other functions of NS1act to inhibit host cell gene expression so as to favor viral gene expression,such as effects on cellular RNA metabolism and export.

NS1is a functionally complex protein and is a central player in the virus’s response to host defense mechanisms and the establishment of ef?cient viral gene expression.Because of its importance to virus replication and virus-host interactions and the fact that it is highly conserved across in?uenza virus A strains(19,62),NS1seems a particularly good target for drug discovery.We report here the identi?cation of chemical com-pounds that inhibit both NS1function and in?uenza virus replication.

MATERIALS AND METHODS

Mammalian cells and viruses.Vero E6,MA104,and293cells were main-tained in Dulbecco modi?ed Eagle medium supplemented with10%fetal bovine serum.MDCK cells were maintained in Iscove medium supplemented with10% fetal bovine serum and2mM L-glutamine.All media and sera were from Invitrogen.For infections,viral stocks were diluted in growth medium supple-mented with0.3%bovine serum albumin,0.22%sodium bicarbonate,and0.25U of TPCK(tolylsulfonyl phenylalanyl chloromethyl ketone)-trypsin(Invitrogen)/ ml.In?uenza viruses A/PR/8/34(PR),A/WSN/33(WSN),and A/Tx/36/91(TX) were gifts from Adolfo Garcia-Sastre,and A/HK/19/68(HK)was a gift from Tom Braciale.The viruses were propagated in10-day-old embryonated chicken eggs at37°C.Mutant delNS1(a gift from Adolfo Garcia-Sastre)was propagated in MDCK cells containing a stably transfected NS1PR gene(a gift from Luis Mar-tinez-Sobrido and Adolfo Garcia-Sastre).The titered stock of respiratory syn-cytial virus(RSV)strain RS?sh(49)was a gift from Gail Wertz.Titers of in?uenza virus stocks were determined by50%tissue culture infective dose (TCID50)analysis on MDCK cells using the hemagglutination assay protocol of Reed and Muench(57).

Plasmids.A full-length NS1cDNA from A/WSN/33(pCAGGS-NS1,a gift from Peter Palese)was used to PCR amplify NS1for cloning into the galactose-inducible yeast expression vector pYES2(Invitrogen)to create pYES-NS1.cDNAs encoding NS1from A/Tx/36/91and A/PR/8/34under the control of chicken?-actin promoter[pCAGGS-NS1(Tx/91)and pCAGGS-NS1(PR/34)] and a reporter plasmid encoding?re?y luciferase under the control of IFN-?promoter(p125-Luc[76])were kindly provided by Adolfo Garcia-Sastre.Severe acute respiratory syndrome(SARS)papainlike protease(PLP)was expressed from a CAGGS plasmid.

Yeast strains and growth.Strain9526-6-2(MAT a his3?1leu2?0lys2?0ura3?0 pdr1::KanMX4pdr3::KanMX4)was a gift from Dan Burke.It was derived by tetrad dissection from two parent strains that had been modi?ed by one step gene replacements.The pdr1::KanMX4was constructed in BY4741(MAT a his3?1leu2?0met15?0ura3?0)and the pdr3::KanMX4was constructed in BY4742(MAT?his3?1leu2?0met15?0ura3?0).PCR-mediated one-step gene replacements,matings and tetrad dissections were performed as described pre-viously(2).Strains9526-6-2/pYES2and9526-6-2/pYES-NS1were generated by transformation of9526-6-2with the plasmids pYES2and pYES-NS1,respec-tively,and were maintained on synthetic complete medium(SC)lacking uracil. For growth experiments and library screening,a single transformed colony was grown overnight,and the cell number was determined by using a Coulter counter (Beckman Coulter Corp.).The cells were diluted to5?105cells/ml in SC lacking uracil and containing raf?nose and2%galactose.A95-?l portion of this culture was added to5?l of preplated test compounds in96-well plates such that the?nal drug concentration was50?M and the?nal dimethyl sulfoxide(DMSO) concentration was1%.The Diversity Set library(National Cancer Institute Developmental Therapeutics Program)was used for the drug screen.It was provided as10mM stocks in100%DMSO.Optical density readings at600nm (OD600)were taken every12h for60h using a Thermomax microplate reader (Molecular Devices).

Virus replication assays.Con?uent cell monolayers were infected at a multi-plicity of infection(MOI)of0.1for48h in the presence or absence of drug. Compounds were added at the beginning of infection and were present through-out the infection.After48h virus titers were determined by TCID50analysis as described previously(57).Growth of RS?sh was quanti?ed by TCID50analysis by scoring green?uorescent protein(GFP)?uorescence at4days postinfection. Reverse transcriptase PCR(RT-PCR).MDCK cells were infected for6h with PR or delNS1at an MOI of2.0in the presence or absence of drug,and the total RNA was isolated by using RNeasy(Qiagen).For?rst-strand cDNA synthesis,2?g of total RNA was primed with random nanomers(New England Biolabs)at a?nal concentration of2?M.Reverse transcription was performed with10U of Moloney murine leukemia virus RT(New England Biolabs)/?l in the presence of 1U of RNase inhibitor/?l.Thereafter,1/20volume of cDNA was used as a template for PCR(30cycles).The following primer pairs were used:canine IFN-?(accession no.XM538679),CCAGTTCCAGAAGGAGGACA and CCT GTTGTCCCAGGTGAAGT;NS1from A/PR/8/34(accession no.J02150),CT TCGCCGAGATCAGAAATC and TGGACCATTCCCTTGACATT;M2from A/PR/8/34(accession no.V01099),ATGATCTTCTTGAAAATTTGC and CT CCAGCTCTATGCTGAC;and canine?-actin(accession no.XM536230),GG CATCCTGACCCTGAAGTA and GGGGTGTTGAAAGTCTCGAA. Luciferase reporter assay.293cells were transfected with0.2?g of p125Luc and0.8?g of pCAGGS-NS1(Tx/91)or pCAGGS-NS1(PR/34)using Polyfect transfection reagent(Qiagen).At16h posttransfection the cells were stimulated with50?g of poly[IC](Sigma-Aldrich)/ml and treated with50?M drug for24h. The cells were lysed with1?passive lysis buffer(Promega),and20?l of the lysate was used to measure luciferase activity using the luciferase assay system (Promega)according to the manufacturer’s protocol.For SARS-CoV PLP ex-periments,the luciferase plasmid alone or together with a SARS PLP-expressing plasmid(100ng/well each plasmid)was transfected into293T cells in triplicate using Fugene6(Roche).At6h postinfection,the cells were treated with each drug and allowed to incubate for an additional18h.At24h posttransfection,500 ng of poly[IC]/well was transfected into cells by using Lipofectamine(Invitrogen) to induce IFN-?gene transcription.At6h after poly[IC]addition,the cells were lysed in Easy-Glo lysis reagent and quantitated.All transfection experiments were conducted in triplicate.

Protein labeling and Western blot analysis.Con?uent monolayers of MDCK cells in35-mm plates were infected at an MOI of0.1with A/HK/19/68in the presence or absence of50?M https://www.wendangku.net/doc/1f2249640.html,pounds were added at the beginning of infection and were present throughout the infection.The cells were labeled with[35S]methionine and[35S]cysteine for the?nal hour of infection. After24h the medium was replaced with Dulbecco modi?ed Eagle medium lacking L-cysteine and L-methionine(Sigma-Aldrich)and containing10%fetal bovine serum.After30min of starvation the cells were labeled for1h with25 mCi of Express35S35S protein labeling mix(Perkin-Elmer)/https://www.wendangku.net/doc/1f2249640.html,beled cells were washed with phosphate-buffered saline and lysed using passive lysis buffer(Pro-mega).The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE)and visualized by autoradiography using a Storm Phosphorimager (GE Healthcare Life Sciences).

For analyzing NS1expression in yeast,cells transformed with pYES-NS1were grown in SC containing raf?nose and lacking uracil to an OD 600of 0.6to 0.7.The cells were then induced by replacing the medium with the identical medium but containing 2%galactose,in the presence or absence of drug.Lysates were prepared after 8h.A total of 20?g of each sample was resolved by SDS-PAGE,followed by Western blotting and probing with a 1:1,000dilution of rabbit polyclonal antiserum ?-NS1(a gift from Peter Palese).To detect the expression levels of NS1in transfected cells,293cells were lysed by using passive lysis buffer and subjected to SDS-PAGE and Western blotting.The blots were probed with ?-NS1at a dilution of 1:1,000.As a loading control,the blots were also probed with a 1:2,000dilution of monoclonal ?-tubulin antibody (Sigma-Aldrich).

Cytotoxicity assay.To determine the cell viability,a trypan blue dye exclusion test was used.MDCK-UK,VeroE6,293,and MA104cells at 3?105cells/ml were seeded in the presence or absence of increasing concentrations of com-pounds and incubated for 48h.Aliquots of trypsin-treated cells were mixed with an equal volume of phosphate-buffered saline containing 0.4%trypan blue.Cells that excluded the dye were counted by using a hemacytometer.The experiment was performed in duplicate.

RESULTS

Screen for compounds that inhibit NS1function.Ward et al.demonstrated that Saccharomyces cerevisiae strains expressing NS1protein from in?uenza virus A exhibit a pronounced slow-growth phenotype (72).We investigated whether an NS1-expressing yeast strain could be used to screen for small mol-ecules that suppress the slow-growth phenotype by inhibition

of NS1function.A test strain was generated that carries null alleles for two genes that control drug ef?ux,PDR1and PDR3,thus allowing ef?cient retention of small molecules (59).This in turn was used to create a strain expressing NS1from A/WSN/33under the control of the GAL1promoter,which is inducible by galactose.Shown in Fig.1A are spot tests of the control and NS1-expressing strains.Growth on medium con-taining galactose resulted in strong growth inhibition of the NS1-expressing strain but not of the control strain,whereas there was no difference between the two strains on glucose-containing medium.

Time course experiments were performed using a 96-well format to establish conditions for the drug screen.Cells were plated at 5?105cells/ml in either medium containing raf?nose as the sole carbon source or medium containing raf?nose plus 2%galactose to induce the expression of NS1.Figure 1B shows growth curves for the control and NS1-expressing strains under these conditions over a 68-h period.The NS1-expressing strain grew signi?cantly more slowly than the control strain in the presence of galactose.These data indicated a time period be-tween 36and 48h,during which a signi?cant growth differen-tial could be exploited in a screen for small molecules that suppress the slow-growth phenotype.

To perform the screen,cells from an overnight culture were plated at 5?105cells/ml in galactose-containing medium

in

FIG.1.Assay development and screening results.(A)Tenfold serial dilutions (left to right)of strains transformed with the indicated expression plasmids were spotted on plates containing either glucose or galactose as the sole carbon source,and the plates were incubated for 3days at 28°C.(B)Next,100?l cultures of the indicated strains at 5?105cells/ml were incubated at 28°C for the times shown.The OD was measured by using a 96-well plate reader.Values shown are with blank (medium alone)subtracted.NS1,9526-6-2/pYES-NS1;pYES,9526-6-2/pYES2;Raf,raf?nose;Gal,galactose.(C)Strain 9526-6-2/pYES-NS1was grown in medium containing raf?nose plus 2%galactose in the presence of 50?M drug or 1%DMSO for the indicated times at 28°C.The ?nal DMSO concentration in the drug-treated cultures was also 1%.(D)Western blot of whole-cell lysates of 9526-6-2/pYES-NS1grown in the absence (uninduced)or presence (induced)of 2%galactose and either 1%DMSO or the indicated compounds at 50?M for 8h.The lysates were separated by SDS-PAGE and blotted for the presence of NS1.The band near the 37-kDa marker is nonspeci?c.

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the presence 50?M drug or 1%DMSO as a control.Approx-imately 2,000compounds from the National Cancer Institute Diversity Set library (see https://www.wendangku.net/doc/1f2249640.html,/branches/dscb /diversity_explanation.html)were screened manually.Optical density readings were taken every 12h for 60h.This screen resulted in 15hits,9of which proved to be reproducible when independent samples were obtained from the NCI.The nine hits produced a ?1.5-fold increase in OD over at least two consecutive OD readings during the time course (data not shown).Of these,four were studied further based on their ability to inhibit in?uenza virus replication (see below).Of the remaining ?ve,two were toxic to MDCK cells and three showed no activity against in?uenza virus replication (not shown).Shown in Fig.1C are individual growth curves for galactose-containing cultures of the NS1-expressing strain treated with each of the positive compounds.Their structures are shown in Fig.2.

One possible mechanism for suppression of the NS1-in-duced growth defect in yeast could be a decrease in NS1protein expression triggered by addition of the drugs.For ex-ample,this could be due to drug effects on transcription from the GAL1promoter,on plasmid replication or metabolism,or on stability of NS1RNA or protein.To investigate whether any of the compounds had such an effect,yeast cells expressing NS1were grown in the presence of drug for 8h and analyzed for protein expression by Western blotting.As shown in Fig.1D,none of the compounds caused a change in the level of NS1protein.These data indicate that in yeast,the positive compounds from the screen act either at the level of NS1function itself to suppress the slow-growth phenotype,or pos-sibly on cellular functions that speci?cally modify or bypass NS1function without altering its expression.

Effects on in?uenza virus replication.To test the effects of the positive compounds on in?uenza virus replication,each

was used to challenge virus replication assays for A/Hong Kong/19/68(HK),A/WSN/33(WSN),and A/PR/8(PR).MDCK cells were infected at an MOI of 0.1and incubated for 48h in the presence of drug or 1%DMSO as control,followed by determination of TCID 50(57).As shown in Fig.3A,all three viral strains were sensitive to each of the four com-pounds,with varying overall sensitivity and concentration de-pendence.The greatest inhibition observed was ?100-fold for virus HK in cells treated with NSC109834,NSC128164,or NSC125044.A ?10-fold inhibition of HK was observed with as little as 10?M NSC109834or NSC125044.The 50%inhibitory concentration for inhibition of virus replication was calculated for each compound and is presented in Table 1.Also presented in Table 1are the selective index (SI)values for each com-pound.

To determine the effect of NSC109834and NSC128164on viral protein synthesis,cells were infected with HK at an MOI of 0.1and incubated in the presence of these compounds for 24h.Protein synthesis was monitored by incorporation of [35S]methionine and [35S]cysteine during the last 45min of infection,followed by analysis using SDS-PAGE.Figure 3B (left)shows that both compounds signi?cantly affected the level of synthesis of the major viral proteins.Similar assays of infected cells treated with NSC125044or NSC95676showed that NSC125044resulted in a decrease in viral protein synthe-sis but that NSC96575produced little or no effect (data not shown).In addition,Western blot analysis was performed on lysates from infected cells.The right panel of Fig.3B shows that the level of viral NP protein was inhibited in cells treated with NSC109834and NSC128164.Since NS1is known to in-hibit the cellular IFN response,the observed decrease in viral protein synthesis may be due to an inhibition of viral reinfec-tion and spread during the 24h experiment.The data in Fig.3A and B demonstrate that the yeast screen is capable of identifying compounds that have signi?cant anti-in?uenza vi-rus activity.Of the nine reproducible positives from the screen,four showed signi?cant antiviral activity and are described here.This suggests a hit rate for the screen of ca.0.2%.

Assays for nonspeci?c effects.Two types of assays were performed to examine the possibility of nonspeci?c effects of the compounds that might explain their ability to inhibit in?u-enza virus replication.First,cell growth assays were performed to determine the effects on cell replication.During a 48-h treatment,which is the period used for the virus replication assays shown in Fig.3,none of the compounds exhibited sig-ni?cant growth toxicity over a dose range of 20to 100?M (see Fig.S1and Table S1in the supplemental material).Minor effects on cell growth were observed when treatment was ex-tended for up to 6days (data not shown).A second assay for nonspeci?c effects was to challenge the replication of an-other negative-strand RNA virus,RSV.As shown in Fig.4none of the compounds had any measurable effect on RSV replication in MA104cells compared to the DMSO control.Similar results were obtained with RSV infection of MDCK cells (not shown).These data demonstrate that the com-pounds do not affect the cell’s ability to support growth of negative-strand RNA virus replication in general and argue that the effects of the compounds on in?uenza virus repli-cation are speci?c for that

virus.

FIG.2.Chemical structures of anti-in?uenza virus compounds.

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Interactions with the cellular IFN system.NS1is a well-characterized inhibitor of the IFN system (14).Blockade of the IFN response by NS1prevents establishment of the antiviral state,which would otherwise signi?cantly limit virus replica-tion.Accordingly,mutant viruses with altered NS1function replicate poorly in cells with an intact IFN system but replicate signi?cantly better in IFN-de?cient cells (18,42).Cells with defects in the IFN system might therefore be expected to be resistant to compounds that inhibit NS1function.To test this,two African green monkey kidney cell lines were compared.Vero E6cells fail to produce IFN,whereas MA104cells are IFN competent (8,12,40).Each was tested for the ability to support replication of virus PR in the presence of NSC109834or NSC128164.Figure 5shows that Vero cells were

completely

FIG.3.Inhibition of virus replication in MDCK cells.(A)Cells were infected with the indicated viruses at an MOI of 0.1and treated with drug or 1%DMSO (shown as 0?M)starting 1h postinfection.The DMSO concentration for all drug-treated cultures was 1%.After incubation for 48h,the supernatants were collected and analyzed for TCID 50by the method of Reed and Muench (57).(B)Protein expression in infected cells.In the left panel,MDCK cells were infected with A/HK/19/68at an MOI of 0.1for 24h,in the presence or absence of the indicated compounds at 50?M.The cells were labeled with [35S]methionine and [35S]cysteine for the ?nal hour of infection.Cell lysates were analyzed by SDS-PAGE and visualized by using a phosphorimager.In the right panel,infected cells were analyzed for total NP protein expression by Western blotting.Blots were also probed for ?-tubulin and ?-actin as loading controls.

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resistant to both compounds,whereas MA104cells were sen-sitive,a ?nding consistent with the idea that the compounds inhibit NS1function.

One function of NS1during infection is inhibition of IFN gene expression through effects on transcriptional activation and mRNA maturation (14,47,65,71).Compounds that block NS1function would therefore be expected to restore induction of IFN-?mRNA in infected cells.To examine this,MDCK cells were infected with either virus PR or with delNS1,a derivative of PR that is deleted for NS1coding sequences (18).As expected,delNS1-infected cells showed a pronounced in-duction of IFN-?as seen by RT-PCR analysis,whereas PR-infected cells contained the same low level of IFN-?mRNA observed in mock-infected cells (Fig.6A).To test the effects of the drugs,cells infected with PR at an MOI of 2.0were treated with each compound for 6h,a period during which an initial round of virus production normally occurs.The cells were harvested for RNA isolation and assayed by RT-PCR for IFN-?mRNA.As shown in Fig.6A,all four compounds trig-gered signi?cant activation of IFN-?mRNA.For three of the compounds (NSC128164,NSC109834,and NSC125044)acti-

vation of IFN-?was roughly the same as that seen when cells were infected with delNS1in the absence of drug and was also as strong as for uninfected cells treated with the IFN-?inducer poly[IC].These data suggest a signi?cant inhibition of NS1function by these compounds.Importantly,none of the com-pounds triggered induction of IFN-??mRNA in the absence of viral infection (Fig.6B).Also shown in Fig.6A are RT-PCR results for three viral RNAs produced during infection:NP,M2,and NS1.Except for NSC125044,a strong reduction for M2and NS1RNA was observed,but no effect was seen for NP RNA.These data indicate the possibility of differential effects of some of the compounds on viral RNA production.

Interestingly,we observed that in delNS1-infected cells there was only a moderate decrease in the level of M2RNA

com-

FIG.4.Effect of anti-in?uenza virus compounds on RSV replication.(A)MA104cells were infected at an MOI of 0.1with the virus RS ?sh (49),in which the SH open reading frame is replaced with that of GFP.The infected cells were treated with the indicated compounds or 1%DMSO (shown as 0?M)starting at 1h postinfection.After 48h the cells were analyzed for GFP expression by ?uorescence microscopy and photographed.Representative ?elds are shown.(B)MA104cells plated in 96-well plates were infected with serially diluted RS ?sh.After 48h,quadruplicate wells were scored for the presence or absence of GFP expression by ?uorescence microscopy,and the data were analyzed for determination of TCID 50

.

FIG.5.A/PR/8replication in drug-treated Vero and MA104cells.Cells were infected A/PR/8at an MOI of 0.1and treated with drug or 1%DMSO (shown as 0?M)starting 1h postinfection.After incuba-tion for 48h,supernatants were collected and analyzed for TCID 50by the method of Reed and Muench (57).

TABLE 1.50%Inhibitory concentrations and selective indexes of

anti-NS1compounds on different in?uenza strains cultured

in MDCK-UK cells a

Anti-NS1compound

IC 50(?M)/SI

PR

HK

WSN

NSC12816414/16.65/44.413/18.5NSC10983410/32.32/200.312/28.2NSC9567612/104.920/62.819/64.5NSC125044

11/12.47/18.912/11.8

a

SI ?CC 50/IC 50.The 50%inhibitory concentration (IC 50)of the compounds was calculated by interpolation of the dose-response curves shown in Fig.3A.

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pared to wild-type virus,whereas in cells infected with wild-type virus and treated with NSC128164,NSC109834,or NSC95676,a more severe inhibition of M2RNA was observed.This result would not be expected if the level of M2RNA expression were a simple function of NS1activity.One possi-bility to explain this result is that the compounds have an NS1-independent activity that results in a decrease in M2RNA expression.To test this,cells were infected with delNS1at an MOI of 2.0and treated with each compound during a 6-h infection.Again,RNA was recovered for RT-PCR analysis.As shown in Fig.6C,the compounds had no effect on M2RNA levels in delNS1-infected cells,indicating that their effect on M2RNA in wild type-infected cells was indeed NS1depen-dent.Furthermore,the compounds had no effect on IFN-?mRNA levels in delNS1-infected cells,a ?nding consistent with the data shown in Fig.6B.The mechanism by which the com-pounds affect M2RNA levels in wild type-infected cells will require further investigation;however,the data are consistent with the possibility that NS1is allosterically regulated by the compounds so as to alter expression of M2and other viral RNAs (see Discussion).The fact that NSC125044had no effect on M2RNA levels despite strong activity in terms of IFN-?mRNA accumulation (Fig.6A)suggests that its mechanism of action differs from that of the other compounds.

A titration experiment was performed to assess the concen-tration dependence of the effect on IFN-?mRNA induction.Figure 6D shows that NSC109834induced IFN-?RNA be-tween 20and 50?M.NSC128164restored induction even at the lowest concentrations tested,with nearly maximal induc-tion between 10and 20?M.Also,in Fig.6are shown the results of RT-PCR analysis of viral RNAs from infected Vero cells challenged by each of the compounds.As expected,there was little or no effect on viral RNA expression in Vero cells due to the lack of IFN expression (Fig.6E).

Drug activity is linked to NS1function.To determine whether the activities of the various compounds are speci?c for NS1function,a transfection experiment was performed.Hu-man 293cells were cotransfected with a luciferase reporter driven by the IFN-?promoter and also expression plasmids encoding the NS1protein from either PR or A/TX/36/91(TX).As shown in Fig.7A,in the absence of NS1expression the IFN-?reporter was fully inducible by poly[IC](second lane),whereas in the presence of either NS1PR8or NS1TX ,induction was almost completely inhibited (“DMSO”lane),as has been shown previously (28).Signi?cantly,each compound ef?ciently restored induction of the IFN-?reporter,as shown in Fig.7A.NSC128164was the most ef?cient,increasing IFN-?promoter activity to maximal levels through inhibition of both NS1PR8and NS1TX .NSC95676also strongly restored induction.NSC109834and NSC125044differentially affected NS1PR8and NS1TX activity,perhaps suggesting a structural difference be-tween these proteins as they relate to the compounds.Impor-tantly,none of the compounds triggered induction of the IFN-?reporter construct in the absence of cotransfected NS1(Fig.7B).This demonstrates that NS1protein is absolutely required for the function of the compounds and is consistent with the idea that NS1is their direct target.These results are also fully consistent with those presented in Fig.6for induction of cellular IFN-?mRNA in virus-infected cells,where induc-tion was only observed in the presence of virus infection.As

an

FIG.6.Drug-dependent restoration of IFN-?mRNA induction in virus-infected cells.(A)MDCK cells were infected with A/PR/8at an MOI of 2.0and incubated in the presence of 50?M concentrations of the indicated compounds for 6h.Cells were harvested for RT-PCR analysis of cellular IFN-?and ?-actin mRNAs,as well as in?uenza virus RNAs corresponding to NP,M2,and NS1sequences.Also shown are RT-PCR products for cells treated with poly[IC]for 6h (second lane)and cells infected with the NS1deletion virus delNS1for 6h (rightmost lane).Mock,uninfected cells.(B)Cells were uninfected,except for the third lane,where cells were infected with PR.Drug treatment and RT-PCR were as described for panel A.(C)Same as panel A except cells were infected with delNS1,as indicated.(D)Cells were treated and analyzed as in panel A,except the drug concentra-tions are as indicated in the ?gure.(E)Vero cells were infected with A/PR/8at an MOI of 2.0and incubated in the presence of 50?M concentrations of the indicated compounds for 6h.Cells were ana-lyzed for the indicated RNAs as in panel A.

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additional control,transfected cells were analyzed for NS1protein expression by Western blotting to determine whether drug treatment altered the cellular levels of NS1.As shown in Fig.7C,treatment with the compounds had no effect on NS1protein levels,indicating that restoration of reporter activity was not due to drug-induced effects on NS1gene expression or protein turnover.In performing the Western blot experiments we successfully reproduced data from Kochs et al.(28)that showed that NS1TX protein dramatically inhibits its own ex-pression in transfection experiments (Fig.7C).Despite the low expression of NS1TX this protein clearly was active in inhibi-tion of IFN-?promoter activity (Fig.7A,DMSO lane).There-fore,we conclude that drug-dependent induction of IFN-??promoter activity in the case of NS1TX ,as for NS1PR ,was due to the inhibition of NS1-dependent function by the compounds under study.In addition,we used the compounds to challenge an independent inhibitor of IFN-?induction,the SARS-CoV PLP (9),as shown in Fig.7D.Consistent with published re-sults,cells cotransfected with the IFN-?reporter and a SARS-CoV PLP expression plasmid were signi?cantly inhibited

for

FIG.7.Speci?c inhibition of NS1-dependent function.(A)293cells were cotransfected with a ?re?y luciferase reporter driven by the human IFN-?promoter and expression constructs encoding the indicated NS1proteins.At 16h after transfection the cells were treated with 50?g of poly[IC]/ml and incubated for an additional 24h prior to harvesting for determination of the luciferase activity.Drug treatment with 50?M concentrations of the indicated compounds was for the ?nal 24h of the experiment.“Con”indicates the luciferase activity of untransfected cells;“DMSO”indicates treatment with 1%DMSO.(B)293cells were transfected with the IFN-?reporter and treated with poly[IC](?rst lane only),1%DMSO,or 50?M concentrations of the indicated anti-NS1compounds in the absence of poly[IC].(C)293cells were transfected with constructs to express the indicated NS1proteins and harvested for Western blot analysis 40h later.Drug treatment with 50?M concentrations of the indicated compounds was for the ?nal 24h of the experiment.Whole-cell extracts were blotted for NS1or ?-tubulin,whose positions are indicated.“Con”indicates the signal from untransfected cells.“DMSO”indicates DMSO control.(D)293T cells were transfected with the IFN-?luciferase plasmid alone or together with a SARS PLP-expressing plasmid.At 6h posttransfection cells were treated with the indicated drugs,incubated for an additional 18h and then treated with poly[IC]for 6h.The cells were then lysed and measured for luciferase activity.

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IFN-?reporter induction by poly[IC](9).However,none of the anti-NS1compounds had any effect in restoring IFN-?induction that had been inhibited by SARS-CoV PLP.These data demonstrate the speci?city of these compounds for in?u-enza virus NS1in restoration of IFN-?induction.

DISCUSSION

It is widely appreciated that global in?uenza pandemics re-present a considerable risk to human health and the economy, and new therapeutic approaches and drug targets are needed to protect the public health,especially against RNA viruses that evolve quickly in response to chemotherapeutic interven-tion(25,26,50,51).In the present study compounds were identi?ed based on their ability to suppress the phenotypic effect of NS1in S.cerevisiae.The precise mechanism of growth inhibition by NS1in yeast is not known,although Ward et al. showed that regions within the N-terminal and C-terminal domains are required for toxicity in yeast(72).Regardless,the fact that the compounds identi?ed in the yeast screen also inhibit virus replication and speci?cally reverse NS1-depen-dent inhibition of IFN-?mRNA induction strongly indicates that the mechanism of growth inhibition in yeast is directly related to the function of NS1during infection.This illustrates an advantage of the yeast-based drug discovery assay used here:it is not necessary to know the precise mechanism(s)of the target in order to identify relevant inhibitory compounds, so long as expression of the target protein induces a phenotypic change in yeast that can be incorporated into an assay for drug discovery,and physiologically relevant models(i.e.,virus infec-tion and IFN-?mRNA induction)can be used as secondary assays.This situation also suggests that compounds with en-tirely different mechanisms of action can be derived from the same screen,assuming that multiple features of the target contribute to the phenotypic effect.Additional advantages of the yeast system include identi?cation of compounds that are able to enter living cells and the fact that the selection is positive for growth,which eliminates compounds that are se-verely toxic,at least in yeasts.

An impressive list of functional domains of NS1has been established that includes regions that mediate RNA binding (10,34,70),binding to RIG-I(20,41,52),inhibition of nuclear RNA export(53,61),binding to CPSF(28,45),and other activities(14).Additional experiments will determine which domain(s)of NS1are involved in the response to the com-pounds under study.A method for high-throughput screening against the RNA-binding activity of NS1was reported recently (39).It is important to emphasize that,based on the design of the yeast screen,direct targets of the compounds can be either NS1itself or possibly cellular proteins.Whereas our results ?rmly establish NS1as a required participant in drug-mediated effects(Fig.6and7),they do not rule out the possibility that the direct target is a cellular protein that regulates NS1or one that can be caused to bypass its activity.This question is the subject of ongoing investigation.

NS1has been shown to inhibit induction of IFN-?by several independent mechanisms.Two of these are transcriptional mechanisms involving the N-terminal domain of NS1and are triggered by binding to dsRNA or to RIG-I(10,20,41,52,71). An additional mechanism is posttranscriptional and is carried out by binding of the“effector”domain of NS1to CPSF(28,

45).As reported by Kochs et al.,both NS1

PR

and NS1

Tx

are able block IFN-?induction in virus-infected cells and can also bind to RIG-I and interfere with activation of IRF3(28).This indicates that both proteins share the capacity to inhibit tran-scriptional activation of IFN-?.Interestingly,Kochs et al.also

found that only NS1

Tx

,but not NS1

PR

,was able to suppress cellular gene expression posttranscriptionally through binding

to CPSF(28).Since we found that both NS1

PR

and NS1

Tx

were sensitive to the effects of the compounds reported here,this suggests that the compounds are acting,at least in part,to counter the transcriptional mechanism(s)carried out by the N-terminal domain of NS1and not the mechanism involving CPSF.We also observed that the compounds were active only in cells that express NS1and that also have an intact IFN system(Fig.5to7).In the absence of NS1,the compounds failed to induce IFN-?mRNA or promoter activity on their own(Fig.6and7).Also,when cells were treated with poly[IC] in the absence of NS1to activate IFN-?transcription or were infected with delNS1,which allowed activation of IFN-?,the compounds did not cause a further increase in activation. These data indicate that the compounds are not simply target-ing cellular components of the IFN-?activation pathway that are poised to activate IFN-?in the absence of NS1.Further-more,our experiments showed that the compounds did not restore IFN-?induction that had been inhibited by the SARS-CoV PLP protein(Fig.7D).This also suggests that they are not acting at the level of a common signaling molecule in the activation of IFN-?transcription.Rather,our data suggest a direct,NS1-dependent function for the drugs.As mentioned above,this could involve direct binding to NS1or to a cellular function that regulates it.

As shown in Fig.6A,three of the compounds(NSC128164, NSC109834,and NSC95676)triggered a signi?cant reduction in viral M2and NS1-speci?c RNAs,as judged by RT-PCR assay.However,NSC125044did not affect viral RNA levels despite triggering a signi?cant increase in IFN-?mRNA(Fig. 6A)and a signi?cant reduction in overall viral production,as the other three compounds also did(Fig.3).These data sug-gest that at least two antiviral mechanisms are at play here. Interestingly,in contrast to the decrease in viral RNAs caused by NSC128164,NSC109834,and NSC95676,complete deletion of NS1sequences in virus delNS1did not cause the same decrease in viral RNA levels(Fig.6A and6C).This indicates that in a wild-type infected cell that is treated with NSC128164, NSC109834,or NSC95676,NS1has a negative effect on viral RNA expression that is not observed in cells infected with the delNS1virus.Since these compounds are only active in the presence of NS1protein and are not simply mimicking the absence of NS1,this suggests that there may be interactions between these compounds and NS1that allosterically regulate the ability of NS1to control transcription,RNA stability,or other processes.As reported previously,several temperature-sensitive mutants of NS1affect viral RNA levels.For instance, an Arg-to-Lys mutation at position25was shown to lead to a signi?cant decrease in M1and HA mRNAs(36).Other studies have implicated NS1in the function or regulation of the viral polymerase complex(38,43),a process that could be affected directly by some of the compounds reported here.On the other hand,another temperature-sensitive mutant of NS1,also with

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a change in the N-terminal domain at amino acid11,showed a drastic decrease in formation of virus particles without large changes in transcription of viral RNAs.Thus,allosteric effects of the compounds on NS1function may trigger a variety of effects that result in reduction of overall virus replication. Therefore,it is anticipated that the compounds identi?ed here may be of use in elucidating these or novel interactions be-tween NS1and viral or host functions,in addition to their potential clinical utility.

ACKNOWLEDGMENTS

We thank Dan Burke,Tom Braciale,Gail Wertz,Luis Martinez-Sobrido,Adolfo Garcia-Sastre,and Peter Palese for many valuable reagents,including cells,viruses,antibodies,and DNA clones.

This study was supported by Public Health Service grant R01AI071341 to D.A.E.

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III-V族化合物半导体-吉林大学课程中心

第6章 III-V族化合物半导体
吉林大学电子科学与工程学院
半导体材料

第六章 III-V族化合物半导体
IIIA元素：B 、Al、Ga、In VA元素： N、P、As、Sb 组合形成的化合物15种（BSb除外） 目前得到实用的III-V族化合物半导体 GaN GaP GaAs GaSb InP InAs InSb 原子序数之和：由小→大 ? 材料熔点：由高→低 ? 带隙宽度：由大→小
吉林大学电子科学与工程学院 半导体材料

元素 B Al Ga In
N BN 直接6.4eV AlN 直接6.2eV GaN 直接3.4eV InN 直接0.7eV
P
As
Sb
BP BAs 间接2.0eV 间接1.5eV AlP AlAs AlSb 间接2.45eV 间接 2.12eV 间接1.6eV GaP GaAs GaSb 间接2.26eV 直接 1.43eV 直接 0.73eV InP InAs InSb 直接1.35eV 直接0.45eV 直接0.18eV
吉林大学电子科学与工程学院
半导体材料

与Si相比，III-V族二元化合物半导体的独特性质
1. 带隙较大，大部分室温时＞ 1.1eV ，因而所制造的 器件耐受较大功率，工作温度更高 2. 大都为直接跃迁型能带，因而其光电转换效率高， 适合制作光电器件，如 LED 、 LD、太阳电池等。 GaP虽为间接带隙，但Eg 较大(2.25eV)，掺入等电 子杂质所形成的束缚激子发光仍可得到较高的发光 效率。是红 (Zn-O 、 Cd-O) 、黄 (Bi) 、绿 (N) 光 LED 的主要材料之一 3. 电子迁移率高，很适合制备高频、高速器件
吉林大学电子科学与工程学院 半导体材料

武汉理工 材料科学基础 课后答案 _第三章

第三章答案 3-2略。 3-2试述位错的基本类型及其特点。 解：位错主要有两种：刃型位错和螺型位错。刃型位错特点：滑移方向与位错线垂直，符号⊥，有多余半片原子面。螺型位错特点：滑移方向与位错线平行，与位错线垂直的面不是平面，呈螺施状，称螺型位错。 3-3非化学计量化合物有何特点？为什么非化学计量化合物都是n型或p型半导体材料？ 解：非化学计量化合物的特点：非化学计量化合物产生及缺陷浓度与气氛性质、压力有关；可以看作是高价化合物与低价化合物的固溶体；缺陷浓度与温度有关，这点可以从平衡常数看出；非化学计量化合物都是半导体。由于负离子缺位和间隙正离子使金属离子过剩产生金属离子过剩（n型）半导体，正离子缺位和间隙负离子使负离子过剩产生负离子过剩（p型）半导体。 3-4影响置换型固溶体和间隙型固溶体形成的因素有哪些？ 解：影响形成置换型固溶体影响因素：（1）离子尺寸：15%规律：1.（R1-R2）/R1>15%不连续。2.<15%连续。3.>40%不能形成固熔体。（2）离子价：电价相同，形成连续固熔体。（3）晶体结构因素：基质，杂质结构相同，形成连续固熔体。（4）场强因素。（5）电负性：差值小，形成固熔体。差值大形成化合物。 影响形成间隙型固溶体影响因素：（1）杂质质点大小：即添加的原子愈小，易形成固溶体，反之亦然。（2）晶体（基质）结构：离子尺寸是与晶体结构的关系密切相关的，在一定程度上来说，结构中间隙的大小起了决定性的作用。一般晶体中空隙愈大，结构愈疏松，易形成固溶体。（3）电价因素：外来杂质原子进人间隙时，必然引起晶体结构中电价的不平衡，这时可以通过生成空位，产生部分取代或离子的价态变化来保持电价平衡。 3-5试分析形成固溶体后对晶体性质的影响。 解：影响有：（1）稳定晶格，阻止某些晶型转变的发生；（2）活化晶格，形成固溶体后，晶格结构有一定畸变，处于高能量的活化状态，有利于进行化学反应；（3）固溶强化，溶质原子的溶入，使固溶体的强度、硬度升高；（4）形成固溶体后对材料物理性质的影响：固溶体的电学、热学、磁学等物理性质也随成分而连续变化，但一般都不是线性关系。固溶体的强度与硬度往往高于各组元，而塑性则较低 3-6说明下列符号的含义：V Na，V Na'，V Cl˙，（V Na'V Cl˙），Ca K˙，Ca Ca，Ca i˙˙

第三章 习题解答

第三章习题解答 3，7，10，11，25 3/113、非化学计量化合物有何特点？为什么非化学计量化合物都是n型或p型半导体材料？ 解答：非化学计量化合物的特点：非化学计量化合物产生及缺陷浓度与气氛性质、压力有关；可以看作是高价化合物与低价化合物的固溶体；缺陷浓度与温度有关，这点可以从平衡常数看出；非化学计量化合物都是半导体。由于负离子缺位和间隙正离子使金属离子过剩，产生金属离子过剩（n 型）半导体，正离子缺位和间隙负离子使负离子过剩，产生负离子过剩（p 型）半导体。 、说明下列符号的含义： 6/113 解答：钠原子空位， 钠离子空位、带一个单位负电荷， 氯离子空位、带一个单位正电荷， 最邻近的Na+空位、Cl-空位形成的缔合中心， Ca2+占据K位置、带一个单位正电荷， Ca原子位于Ca原子位置上， Ca2+处于晶格间隙位置。 1

2 7/113、写出下列缺陷反应式：（l ）NaCl 溶入CaCl 2中形成空位型固溶体； （2）CaCl 2溶入NaCl 中形成空位型固溶体；（3）NaCl 形成肖特基缺陷； （4）AgI 形成弗伦克尔缺陷（Ag +进入间隙）。 解答： （l ）NaCl 溶入CaCl 2中形成空位型固溶体 ?++??→?Cl Cl Ca CaCl V Cl Na' NaCl 2 （2）CaCl 2 溶入NaCl 中形成空位型固溶体 'N a Cl N a N aCl 2V Cl 2Ca CaCl ++??→?? （3）NaCl 形成肖特基缺陷 ?+→Cl N a 'V V O （4）Agl 形成弗伦克尔缺陷（Ag +进入间隙） A g 'i A g V Ag Ag +→? 10/113、MgO 晶体的肖特基缺陷生成能为84kJ/mol ，计算该晶体1000K 和1500K 的缺陷浓度。（答：6.4×10-3，3.5×10-2）。 解答： n/N = exp(-E/2RT)，R=8.314， T=1000K ：n/N=6.4×10-3； T=1500K ：n/N=3.5×10-2。

材料科学基础第三章答案

习题：第一章第二章第三章第四章第五章第六章第七章第八章第九章第十章第十一章答案：第一章第二章第三章第四章第五章第六章第七章第八章第九章第十章第十一章 3-2 略。 3-2试述位错的基本类型及其特点。 解：位错主要有两种：刃型位错和螺型位错。刃型位错特点：滑移方向与位错线垂直，符号⊥，有多余半片原子面。螺型位错特点：滑移方向与位错线平行，与位错线垂直的面不是平面，呈螺施状，称螺型位错。 3-3非化学计量化合物有何特点？为什么非化学计量化合物都是n型或p型半导体材料？ 解：非化学计量化合物的特点：非化学计量化合物产生及缺陷浓度与气氛性质、压力有关；可以看作是高价化合物与低价化合物的固溶体；缺陷浓度与温度有关，这点可以从平衡常数看出；非化学计量化合物都是半导体。由于负离子缺位和间隙正离子使金属离子过剩产生金属离子过剩（n型）半导体，正离子缺位和间隙负离子使负离子过剩产生负离子过剩（p型）半导体。 3-4影响置换型固溶体和间隙型固溶体形成的因素有哪些？ 解：影响形成置换型固溶体影响因素：（1）离子尺寸：15%规律：1.（R1-R2）/R1>15%不连续。 2.<15%连续。 3.>40%不能形成固熔体。（2）离子价：电价相同，形成连续固熔体。（ 3）晶体结构因素：基质，杂质结构相同，形成连续固熔体。（4）场强因素。（5）电负性：差值小，形成固熔体。差值大形成化合物。 影响形成间隙型固溶体影响因素：（1）杂质质点大小：即添加的原子愈小，易形成固溶体，反之亦然。（2）晶体（基质）结构：离子尺寸是与晶体结构的关系密切相关的，在一定程度上来说，结构中间隙的大小起了决定性的作用。一般晶体中空隙愈大，结构愈疏松，易形成固溶体。（3）电价因素：外来杂质原子进人间隙时，必然引起晶体结构中电价的不平衡，这时可以通过生成空位，产生部分取代或离子的价态变化来保持电价平衡。 3-5试分析形成固溶体后对晶体性质的影响。 解：影响有：（1）稳定晶格，阻止某些晶型转变的发生；（2）活化晶格，形成固溶体后，晶格结构有一定畸变，处于高能量的活化状态，有利于进行化学反应；（3）固溶强化，溶质原子的溶入，使固溶体的强度、硬度升高；（4）形成固溶体后对材料物理性质的影响：固溶体的电学、热学、磁学等物理性质也随成分而连续变化，但一般都不是线性关系。固溶体的强度与硬度往往高于各组元，而塑性则较低， 3-6说明下列符号的含义：V Na，V Na'，V Cl˙，（V Na'V Cl˙），Ca K˙，Ca Ca，Ca i˙˙解：钠原子空位；钠离子空位，带一个单位负电荷；氯离子空位，带一个单位正电荷；最邻近的Na+空位、Cl-空位形成的缔合中心；Ca2+占据K.位置，带一个单位正电荷；Ca原子位于Ca原子位置上；Ca2+处于晶格间隙位置。 3-7写出下列缺陷反应式：（l）NaCl溶入CaCl2中形成空位型固溶体；（2）CaCl2溶入NaCl中形成空位型固溶体；（3）NaCl形成肖特基缺陷；（4）Agl形成弗伦克尔缺陷（Ag+进入间隙）。

化合物半导体项目实施方案

化合物半导体项目实施方案 规划设计/投资分析/产业运营

化合物半导体项目实施方案 化合物半导体多指晶态无机化合物半导体，即是指由两种或两种以上元素以确定的原子配比形成的化合物，并具有确定的禁带宽度和能带结构等半导体性质。化合物半导体包括晶态无机化合物(如III-V族、II-VI族化合物半导体)及其固溶体、非晶态无机化合物(如玻璃半导体)、有机化合物(如有机半导体)和氧化物半导体等。通常所说的化合物半导体多指晶态无机化合物半导体。 该化合物半导体项目计划总投资10693.17万元，其中：固定资产投资8104.59万元，占项目总投资的75.79%；流动资金2588.58万元，占项目总投资的24.21%。 达产年营业收入25737.00万元，总成本费用19973.71万元，税金及附加227.03万元，利润总额5763.29万元，利税总额6785.26万元，税后净利润4322.47万元，达产年纳税总额2462.79万元；达产年投资利润率53.90%，投资利税率63.45%，投资回报率40.42%，全部投资回收期3.97年，提供就业职位398个。 坚持“社会效益、环境效益、经济效益共同发展”的原则。注重发挥投资项目的经济效益、区域规模效益和环境保护效益协同发展，利用项目承办单位在项目产品方面的生产技术优势，使投资项目产品达到国际领先

水平，实现产业结构优化，达到“高起点、高质量、节能降耗、增强竞争力”的目标，提高企业经济效益、社会效益和环境保护效益。 ......

化合物半导体项目实施方案目录 第一章申报单位及项目概况 一、项目申报单位概况 二、项目概况 第二章发展规划、产业政策和行业准入分析 一、发展规划分析 二、产业政策分析 三、行业准入分析 第三章资源开发及综合利用分析 一、资源开发方案。 二、资源利用方案 三、资源节约措施 第四章节能方案分析 一、用能标准和节能规范。 二、能耗状况和能耗指标分析 三、节能措施和节能效果分析 第五章建设用地、征地拆迁及移民安置分析 一、项目选址及用地方案

第三章 半导体中的电子状态

第三章 半导体中的电子状态 半导体的许多物理性质与其内部电子的运动状态密切相关。本章扼要介绍一些有关的基本概念。 §3-1 电子的运动状态和能带 为了便于理解半导体中的电子运动状态和能带的概念，先复习一下孤立原子中的电子态和自由空间中的电子态概念。 一．原子中的电子状态和能级。原子是由带正电荷的原子核和带负电荷的电子组成的，原子核的质量远大于电子的质量。因此，可认为电子是在原子核的库仑引力作用下绕着原子核运动。电子绕原子核运动遵从量子力学规律，处于一系列特定的运动状态，这些特定状态称量子态或电子态。在每个量子态中，电子的能量（能级）是确定的。处于确定状态的电子在空间有一定的几率分布。在讨论电子运动时，也常采用经典力学的“轨道”概念，不过其实际含义是指电子在空间运动的一个量子态和几率分布。对于原子中的电子，能级由低到高可分为E 1﹑E 2﹑E 3 ﹑E 4..等，分别对应于1s ﹑2s ﹑2p ﹑3s …等一系列量子态。如图3-1所示，内层轨道上的电子离原子核近，受到的束缚作用强，能级低。越往外层，电子受到的束缚越弱，能级越高。总之，在单个原子中，电子运动的特点是其运动状态为一些局限在原子核周围的局域化量子态，其能级取一系列分立值。 二．自由空间中的电子态和能级。在势场不随位置变化的自由空间中，电子的运动状态满足下面的定态薛定格方程 )()(222 r E r m ψψ=?- （3-1） 该方程的解为平面波： r k i k e V r ?=1)(ψ )(22)(2222 22z y x k k k m m k k E ++== （3-2） 其中，)(r k ψ称波函数，)(k E 称能量谱值或本征值，V 为空间体积，k 为平面波

化合物半导体项目立项报告

化合物半导体项目立项报告 投资分析/实施方案

报告说明— 该化合物半导体项目计划总投资16675.32万元，其中：固定资产投资12642.31万元，占项目总投资的75.81%；流动资金4033.01万元，占项目总投资的24.19%。 达产年营业收入37009.00万元，总成本费用28082.38万元，税金及附加320.24万元，利润总额8926.62万元，利税总额10478.12万元，税后净利润6694.97万元，达产年纳税总额3783.16万元；达产年投资利润率53.53%，投资利税率62.84%，投资回报率40.15%，全部投资回收期 3.99年，提供就业职位829个。 化合物半导体多指晶态无机化合物半导体，即是指由两种或两种以上元素以确定的原子配比形成的化合物，并具有确定的禁带宽度和能带结构等半导体性质。化合物半导体包括晶态无机化合物(如III-V族、II-VI族化合物半导体)及其固溶体、非晶态无机化合物(如玻璃半导体)、有机化合物(如有机半导体)和氧化物半导体等。通常所说的化合物半导体多指晶态无机化合物半导体。

目录 第一章基本情况 第二章项目投资单位 第三章项目建设背景及必要性分析第四章建设内容 第五章选址分析 第六章工程设计 第七章项目工艺技术 第八章环境保护、清洁生产 第九章项目职业安全 第十章项目风险评价 第十一章节能可行性分析 第十二章项目实施安排方案 第十三章项目投资方案分析 第十四章项目经济评价 第十五章项目评价结论 第十六章项目招投标方案

第一章基本情况 一、项目提出的理由 化合物半导体多指晶态无机化合物半导体，即是指由两种或两种以上 元素以确定的原子配比形成的化合物，并具有确定的禁带宽度和能带结构 等半导体性质。化合物半导体包括晶态无机化合物(如III-V族、II-VI族 化合物半导体)及其固溶体、非晶态无机化合物(如玻璃半导体)、有机化合 物(如有机半导体)和氧化物半导体等。通常所说的化合物半导体多指晶态 无机化合物半导体。 二、项目概况 （一）项目名称 化合物半导体项目 （二）项目选址 xxx循环经济产业园 对周围环境不应产生污染或对周围环境污染不超过国家有关法律和现 行标准的允许范围，不会引起当地居民的不满，不会造成不良的社会影响。项目选址应符合城乡建设总体规划和项目占地使用规划的要求，同时具备 便捷的陆路交通和方便的施工场址，并且与大气污染防治、水资源和自然 生态资源保护相一致。对各种设施用地进行统筹安排，提高土地综合利用