CN1738831A - Process for designing inhibitors of influenza virus non-structural protein 1 - Google Patents

Abstract

Disclosed are methods and compositions useful in identifying inhibitors of influenza virus, such as influenza A and B virus. Also disclosed are methods for preparing compositions for administration to animals, including humans infected with or to protect against influenza virus.

Description

The method of design of influenza virus non-structural protein 1 inhibitor

The cross reference of related application

The application requires the right of priority of following provisional application: 60/425,661, and the applying date is on November 13rd, 2002; 60/477,453, the applying date is on June 10th, 2003, and its content at this as a reference.

Government-funded

Research fund part is subsidized by national health institute, and contract number is Nos.GM47014 and AI11772.

Background technology

Influenza virus is a kind of major issue that concerns human health.It can cause and is called as the communicable acute respiratory disease of having of influenza.According to estimates, being very popular of 1918-1919 " spanish influenza " worldwide causes 500,000,000 people morbidity, and the result has 2,000 ten thousand people's death (Robbins, 1986).The hereditary determinant of virus virulence in 1918 does not still obtain identifying do not have special clinical prevention measure or treatment means effectively to tackle it yet and take place once more, and referring to Tumpey etc., PNAS USA 99 (15): 13849-54 (2002).Potential threat 1918 or that similar influenza virus occurs once more causes that great concern is not strange, and no matter it is to cause by natural cause or by bioterrorism.Even in the non-infectious time, influenza virus is infected and only causes about 20,000 to 30,000 people's death (Wright﹠amp in the U.S. every year; Webster, (2001) Orthomyxoviruses.In " Fields Virology, 4th Edition " (D.M.Knipe, and P.M.Howley, Eds.) pp.1533-1579.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA).In addition, millions of losses that cause people's productivity and quality of life owing to the light symptoms that overcomes disease in several days or several weeks are still arranged.Another complicated factors is that influenza A virus will experience lasting antigenic determinant variation, thereby causes to separate new bacterial strain every year.Simply saying, is exactly the resisiting influenza virus reagent that need to continue new kind.

Influenza virus is unique member of orthomyxoviridae family, and is proteic antigenic different with matrix (M) according to their nucleoprotein (NP), is divided into 3 different types (A, B and C) (Pereira, (1969) Progr.Molec.Virol.11:46).Orthomyxovirus is the animal virus that cyst membrane is arranged of about 100 nanometers of diameter.Influenza virus is by containing the genomic inner core protein core of single stranded RNA (spiral helicine nucleocapsid), and inside is lined with the outer lipoprotein cyst membrane of stromatin (M).The genome of sectional influenza A virus is made up of the single stranded RNA s of 10 polypeptide of coding of the negative polarity of 8 (influenza C virus is 7) molecule linearities, and these 10 polypeptide comprise: the nucleoprotein (NP) of RNA polymerase protein (PB2, PB1 and PA) that RNA instructs and formation nucleocapsid; Stromatin (M1, M2); Two surface glycoproteins outstanding from the lipoprotein cyst membrane: erythrocyte agglutination element (HA) and neuraminidase (NA); And Nonstructural Protein, its function is set forth (NS1 and NS2) below.Genomic transcribe and be replicated in the nuclear carry out, assemble by the blastogenesis on plasma membrane.Virus can be during polyinfection once more allocation base because of.

The RNA polymerase compositions that duplicate and transcribe the albumen that needs 4 encoding virals: NP and 3 viral RNA dependences of Influenza Virus RNA, and PB1, PB2 and PA (Huang et al., 1990, J.Virol.64:5669-5673).NP is the primary structure composition of virosome, and itself and geneome RNA interact, and is used for resisting termination (Beaton﹠amp between synthesis phase at RNA; Krug, 1986, Proc.Natl.Acad.Sci.USA 83:6282-6286).NP also is essential (Shapiro ﹠amp in the prolongation process of RNA chain; Krug, 1988, J.Virol.62:2285-2290), but be not the RNA start of chain necessary (Honda, et al, 1988, J.Biochem.104:1021-1026).

Influenza virus is adsorbed onto on the sialyloligosaccharide of the glycoprotein of cytolemma and glycolipid class by HA.After the endocytosis of virosome, the conformational change of HA molecule occurs in the endosome of cell, promotes film to merge, and has so just caused the shelling of virus particle.Nucleocapsid is moved to nucleus, and wherein to transcribe be the important initial period that infects to the virus mRNA of carrying out.Virus mRNA is transcribed by the mechanism of a uniqueness, and wherein viral nucleic acid restriction endonuclease cutting comes from the 5 ' end of " having cap " of the xenogenesis mRNA of cell, then it is transcribed the viral RNA template as primer by the transcriptase of virus.Stop transcribing at the 15-22 base place of distance template end, at the signal of this oligonucleotide (U) sequence as poly (A) tail that adds the non-dependence of template.Like this in Zhi Bei 8 virus mRNA molecules, there are 6 to be monocistron, directly be transcribed into the albumen and the virus polymerase protein of HA, NA, NP representative, PB2, PB1 and PA (influenza virus separates from people, Mammals and birds, classifies according to their surface glycoprotein, erythrocyte agglutination element (HA) and neuraminidase (NA)).

Other two transcriptons are through shearing, and each transcripton all produces 2 mRNA, obtain M1, M2, Nonstructural Protein-1 (NS1) and Nonstructural Protein-2 (NS2) with different reading frame translations.Eukaryotic cell is resisted virus infection by the histone that generation comprises Interferon, rabbit.NS1 albumen promotes duplicating of influenza virus by the Interferon, rabbit that suppresses to produce in the host bacterium and infects.The proteic length of the NS1 of influenza A virus is variable (Parvinetal., (1983) Virology 128:512-517), and can tolerate big section of its C-terminal deletion and do not influence its functional integrity (Norton et al., (1987) 156 (2): 204-213).NS1 albumen contains two functional domains, promptly in conjunction with the territory and the effector domain of double-stranded RNA (dsRNA).Effector domain is positioned at proteic C-terminal zone.Its function has obtained well determining comparatively speaking.Specifically, realize nRNA output function (Qian et al., (1994)) J.Virol.68 (4) by interacting with host's nucleoprotein in the effect zone: 2433-2441).

The proteic dsRNA of NS1A is positioned its N-terminal (Qian et al., 1994) in conjunction with the territory.Aminoterminal fragment, it forms [NS1A (1-73)] by 73 initial aminoterminal amino acid, has the dsRNA binding characteristic (Qian et al., (1995) RNA 1:948-956) of full-length proteins.The NMR solution of NS1A (1-73) and X-ray crystal structure are presented at that its six coiled strands with uniqueness are folded to form symmetric homodimer (Chien et al., (1997) Nature Struct.Biol.4:891-895 in the solution; Liuet al., (1997) Nature Struct.Biol.4:896-899).Each polypeptide chain in NS1A (1-73) territory all is made up of 3 alpha-helixs, and corresponding fragment is Asn4-Asp24 (spiral 1), Pro ³¹-Leu ⁵⁰(spiral 2), and Ile ⁵⁴-Lys ⁷⁰(spiral 3).The initial analysis of NS1A (1-73) surface characteristic shows 2 possible nucleic acid binding sites, the stretching, extension in the solvent of being exposed to that comprises most of spiral of forming by basic side chain 2 and 2 ', another is in the opposite location of molecule, the lysine residue (Chien etal., 1997) that comprises some spirals 3 and spiral 3 '.The rite-directed mutagenesis experiment of carrying out subsequently shows two basic aminoacids (Arg in second alpha-helix ³⁸And Lys ⁴¹) side chain is that the dsRNA of complete dimer protein is in conjunction with the necessary unique amino acid side chain of activity (Wang et al., 1999RNA 5:195-205).These researchs prove that also the dimerization in NS1A (1-73) territory is that dsRNA is in conjunction with necessary.Yet, bypass in conjunction with dsRNA and do not talk (for example, Hatada﹠amp; Futada, (1992) J.Gen.Virol., Vol.73 (12): 3325-3329; Lu et al., (1995) Virology, 214:222-228; Wang et al., (1999)), the precise function in conjunction with the territory of dsRNA also is not determined.

The present invention's general introduction

The present invention has used the applicant about NS1 albumen, and particularly the dsRNA of albumen N-terminal portions is the discovery that how to participate in the course of infection of influenza virus in conjunction with the territory.The applicant find the proteic RNA of NS1A in conjunction with the territory to influenza A virus duplicate with pathogenic be vital.The applicant finds when the combining with dsRNA in host cell in conjunction with the territory of NS1A, and the virus defense system that host cell can't activate it suppresses the generation of viral protein.NS1A causes enzyme, double-stranded RNA activated protein kinase (" PKR ") to keep the non-activity state in conjunction with dsRNA, to such an extent as to it can not catalysis translation initiation factor eIF2 α phosphorylation, otherwise this factor can suppress the synthetic of viral protein and duplicate.Other people report in the past shows that the amino acid that participates in inhibition PKR does not comprise the necessary amino acid in conjunction with dsRNA.Report is opposite is with these, and the applicant also finds two amino-acid residues (NS1A just: arginine 38 (R in the NS1 albumen of influenza A and B virus ³⁸) and Methionin 41 (K ⁴¹); NS1B: arginine 50 (R ⁵⁰) and arginine 53 (R53)) be the Key residues in the RNA combination, also relevant with dsRNA in this way in conjunction with the ability of the releasing host cell defence in territory.The applicant finds that NS1A or NS1B and dsRNA have a structure separation surface, and define the Structural Characteristics at interface according to above-mentioned, and it is the target spot of medicinal design.The applicant has invented the detection method of interactional feature between a series of definite NS1A or NS1B and the dsRNA, is used among a small circle and/or this interactional inhibitor of high flux screening.The applicant also finds a kind of aminoterminal fragment, and it forms [NS1B (1-93)] by N-terminal initial 93 amino acid, has the proteic dsRNA binding characteristic of total length NS1 of influenza B virus.

One aspect of the present invention relates to the authentication method with the active compound of inhibition influenza virus, and it comprises:

A) preparation reaction system, it comprises influenza virus NS1 albumen or its dsRNA in conjunction with the territory, combines the territory with above-mentioned protein bound dsRNA or its, and candidate compound and;

B) combination degree between detection NS1 albumen and dsRNA, it is in the presence of this compound, and compared with the control, the binding ability between NS1 albumen and dsRNA reduces the indication compound has the activity of inhibition to influenza virus.Influenza is had the active compound of inhibition carry out further experiment what identify to determine whether they are suitable as medicine.In this way, influenza virus is duplicated effective inhibitors can in experimentation on animals subsequently, determine, also can be used for treatment (preventing disease or other) and comprise the influenza infection of people's animal.

Accordingly, another aspect of the present invention relates to the authentication method with the active compound of inhibition influenza virus, comprises:

A) reaction system of preparation, it comprises a kind of influenza virus NS1 albumen or its dsRNA in conjunction with the territory, with the territory that combines of above-mentioned protein bound dsRNA or its, and candidate compound and;

B) combination degree between detection NS1 albumen and dsRNA, wherein in the presence of this compound, compared with the control, the binding ability between NS1 albumen and dsRNA reduces the indication compound has the activity of inhibition to influenza virus; With

C) having of identifying detection b) suppresses active compound in external inhibition degree to the influenza virus growth.

In some embodiments, this method further needs d) determine c) in identify have an inhibition degree that the compound in the growth of vitro inhibition influenza virus duplicates the influenza virus among the non-human animal.

In addition, another aspect of the present invention relates to a kind of preparation of compositions method that influenza virus is duplicated that suppresses in vivo and in vitro, and it comprises:

A) reaction system of preparation, it comprises influenza virus NS1 albumen or its dsRNA in conjunction with the territory, combines the territory with above-mentioned protein bound dsRNA or its, and candidate compound;

B) combination degree between detection NS1 albumen and dsRNA, wherein in the presence of this compound, compared with the control, the binding ability between NS1 albumen and dsRNA reduces the indication compound has the activity of inhibition to influenza virus;

C) having of identifying detection b) suppresses active compound in external inhibition degree to the influenza virus growth;

D) compound of identifying detection c) in the growth of vitro inhibition influenza virus suppresses the degree of duplicating of influenza virus in the non-human animal;

E) with d) in the compound that in inhuman animal, suppresses virus replication identified prepare with the preparation composition with carrier to suppress effective dose.

In aspect each of the invention described above, some embodiments need be used fluorescence molecule mark NS1 albumen or dsRNA, determine the bonded degree by FRET (fluorescence resonance energy transfer) or fluorescence polarization then.In other embodiments, contrast is dsRNA and NS1 albumen or lacks R ³⁸And/or R ⁴¹The dsRNA of amino-acid residue is in conjunction with the combination degree between the territory.The authentication method that other embodiment needs influenza virus NS1 albumen/dsRNA mixture to form.Yet, still have other embodiment need utilize the method screening of influenza virus NS1 albumen/dsRNA mixture formation or optimize inhibitor.These embodiments comprise displacement disturbance of NS1 albumen nmr chemical or RNA gel-filtration sedimentation equilibrium, and the effective screening that utilizes proteic structure of NS1 and NS1-RNA mixture model to carry out.

Another aspect of the present invention relates to the composition that contains reaction mixture, and it comprises: influenza virus NS1 albumen or its dsRNA binding fragment, and with above-mentioned protein bound dsRNA.In some specific embodiments, NS1 albumen is NS1A albumen, or its dsRNA binding fragment, 73 amino-acid residues of albumen n end.In other embodiments, NS1 albumen is NS1B albumen or its dsRNA binding fragment, 93 amino-acid residues of albumen n end.In the other embodiment, composition further contains the tested compound of the active material standed for of detected inhibition to influenza virus.

Another aspect of the present invention relates to the method that evaluation can be used for treating the compound of influenza infection, it is included in and utilizes NS1 albumen or its dsRNA structure in conjunction with territory, NS1A (1-73) or NS1B (1-93) in the medicament screening experiment, and the three-dimensional equivalents of NS1-RNA mixture model.

These and other aspect of the present invention will and describe in detail with reference to figure below and better be set forth.

The file of this patent contains at least one width of cloth cromogram.The duplicate of the wounded figure of this patent will through require and pay necessary fee after provide by patent and trademark office.

The accompanying drawing summary

The different duplex of Fig. 1 is in conjunction with the gel Displacement Analysis of NS1A (1-73) ability.This experiment be under the condition of standard, utilize specified ³²The double chain nucleotide of P-mark (1.0nM) and (+) arranged, do not have to carry out under the NS1A (1-73) of (-) 0.4 μ M.

The gel filtration chromatography figure of Fig. 2 different duplex in the presence of NS1A (1-73): (A) dsRNA; (B) RNA-DNA crossbred; (C) DNA RNA hybrid; (D) dsDNA.First peak in (A) derives from NS1A (1-73)-dsRNA mixture, and the main peak between 20 minutes to 30 minutes is all corresponding to duplex.

Gel filtration chromatography (A) the 4 μ M of the NS1A of Fig. 3 purifying (1-73)-dsRNA mixture, the fresh composite sample of 100 μ l; (B) 4 μ M, the composite sample of 100 μ l preparation after one month.

The sedimentation equilibrium of Fig. 4 (A) during according to 16000 rev/mins determined the stoichiometry of 3 samples, and load sample concentration is the individual absorbance units of 0.6 (), 0.3 (△) and 0.5 (not showing not avoid data point overlapping).Solid line is hypothesis dsRNA: when the NS1 mixture is 1: 1 stoichiometry, and the associating match of three groups of data; Illustration shows the residual at random figure of match.The line hypothesis dsRNA of band point: the NS1 mixture is 1: 2 stoichiometry and drawing.(mixture had and the shown concentration profile much at one of line of being with point in 2: 1, and this is because the molecular weight (referring to infra) of dsRNA and the proteic reduction much at one of NS1).(B): 3 samples (on seeing) are at 16000 (), the dissociation constant of the sedimentation equilibrium of estimating during 22000 (zero) and 38000 (△) rpms speed.Data when this has demonstrated that load sample concentration is 0.5 absorbance units.Solid line is an overall fit of utilizing an ideal NONLIN list-dimer model, utilizes Eq.7 to calculate dissociation constant according to fitting result.Illustration shows the residual figure of match.

Fig. 5 (A) 2.0mM homogeneous ¹⁵The NS1A of N-enrichment (1-73) is at 20 ℃, the 95%H that contains 50mM ammonium acetate and 1mM sodiumazide of pH6.0 ₂O/5%D ₂Two dimension in the solution of O ¹H- ¹⁵The HSQC collection of illustrative plates of N.The peak that intersects is located with resonance separately and is marked, wherein the code and the sequence number of show amino acid letter.Also demonstrate simultaneously the NH of the NH resonance of tryptophane side chain and glutamine and l-asparagine side chain ₂Resonance.Localized arginic N ^ε-H ^εThe position of resonance peak from the upfield F1 ( ¹⁵N) dimension has been passed through folding.(B) with ¹H ^N- ¹⁵The hsqc spectrum of N is represented ¹⁵NS1A of N-enrichment (1-73) and 16-bp dsRNA be at pH6.0, do not form mixture (redness) under 20 ℃ of conditions and form the coverage diagram of mixture (blueness).Mark is corresponding to the amide backbone location at the intersection peak of the good discrimination of free protein.

The result that the banded pictorialization chemical shift disturbance of Fig. 6 (A) NS1A (1-73) is measured.In the NMR of NS1A (1-73)-dsRNA mixture spectrum, produce displacement turbulent NS1A (1-73) residue and mark with blue-greenish colour, to its amide group ¹⁵N and ¹The residue that the chemical shift of H does not have to change marks with pink, and the white representative is because the chemical shift location of the residue that can't differentiate in the two-dimentional hsqc spectrum that the intersection peak that overlaps each other causes.(B) side chain that shows among Fig. 6 B this also use underlined alkaline residue list.The conjugated antigen determinant of attention NS1A (1-73) and dsRNA seems in the bottom of structure.

The NS1A of Fig. 7 purifying (1-73)-dsRNA mixture (A), the mixture of duplex and NS1A (1-73): RNA-DNA crossbred (B), and the CD of DNA RNA hybrid (C) spectrum.Orange: the experiment CD spectrum of mixture (duplex and albumen dimer molar ratio are 1: 1).Red: duplex self.Blue: NS1A (1-73) self.Green: total spectrum of the duplex of calculating and NS1A (1-73).

The model of the dsRNA binding characteristic of Fig. 8 NS1A (1-73).The experiment that this model designs checking hint hypothesis is useful.The base pair of phosphate backbone and dsRNA is respectively with orange and yellow demonstration.All arginine and lysine side-chains all mark with green.

Implement best way of the present invention

The invention provides design from influenza A and the proteic dsRNA of B virus N S1 method in conjunction with the special inhibitor in territory.The proteic dsRNA of influenza A virus NS1 is in conjunction with the aminoacid sequence, particularly R in territory ³⁸And K ⁴¹Residue comes down to guard.The proteic multisequencing comparison of the NS1 of the various strains of influenza A virus is described in table 1.

In addition, exemplary, the proteic aminoacid sequence of the NS1 of the various strains of influenza A virus is listed below.

The proteic aminoacid sequence of the NS1 of influenza A virus A/Udorn/72:

1 MDPNTVSSFQ VDCFLWHVRK RVADQELGDA PFLDRLRRDQ KSLRGRGSTL GLDIETATRA

61 GKQIVERILK EESDEALKMT MASVPASRYL TDMTLEEMSR EWSMLIPKQK

VAGPLCIRMD

121 QAIMDKNIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT

AEDVKNAVGV

181 LIGGLEWNDN TVRVSETLQR FAWRSSNENG RPPLTPKQKR EMAGTIRSEV

The proteic aminoacid sequence of NS1 of influenza A virus A/ goose/Guangdong/3/1997 (H5N1):

1 MDSNTITSFQ VDCYLWHIRK LLSMSDMCDA PFDDRLRRDQ KALKGRGSTL GLDLRVATME

61 GKKIVEDILK SETNENLKIA IASSPAPRYV TDMSIEEMSR EWYMLMPRQK

ITGGLMVKMD

121 QAIMDKRIIL KANFSVLFDQ LETLVSLRAF TESGAIVAEI SPIPSVPGHS

TEDVKNAIGI

181 LIGGLEWNDN SIRASENIQR FAWGIRDENG GPSLPPKQKR YMAKRVESEV

The proteic aminoacid sequence of NS1 of influenza A virus A/QUAIL/NANCHANG/12-340/2000 (H1N1):

1 ELGDAPFLDR LRRDQKSLKG RGSTLGLNIE TATCVGKQIV ERILKEESDE AFKMTMASAL

61 ASRYLTDMTI EEMSRDWFML MPKQKVAGPL CVRMDQAIMD KNIILKANFS

VIFDRLETLT

121 LLRAFTEEGA IVGEISPLPS LPGHTNEDVK NAIGVLIGGL EWNDNTVRVS ETL

Influenza A virus gi|577477|gb|AAA56812.1|[577477] the proteic aminoacid sequence of NS1:

1 MDSNTVSSFQ VDCFLWHVRK RFADQEMGDA PFLDRLRRDQ KSLGGRGSTL GLDIETATRA

61 GKQIVEPILE EESDEALKMT IASAPVSRYL PDMTLEEMSR DWFMLMPKQK

VAGSLCIRMD

121 QAIMDKNITL KANFSIIFDR LETLILLRAF TEEGAIVGEI SPVPSLPGHT

DEDVKNAIGV

181 LIGGLEWNDN TVRDSETLQR FAWRSSNEDR RPPLPPKQKR KMARTIESEV

Influenza A virus gi|413859|gb|AAA43491.1|[413859] the proteic aminoacid sequence of NS1:

1 MDSNTVSSFQ VDCFLWHVRK RFADQERGDA PFLDRLRRDQ KSLRGRGSTL GLDIETATCA

61 GKQIVERILK EESDEALKMT IASVPASRYL TDMTLEEMSR DWFMLMPKQK

VAGSLCIRMD

121 QAIMDKNIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT

DEDVKNAIGV

181 LIGGLEWNDN TVRVSETLQR FAWRSSNEDG RPPFPPKQKR KMARTIESEV

Influenza A virus gi|325085|gb|AAA43684.1|[325085] the proteic aminoacid sequence of NS1:

1 MDSNTVSSFQ VDCFLWHVPK RFADQKLGDA PFLDRLRRDQ KSLRGRASTL GLDIETATRA

61 GKQIVERILE EESNEALKMT IASVPASRYL TDMTLEEMSR DWFMLMPKQK

VAGSLCIRMD

121 QAIMEKSIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLHSLPGHT

DEDVKNAVGV

181 LIGGLEWNGN TVRVSENLQR FAWRSRNENE RPSLPPKQKR EVAGTIRSEV

Influenza A virus gi|324876|gb|AAA43572.1|[324862] the proteic aminoacid sequence of NS1:

1 NTVSSFQVDC FLWHVRKRFA DQELGDAPFL DRLRRDQKSL RGRGSTLGLD IETATRAGKQ

61 IVERILVEES DEALKMTIVS MPASRYLTDM TLEEMSRDWF MLMPKQKVAG

SLCIRMDQAI

121 MDKNIILKAN FSVISDRLET LILLRAFTEE GAIVGEISPL PSLPGHTDED

VKNAIGDLIG

181 GLEWNDNTVR VSETLQRFAW RSSNEDGRPL LPPKQKRKMA RTIESEV

Influenza A virus gi|324862|gb|AAA43553.1|[324862] the proteic aminoacid sequence of NS1:

1 MDPNTVSSFQ VDCFLWHVRK QVADQELGDA PFLDRLRRDQ KSLRGRGSTL GLNIETATRV

61 GKQIVERILK EESDEALKMT MASAPASRYL TDMTIEEMSR DWFMLMPKQK

VAGPLCIRMD

121 QAIMDKNIIL KANFSVIFDR LETLILLRAF TEAGAIVGEI SPLPSLPGHT

NEDVKNAIGV

181 LIGGLEWNDN TVRVSKTLQR FAWRSSDENG RPPLTPK

Influenza A virus gi|324855|gb|AAA43548.1|[324855] the proteic aminoacid sequence of NS1:

1 NTVSSFQVDC FLWHVLKRFA DQELGDAPFL DRLRRDQKSL RGRGSTLGLD IETATRAGKQ

61 IVERILEEES DEALKMNIAS VPASRYLTDM TLEEMSRDWF MLMPKQKVAG

SLCIRMDQAI

121 MDKNIILKAN FSVIFDRLET LILLRAFTEE GAIVGEISPL PSLPGHTDED

VKNAIGILIG

181 GLEWNDNTVR VSETLQRFAW RSSNEDGRPP LPPKQKWKMA RTIEPEV

Influenza A virus gi|324778|gb|AAA43504.1|[324778] the proteic aminoacid sequence of NS1:

1 NTVSSFQVDC FLWHVRKRFA DLELGDAPFL DRLCRDQKSL RGRSSTLGLD IETATRAGKQ

61 IVERILEEES DETLKMTIAS APAFRYPTDM TLEEMSRDWF MLMPKQKVAG

SLCIRMDQAI

121 MDKNIILKAN FSVIFDRLET LILLRAFTEE GAIVGEISPL PSLPGHTNED

VKNAIGDLIG

181 GLEWNDNTVR VSETLQRFAW RSSNEGGRPP LPPKQKRKMA RTIESEV

The proteic aminoacid sequence of the NS1 of influenza A virus A/PR/8/34:

1 MDSNTITSFQ VDCYLWHIRK LLSMRDMCDA PFDDRLRRDQ KALKGRGSTL GLDLRVATME

61 GKKIVEDILK SETDENLKIA IASSPAPRYI TDMSIEEISR EWYMLMPRQK

ITGGLMVKMD

121 QAIMDKRITL KANFSVLFDQ LETLVSLRAF TDDGAIVAEI SPIPSMPGHS

TEDVKNAIGI

181 LIGGLEWNDN SIRASENIQR FAWGIRDENG GPPLPPKQKR YMARRVESEV

The proteic aminoacid sequence of NS1 of influenza A virus A/ turkey/Oregon/71 (H7N5):

1 MDSNTITSFQ VDCYLWHIRK LLSMRDMCDA PFDDRLRRDQ KALKGRGSTL GLDLRVATME

61 GKKIVEDILK SETDENLKIA IASSPAPRYI TDMSIEEISR EWYMLMPRQK

ITGGLMVRPL

121 WTRG

The proteic aminoacid sequence of NS1 of influenza A virus A/ Hong Kong/1073/99 (H9N2):

1 MDSNTVSSFQ VDCFLWHVRK RFADQELGDA PFLDRLRRDQ KSLRGRGSTL GLDIRTATRE

61 GKHIVERILE EESDEALKMT IASVPASRYL TEMTLEEMSR DWLMLIPKQK

VTGPLCIRMD

121 QAVMGKTIIL KANFSVIFNR LEALILLRAF TDEGAIVGEI SPLPSLPGHT

DEDVKNAIGV

181 LIGGLEWNDN TVRVSETLQR FTWRSSDENG RSPLPPKQKR KVERTIEPEV

The proteic aminoacid sequence of NS1 of influenza A virus A/Fort Monmouth/1/47-MA (H1N1):

1 MDPNTVSSFQ VDCFLWHVRK RVADQELGDA PFLDRLRRDQ KSLKGRGSTL GLNIETATRV

61 GKQIVERILK EESDEALKMT MASAPASRYL TDMTIEEMSR DWFMLMPKQK

VAGPLCIRMD

121 QAIMDKSIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT

NEDVKNAIGV

181 LIGGLEWNDN TVRVSKTLQR FA

The change strain of influenza B virus also have similar dsRNA in conjunction with the territory.The proteic multisequencing comparison of the NS1 of the various change strains of influenza B virus is described in table 2.

In addition, only by example, the proteic aminoacid sequence of NS1 of the various change strains of influenza B virus is listed below.

The proteic aminoacid sequence of NS1 of influenza B virus (B/Lee/40):

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERFSWQRAL DYPGQDRLHR LKRKLESRIK

61 THNKSEPENK RMSLEERKAI GVKMMKVLLF MDPSAGIEGP EPYCVKNPST

SKCPNYDWTD

121 YPPTPGKYLD DIEEEPENVD HPIEVVLRDM NNKDARQKIK DEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGD KSLSTLHRLN AYDQNGGLVA KLVATDDRTV

EDEKDGHRIL

241 NSLFERFDEG HSKRIRAAET AVGVLSQFGQ ERRLSPEEGD N

The proteic aminoacid sequence of NS1 of influenza B virus B/Memphis/296:

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN

SNCPKYNWTD

121 YPSTPGRCLD DIEEEPEDVD GPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRIN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERLNEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

Influenza B virus gi|325264|gb|AAA43761.1|[325264] the proteic aminoacid sequence of NS1:

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MNPSAGIEGF EPYCMKNSSN

SNCPNCNWTD

121 YPPTSGKCLD DIEEEPENVD DPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERFNEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

Influenza B virus B/Ann Arbor/1/66[gi|325261|gb|AAA43579.1|[325261]] the proteic aminoacid sequence of NS1:

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSSQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MNPSAGIEGF EPYCMKNSSN

SNCPNCNWTD

121 YPPTPGKCLD DIEEEPENVD DPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERFNEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

Influenza B virus gi|325256|gb|AAA43756.1|[325256] the proteic aminoacid sequence of NS1:

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERFSWQRAL DYPGQDRLHR LKRKLESRIK

61 THNKSEPENK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCVKNPST

SKCPNYDWTD

121 YPPTPGKYLD DIEEEPENVD HPIEVVLRDM NNKDARQKIK DEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGD KSLSTLHRLN AYDQNGGLVA KLVATDDRTV

EDEKDGHRIL

241 NSLFERFDEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

The proteic aminoacid sequence of NS1 of influenza B virus (B/Shangdong/7/97):

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN

SNYPKYNWTD

121 YPSTPGRCLD DIEEETEDVD DPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERLNEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

The proteic aminoacid sequence of NS1 of influenza B virus (B/Nagoya/20/99):

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN

SNYPKYNWTN

121 YPSTPGRCLD DIEEETEDVD DPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERLNEG HPKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

The proteic aminoacid sequence of NS1 of influenza B virus (B/Saga/S172/99):

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN

SNCPKYNWTD

121 YPSTPGRCLD DIEEEPEDVD GPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVL8LRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERLNEG HSKPIRAAET AVGVLSQFGQ EHRLSPEEGD N

The proteic aminoacid sequence of NS1 of influenza B virus (B/Kouchi/193/99):

1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK

61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN

SNCPKYNWTD

121 YPSTPGRCLD DIEEEPEDVD GPTEIVLRDM NNKDARQKIK EEVNTQKEGK

FRLTIKRDIR

181 NVLSLRVLVN GTFLKHPNGY KSLSTLHRLN AYDQSGRLVA KLVATDDLTV

EDEEDGHRIL

241 NSLFERLNEG HSKPIRAAET AMGVLSQFGQ EHRLSPEEGD N

Like this, utilize disclosed any NS1 albumen in the invention or it with the dsRNA bonded fragment (R that complete NS1A is arranged ³⁸, K ⁴¹Residue has the R of complete NS1B ⁵⁰, R ⁵³Residue) can identify that influenza A virus and influenza B virus strain are had the active compound of inhibition.

It is that nature exists that the present invention does not need protein.Also can use the proteic analogue of NS1, as long as it has the dsRNA binding specificity of the suitable function of albumen that exists with nature.Representational analogue comprises proteic fragment, for example, dsRNA in conjunction with the territory.Except the proteic fragment of NS1, analogue can also be by replacing, and leaves out or increase one or more amino acid and distinguish to some extent with naturally occurring albumen.For example, the amino-acid residue that function is suitable can change sequence by the residue of replacing in the sequence.Such replacement can be selected from other the amino acid that belongs to similar; For example, nonpolar (hydrophobic) amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met); Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, l-asparagine and glutamine; And positive charge (alkalescence) amino acid comprises arginine, Methionin and Histidine; Negative charge (acidity) amino acid comprises aspartic acid and L-glutamic acid.Can change the R of NS1A ³⁸And K ⁴¹Residue, but certain limitation is arranged.For example, the applicant determines to replace R with lysine residue ³⁸Do not observe the influence that is combined with to RNA, yet, just destroyed this when replacing in conjunction with active with L-Ala, show that the basic aminoacids side chain (for example, Methionin or arginine) at this position positive charge is that these dsRNA-protein-interactings are necessary; With any one replacement of remaining 17 natural common amino acids,, can both predict active destroyed as L-Ala.Yet R in preferred embodiments, ³⁸And K ⁴¹Residue is complete.Above-mentioned explanation is to the R of NS1B ⁵⁰And R ⁵³Residue is suitable for equally.For purpose of the present invention, term " dsRNA is in conjunction with the territory " is meant and comprises that the albumen that exists with nature is combining the proteic analogue of NS1 that suitable function is arranged aspect the dsRNA.

NS1 albumen of the present invention can be prepared according to existing handbook.Influenza virus NS1 albumen or its dsRNA can obtain from natural origin in conjunction with the territory, for example, utilize protein separation technology known in the art, respectively purifying from the cell of influenza infection and virus; (for example see Sambrook et al., 1989 by recombinant DNA technology production known in the art, Molecular Cloning:A Laboratory Manual, Cold Spring HarborLaboratories Press, Cold Spring Harbor, N.Y.); And/or utilize all or part of chemosynthesis of technology known in the art; For example, polypeptide can be synthetic by solid phase technique, cuts from resin, utilizes preparation property high performance liquid phase purifying (for example to see Creighton, 1983, Protein:Structures and Molecular Principles, W.H.Freeman﹠amp; Co., N.Y., pp.50-60).At Qian et al., RNA1 (9): 948-56 (1995) and Chien et al. have reported the Biotechnology Compose Experiment method of the polypeptide that NS1A1-73 amino acid limits in (1997), and it has or do not have and is fit to the isotopic labeling that NMR analyzes.The composition of synthetic polypeptide can be determined by amino acid analysis or order-checking; For example, utilize Edman edman degradation Edman (for example seeing Creighton, 1983, supra atpp.34-49).

Another discovery of applicant is that NS1A (1-73) dsRNA of influenza virus non-structural protein 1 combines the primary categories in territory in conjunction with territory and the dsRNA that finds in most of eucaryons and former nucleoprotein, and promptly dsRBMs is different.The albumen that contains the dsRBM territory comprises eukaryotic protein kinases R (PKR) (Nanduri et al., 1998), this kinases plays a crucial role in the cell anti-virus reaction, Drosophila melonogaster Staufen (Ramos et al., 2000), with Escherichia coli RnaseIII (Kharrat et al., 1995).It is folding that the dsRBM territory comprises a monomeric alpha-beta-β-β-α.Two ditches and the intervenient major groove (Ryter﹠amp of this territory leap target dsRNA determined in structural analysis; Schultz, 1998).The several amino acid in dsRBM territory participates in directly with the interaction between the vehicular phosphodiester backbone of water, ribose 2 '-OH group and a spot of base.This bonded result is that typical A-type dsRNA duplex twists when mixture forms.This combination is stronger comparatively speaking, K _dAbout 1 nmole.Like this, method of the present invention has been utilized the special phenomenon that takes place between viral protein in the eukaryotic cell that is present in infection and the dsRNA.Therefore, the method according to this invention compounds identified does not perhaps influence normal cell function.

The applicant finds that also the proteic RNA of NS1A is by stop the activation of PKR in conjunction with dsRNA in conjunction with one of the intracellular function in territory.The applicant produces the proteic reorganization of coding NS1A A/Udorn/72 virus, and it has only lacked the RNA combined function.Because 38 R (R ³⁸) and 41 K (K ⁴¹) be unique RNA in conjunction with necessary amino acid, we replace one of them or two amino acid with A.The virus of these 3 sudden changes has all slackened virulence: R greatly ³⁸And K ⁴¹Mutated viruses forms the needle-like plaque, and dual sudden change (R38/K41) can not form the visible plaque.During the high multiple infection of any one mutated viruses to the A549 cell, PKR is activated, and eIF2a is by phosphorylation, and viral protein is synthetic to be suppressed.Surprisingly, after PKR was activated, PKR was degraded.The dual sudden change of R38/K41 is the most effective inducing aspect the PKR activation.

NS1A (1-73) is in conjunction with dsRNA, but debond dsDNA or RNA/DNA crossbred.The NS1A albumen of NS1A (1-73) and total length has shown with the non-sequence-specific of double-stranded RNA s (dsRNAs) and has combined (Lu et al., (1995) Virology 214,222-228, Qianet al., (1995) RNA 1,948-956, wang et al., 1999), but up to the present invention, determine that never can NS1A (1-73) or NS1A albumen combine with RNA-DNA crossbred or double-stranded DNA.The applicant is hatched the duplex of NS1A (1-73) and four 32P-marks together: the dsRNA of 16-bp (RR), dsDNA (DD) and two RNA-DNA heteroduplex bodies (RD and DR).Then these mixtures are gone up at homemade 15% polyacrylamide gel (Fig. 1) and analyzed.The same (Roberts and Crothers (1992) Science 258,1463-1466 with other people report; Ratmeyer et al., (1994) Biochemistry33,5298-5304; Lesnik and Preier (1995) Biochemistry 34,10807-10815), the applicant has observed the migration pattern (the fastest-the slowest) of free duplex on homemade gel: DD＞DR/RD＞RR (swimming lane is respectively 1,3,5 and 7).The more important thing is, find that only dsRNA and NS1A (1-73) form mixture, produce one 30% gel displacement (swimming lane 2), yet, all other two strands all can not and protein binding (swimming lane 4,6 and 8).These data show that under such condition NS1A (1-73) can the specific conformation and/or the constitutional features that are different from dsDNA (Type B conformation) or RNA/DNA crossbred (between the A/B conformation) of discerning dsRNA (A type conformation).

The length of dsRNA and nucleotide sequence are not crucial.In a few thing example described herein, method of the present invention can utilize the dsRNA of the synthetic 16-base pair (bp) of a weak point to operate, and comes the key feature of the interactional mode of identification of protein RNA.This dsRNA molecule has a sequence that comes from the dsRNA bound substrates of normally used 29bp, can justice and the annealing of antisense transcripton being arranged and prepare (Qian etal., 1995) on a small quantity the polylinker of plasmid pGEM1.According to the measurement means of sedimentation equilibrium, the dsRNA two strands of NS1A (1-73) and this synthetic 16-bp combines stoichiometry be approximately 1: 1 (1 protein dipolymer and a dsRNA duplex molecule), its bimolecular dissociation constant (K in solution _d) in micro-molar range.The applicant advises that it can use as the dsRNA substrate molecule that is fit in the high-throughput binding analysis.The dsRNA-conjugated antigen determinant that nmr chemical displacement disturbance experiments shows NS1A (1-73) is relevant with antiparallel spiral 2 and 2 ', with the front pass through rite-directed mutagenesis study shown in the same (Wang et al., 1999).Circular dichroism (CD) spectrum of the NS1A of purifying (1-73)-dsRNA mixture is similar with total CD spectrum of free dsRNA and NS1A (1-73), explanation is as the bonded result, and albumen or its A-type target dsRNA are rare or do not change.In addition, owing to show that NS1A (1-73) does not neither combine with DNA-RNA heteroduplex body with corresponding D NA-DNA duplex yet, as if NS1A (1-73) discern the conformational characteristic of the A-type RNA of specific standard, like this, another important mode, method of the present invention are imitated the interaction between influenza virus NS1 albumen and its host accurately.

Method of the present invention helps using under the situation of external high throughput analysis.In this embodiment of the present invention, analysis system can use one or use the FRET (fluorescence resonance energy transfer) of standard simultaneously or the dsRNA molecule of fluorescence polarization method and mark, perhaps NS1A or NS1A (1-73), or NS1B or NS1B (1-93) molecule, detect the two interchain of these target proteins and various dsRNA and interact, measure binding affinity.According to the proteic structure of the NS1 that discloses above, these analyses will be used for SCREENED COMPOUND to identify the interactional molecule that suppresses between target NS1 and RNA substrate.

According to the present invention, can check various influenza virus is had suppress active compound, comprise at random and compound library deflection.The compound library of deflection is the site that utilizes target NS1-RNA substrate interaction, for example, designs according to the special constitutional features of the inference of having delivered as a result.For example see Chien et al., Nature Struct.Biol.4:891-95 (1997); Liu et al., Nature Struct.Biol.4:896-899 (1997); With Wang et al., RNA 5:195-205 (1999).

Disturb the screening method of the interactional compound between NS1A albumen and the needed dsRNA of virus replication: the NS1 albumen of influenza virus or its dsRNA sometimes are called " binding partners " in conjunction with the territory in this article with its interaction and bonded dsRNA.Any interactional ability that all can be used to check between compound interference binding partners in many analysis systems.Yet screen the fast high-flux analysis of a large amount of compounds, include but are not limited to aglucon (natural or synthetic), also preferred polypeptide or little organic molecule.Should be by the interactional compound between the interference binding partners of this method evaluation by experiment based on cell levels, the system of animal model and the patient who describes in this article further estimate its antiviral activity.Be used for identifying and disturb influenza virus NS1 albumen or its dsRNA in conjunction with the territory, comprise that with the basic principle of the analysis system of the compound of dsRNA interphase interaction preparation under certain condition contains the reaction mixture that influenza virus NS1 albumen or its dsRNA combine territory and dsRNA, reaction is carried out time enough and is allowed two binding partners to interact and combination, thereby forms a mixture.In order to check the inhibition activity of compound, carry out under the condition of the existence of the compound that is reflected at and is not verified, for example, detected compound can join in the reaction mixture at the beginning, or adding influenza virus NS1 albumen, or its dsRNA is in conjunction with adding after for some time after territory and the dsRNA; Contrast is to hatch not having detected compound or have under the condition of placebo.Detect influenza virus NS1 albumen or its dsRNA then in conjunction with the territory, and the mixture that forms between dsRNA.In control reaction, form mixture, do not show that compound disturbs influenza virus NS1 albumen or its dsRNA in conjunction with the interaction between territory and dsRNA but in containing the reaction mixture of detected compound, form mixture.

Another aspect of the present invention comprises that effective screening can be used to treat the method for the compound of influenza infection, is included in and utilizes influenza virus NS1 albumen or its dsRNA in conjunction with the structure of territory NS1A (1-73) or NS1B (1-93) and the three-dimensional coordinator of NS1-RNA mixture model in the drug screening analysis.

Another aspect of the present invention comprises that the three-dimensional coordinator design compound library that utilizes the mixture model is in order to method for screening.

Accordingly, the invention provides the method for identifying compound and the medicine can be used to treat influenza infection.Such embodiment comprises the method in conjunction with the compound of the inhibitor in territory as influenza virus NS1 albumen or its dsRNA of identifying, and contains and come from influenza A or B virus N S1 albumen or its dsRNA database in conjunction with the three-dimensional coordinator in territory.The preferably screening of carrying out with the computer simulation coupling.

In one embodiment, the potential compound is to screen by carrying out the rational drug design according to influenza virus NS1 albumen or its dsRNA in conjunction with the three-dimensional coordinator in territory.As mentioned above, the screening of preferably carrying out with the computer simulation coupling.The potential compound combines the territory with influenza virus NS1 albumen or its dsRNA then, and the dsRNA contact, disturbs the combination between them, determines that then compound suppresses bonded ability (for example measuring).When influenza virus NS1 albumen or its dsRNA reduced in conjunction with territory and dsRNA binding ability, the potential compound was accredited as and suppresses the bonded compound.Perhaps, the potential compound contacts and/or is added to the cell culture fluid of influenza infection, has measured the growth of viral cultures.When the growth of viral cultures reduced, the potential compound was accredited as the compound that suppresses the viral growth ability.

In a preferred embodiment, method further comprise the molecular replacement analysis and two generation drug candidate design, this medicine is that the rational drug design by the three-dimensional coordinator that utilizes medicine is screened.The preferably screening of carrying out with the computer model coupling.Utilize the biochemical method of the standard of giving an example in a large amount of drug screening analysis, to check drug candidate then herein.The inhibitor storehouse that proteic three-dimensional coordinator of NS1A and NS1A-dsRNA mixture model or NS1B-dsRNA mixture model are used for screening for (a) design in these examples of the present invention, (b) reasonably optimizing of lead compound and (c) virtual screening of potential inhibitor method is provided.

In the method for the present invention that might use other analyzed in composition and the various forms part below and described.

Analyze to form: one of the binding partners that uses in the analysis system direct or indirect mark can be measured the combination degree between NS1 albumen or dsRNA bound fraction and dsRNA.According to the analytical form of following detailed description, combination degree can be with influenza virus NS1 albumen or its dsRNA in conjunction with the territory, and the form of the complexing action between dsRNA, or in the presence of candidate compound, the dissociation degree of the mixture that forms is measured in advance.Can use be fit to the mark system any one including but not limited to as ¹²⁵The radio isotope of I; When being exposed to substrate, produce detectable enzyme mark system than chrominance signal or light; And fluorescent mark.

The NS1 albumen or its dsRNA that utilize recombinant DNA technology to produce influenza virus help the through engineering approaches fusion rotein in conjunction with the dsRNA binding partners in territory and the analysis, and described fusion rotein can promote mark, fixing and/or detection.For example, NS1 albumen of influenza virus or its dsRNA in conjunction with the encoding sequence in territory can with have enzymic activity or merge as the heterologous protein of enzyme substrates, to promote mark and detection.Fusion construct should be designed so that the allos component of fusion product do not disturb NS1 albumen of influenza virus or its dsRNA to combine with dsRNA in conjunction with the territory.

Indirect labelling comprises and uses the 3rd albumen, the antibody of mark for example, and the NS1 albumen of itself and influenza virus, or its dsRNA is in conjunction with the territory specific combination.The fragment that this antibody produces including but not limited to polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library.

In order to produce antibody, with the NS1 albumen of influenza virus, or its dsRNA is in conjunction with the various host animals of territory injecting immune.Such host animal can be including but not limited to rabbit, and mouse and rat are only pointed out.Kind according to the host, can use various adjuvants to increase immune response, including but not limited to Freund ' s (fully with incomplete), mineral coagulant such as aluminium hydroxide, tensio-active agent such as lysolecithin, Pluronic polyols, polyanion, peptide, oil breast, people's adjuvant that keyhole limpet hemocyanin, dinitrophenol and potential have use value is BCG (bacille Calmette-Guerin vaccine) and corynebacterium parvum vaccine for example.

Can utilize the technology of any cultured continuously cells produce antibody molecule to prepare monoclonal antibody.These technology are including but not limited to the hybridoma technology (Nature that is described first by Kohler and Milstein, 1975,256:495-497), people B-quadroma technology (Kosboret al., 1983, Immunology Today, 4:72, Cote et al., 1983, Proc.Natl.Acad.Sci., 80:2026-2030) and EBV-hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp.77-96).In addition, the technology of the production of development " embedding and antibody " also can be used (Morrison et al., 1984, Proc.Natl.Acad.Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454), promptly the gene of the antibody molecule of the antigen-specific that is fit to by will coming from mouse and the gene splicing that comes from people's suitable biologically active antibody molecule are in the same place.Perhaps, can adopt the technology of manufacture order chain antibody (U.S.Pat.No.4,946,778) to produce specific NS1 albumen, or its dsRNA is in conjunction with the single-chain antibody in territory at influenza virus.

The antibody fragment that can discern the specific antigen determinant can be by known technology production.For example, such fragment includes but are not limited to: the F by producing with the pepsin digested antibody molecule (ab ') 2 fragments and the Fab fragment that produces by reduction F (ab ') 2 segmental disulphide bridgeses.Perhaps, can make up the Fab expression library (Huse et al., 1989, Science 246:1275-1281) comes the specific mono-clonal Fab fragment that has expection is carried out being easy to fast detect.

Analytical form: can analyze with heterogeneous or homogeneous form.Heterogeneous analysis comprises one of binding partners is anchored on the solid phase that the detection reaction end is anchored on the mixture on the solid phase.In the homogeneous analysis, entire reaction is carried out in liquid phase.In any one method, can change the different information that the order that adds reactant obtains tested compound.For example, the compound of binding partners interphase interaction is disturbed in check, for example by competition, can react in the presence of detected substrate and identifies; Just in advance or simultaneously detected substrate and influenza virus NS1 albumen or its dsRNA are joined in the reaction mixture in conjunction with territory and dsRNA.On the other hand, destroy the compound of preformed mixture, for example compound has higher binding constant to replace one of them binding partners from mixture, can identify by add detection compound in forming the later reaction mixture of mixture.Below various forms will be described simply.

In heterogeneous analysis system, one of binding partners, for example, influenza virus NS1 albumen or its dsRNA be in conjunction with the territory, or dsRNA is anchored at solid phase surface, the binding partners that is not anchored with direct or indirect method mark.In fact, use microwell plate more convenient.The part of grappling can be by non-covalent or covalent attachment with fixing.Perhaps, can use influenza virus NS1 albumen or its dsRNA are anchored on influenza virus NS1 albumen or its dsRNA on the solid phase surface in conjunction with the territory in conjunction with the special antibody of fixed in territory.Can prepared beforehand and this surface of storage.

In order to analyze, with the binding partners of fixed part join with or the surface that covers without detection compound.After reaction is finished, remove responseless component (for example by rinsing), the mixture of any formation will be fixed on solid phase surface.Can make and detect the mixture that is anchored on solid phase surface in many ways.When binding partners during, detect the sign that is fixed on the surface and show and formed mixture by prior mark.When binding partners during, can use indirect labelling to detect the mixture that is anchored on solid phase surface not by prior mark; For example, use the traget antibody special (conversely, directly traget antibody or with the antibody indirect traget antibody of the anti-Ig of mark) to binding partners.According to the addition sequence of reactive component, can detect and suppress the tested compound that mixture formed or destroyed the mixture that forms in advance.

Alternatively, can be in liquid phase deposit under the condition that is in or be not in and react, reaction product is separated from unreacted component and detect mixture at tested compound; For example, use influenza virus NS1 albumen or its dsRNA, come the alloy that forms in the grappling solution in conjunction with the special fixed antibody in territory.Once more, according to the addition sequence of reactive component in the liquid phase, can detect and suppress the tested compound that mixture formed or destroyed the mixture that forms in advance.

Other embodiment of the present invention can be used homogeneous analysis.In this method, influenza virus NS1 albumen that preparation forms in advance or its dsRNA are in conjunction with the mixture of territory and dsRNA, wherein one of binding partners is labeled, but the signal that mark produces (is for example seen because of the formation cancellation of mixture, the U.S.Pa t.No.4 of Rubenstein, 109,496, utilize this method to carry out immunoassay).The generation that affiliation causes the signal on the background that adds with the detection material of one of the competition of the mixture of prior formation and displacement binding partners.In this way, can identify destruction influenza virus NS1 albumen or its dsRNA interactional analyte in conjunction with territory and dsRNA.

For example, the recombinant DNA technology of describing before specific example is to use prepares influenza virus NS1 albumen or its dsRNA in conjunction with being used for fixing of territory.Utilize fusion vector pGEX-5X-1, its encoding domain can be fused in glutathione-S-transferase (GST) gene, and the combination that keeps it by this way in the fusion rotein that produces is active.Can be used for producing to NS1 or the special monoclonal antibody of NS1 fragment by above-mentioned and method influenza virus purification NS1 albumen this area routine or its dsRNA in conjunction with the territory.This antibody can be used emitting isotope ¹²⁵The I mark, for example, by the method for this area routine.In heterogeneous analysis, for example the GST-NS1 fusion rotein can be anchored on gsh-sepharose 4B.Deposit under the condition that is in or be not at tested compound then and add dsRNA so that dsRNA partly interacts with the NS1 of fusion rotein and combines.After tested compound adds, can the unconjugated material of flush away, in system, add mark the special monoclonal antibody of NS1 so that its be attached on the compound binding partners.By the amount detection NS1 of radioactivity residual on measurement gsh-sepharose 4B and the interaction between dsRNA.Tested compound can cause that to interactional successfully inhibition the radioactivity that measures reduces.

When perhaps in liquid phase, not having solid gsh-sepharose 4B, GST-NS1 fusion rotein and dsRNA are mixed.Tested compound can add during binding partners interacts or afterwards.Then mixture is added to the unconjugated material of gsh-sepharose 4B and flush away.By the amount detection NS1 of radioactivity residual on measurement gsh-sepharose 4B and the interactional inhibition degree between dsRNA.

According to the present invention, the given compound that suppresses a kind of virus can be used for detecting the comprehensive antiviral activity to different influenza viruses on a large scale.For example, be not limited to this method,, can detect by combination suppresses the different change strains of the compound of influenza A virus NS1 and dsRNA interphase interaction to influenza A virus and influenza B virus with the NS1 binding site according to hereinafter said analysis.

For potential lead compound in the screening of medicaments research and development, that has identified can further verify the inhibition ability that they duplicate influenza virus at first in tissue culture then to the interactional inhibitor between target NS1 and RNA substrate in animal model experiment.Utilize height and low infection multiplicity to determine the minimum concentration that effective inhibition influenza virus of each inhibitor is duplicated.

The viral growth experiment: the virustatic ability of the inhibitor of determining in the aforesaid analysis system can be by the index analysis of plaque formation or other viral growth, for example TCID50 or the growth in chick embryo allantois.In these experiments,, when infecting or after infecting, tested compound is joined in the tissue culture medium (TCM) with the suitable clone or the embryonated egg of influenza infection of wild-type.The effect of tested compound forms quantitatively by virion and estimates, erythrocyte agglutination element (HA) titer of being measured by the chick embryo allantoic liquid supernatant of cells infected or infection; By presenting of viral plaque; Or do not present under the situation of plaque phenotype, by TCID50 or the growth index in chick embryo allantois, or the erythrocyte agglutination element is analyzed and is estimated.Inhibitor can suppress the HA titer or plaque forms by tested compound, or reduces the ability of the cytopathy effect of the cell of virus infection or chick embryo allantois, or the ability that forms of the virion of measuring in being reduced in that erythrocyte agglutination is plain and analyzing and estimating.

Animal model experiment: can be used for carrying out follow-up experimentation on animals to the most resultful inhibitor of virus replication by what method of the present invention was identified.The ability that inhibitor stops influenza virus to be duplicated can be analyzed in influenza host's natural or that transform animal model.These animals can comprise as pig, ferret, mouse, monkey, the Mammals of horse and primates, or birds.The same with at this detailed description, these animal models can be used to determine the LD in the animal ₅₀And ED ₅₀, these data can be used to obtain the therapeutic index of NS1A (1-73) or NS1B (1-93) and the interactional inhibitor of dsRNA.

NS1 albumen that the inhibitor of determining through high flux screening characterizes or its dsRNA help the optimization design lead compound in conjunction with the binding site of field surface.These signs can be utilized chemical shift of NMR disturbance NMR and NMR resonance location and carry out.NMR can determine the binding site of micromolecular inhibitor to RNA.Location to these binding sites can provide data for the optimization of uniting the design of a lot of beginning inhibitor guide things and guide's thing.

Medication preparation and medication: the compound of the inhibition virus replication of having identified can allow patient take under the effective dosage of treatment and treat virus infection.The treatment effective dose is meant the amount of the compound that enough causes the alleviation of virus infection symptom.

The therapeutic efficiency of this compounds and toxicity can be determined by the pharmacy program of standard in cell cultures or the laboratory animal, for example, determine LD ₅₀(making the dosage of 50% animal dead) and ED ₅₀(making the effectively dosage of treatment of 50% animal).Toxicity and the effective dose ratio of treatment are therapeutic indexs, can be expressed as ratio LD50/ED ₅₀The preferred compound that shows big therapeutic index.When using the compound of performance toxic side effect, one of the design of being careful navigates to the delivery system of infection site with this targeting compounds, reduces medicine to greatest extent to the cells injury that do not infect and reduce side effect.

The data that obtain from cell culture assays and zooscopy can be used for calculating the dosage range that uses at the person.Comprise preferably seldom or do not have a toxic ED ₅₀The circulation composition scope in compound dosage.Dosage can change in this scope according to the formulation and the administering mode that use.Any compound that uses in the method for the present invention can be estimated effective dose from initial cell culture experiments.In animal model, calculate dosage to realize being included in the IC that determines in the cell cultures ₅₀The circulating plasma concentration range of (just, the acquisition half is maximum to be infected, or the maximum tested compound concentrations that suppresses of half).These information can be used to the using dosage among more accurate definite mankind.Can measure the level in the blood plasma, for example, pass through high performance liquid chromatography.

Pharmaceutical composition used according to the invention can be prepared with one or more physiology acceptable carriers or vehicle with traditional mode.

Like this, the physiologically acceptable salt of compound and they and solvation salt can be mixed with by sucking or be blown into the administering mode medication of (or through port or nose) or oral, oral cavity, non-enteron aisle or rectum.

For administering mode by sucking, the compound that the present invention uses can spray the form that presents with aerosol easily, with the propelling agent that is fit to for example, Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas are carried from forcer or jet-type spraying gun.Under the situation of applying pressure aerosol, can carry the gas of some amount to determine dose unit by a valve is provided.The capsule of for example gelatin that uses in the administering mode that sucks or be blown into and explosive barrel can be made into and contain compound and as the powdered mixture of the suitable powder-base of lactose or starch.

For oral administering mode, pharmaceutical composition by conventional method with acceptable vehicle such as binding agent in the pharmacy (for example, the W-Gum of pre-gelatinization, polyvinylpyrrolidone or hypromellose); Weighting agent (for example, lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (for example, Magnesium Stearate, talcum or silica); Disintegrating agent (for example, yam starch or sodium starch glycollate); Or wetting agent (for example, Sodium Lauryl Sulphate BP/USP) is prepared into as tablet or capsular form.Available methods known in the art are given tablet coating.The liquid preparation of oral pharmaceutical can adopt, as solution, and the form of syrup or suspension, or they occur with the form of dryed product but water or other suitable medium are made into before use.Can add acceptable additive in the pharmacy by conventional methods, for example suspension agent (for example, sorbitol syrups, derivatived cellulose or hydrogenant edible fat); Emulsifying agent (for example, Yelkin TTS or Sudan Gum-arabic); Non-hydrophily Jie (for example, Prunus amygdalus oil, grease, alcohol or fractionated vegetables oil); And sanitas (for example, methyl p-hydroxybenzoate or propylparaben or Sorbic Acid) preparation liquid preparation.Also can contain an amount of buffering salt in the preparation, seasonings, pigment and sweeting agent.

The preparation of oral administration can be suitable the may command releasing pattern that is mixed with active compound.

The composition of orally administering can adopt usual manner to be mixed with the form of tablet or cough syrup.

Compound can be mixed with the parenterai administration form by injection, for example, injects or inculcates continuously by bolus.The preparation that is used for injecting can present with unit dosage form, for example, and in ampoule that is added with sanitas or multi-dose container.Composition can adopt suspension, and solution, or the form of the milk sap of oily medium or water medium for example can contain vehicle, suspends, stable and/or dispersion agent.Perhaps, active component with powdered form and the medium that is fit to for example, apyrogenic sterilized water is made into before use.

Compound also can be made into composition such as the suppository or the retention enema of rectal administration, for example, contains traditional suppository base as theobroma oil or other glyceryl ester.

Except aforesaid preparation, compound also can be made the storage formulation.This prolonged action preparation can be by transplanting (for example subcutaneous or intramuscular) or passing through administered intramuscular.Like this, for example, compound can with suitable polymerization or hydrophobic material (for example as the emulsion in the acceptable oil) or ion exchange resin, or with inert (Sparingly) soluble derivative, for example, the form of inert soluble salt is prepared.

If desired, composition can be present in packing or the divider, and it can comprise the one or more unit dosage forms that contain active ingredient.For example this packing can comprise metal or plastic tab, for example blister packaging.This packing or divider and medication instruction book are put together.

The present invention is not limited to described in this article embodiment, only otherwise depart from the scope of the present invention and can modify or change.

The preparation of embodiment 1 protein sample:, under 37 ℃ condition, cultivate, then at cell density OD with expression vector pET11a Transformed E .coli BL21 (DE3) cell culture of coding NS1A (1-73) ₆₀₀, containing the homogeneous enrichment at=0.6 o'clock with 1mM IPTG ¹⁵NH ₄Cl and ₁₃C ₆(Janssonet al., (1996) J.Biomol.NMR 7 induced 5 hours in 131-141.)-glucose as the MJ minimal medium of unique nitrogenous source and carbon source respectively.Cell is through ultrasonication, then at 4 ℃, and under the condition of 100,000 * g centrifugal 1 hour.The method of describing according to other places then, use the ion-exchange of Pharmacia FPLC system and gel permeation chromatography method from supernatant purifying protein (Qian et al., (1995) RNA 1,948-956.).About 5 milligrams of every liter of nutrient solution of ultimate production of the NS1A of purifying (1-73).Proteic concentration is to utilize 5750M ^-1Gm ^-1Monomer molar optical extinction coefficient (ξ 280) is at the absorbancy (A of 280nm ₂₈₀) and determine.

Synthetic and the purifying of embodiment 2RNA oligomer: phosphoramidite chemistry (the Wincott et al. that utilizes standard, (1995) Nucleic Acids Res.23,2677-2684) at DNA/RNA synthesizer Model 392 (Applied Biosystem, InC.) 16-Nucleotide (16-nt) RNA, CCAUCCUCUACAGGCG (justice is arranged) and the CGCCUGUAGAGGAUGG (antisense) of two strands of chemosynthesis (ss).Then these two RNA oligomer are passed through the desalination of Bio-Rad Econo-Pac 10DG post, by 20% (w/v) polyacrylamide, the preparative gel electrophoresis purifying of the urea-denatured glue of 7M.To cut by the suitable product band that the UV image is observed, crushing is extracted 90mM Tris-boric acid with product under the condition of the shaken over night of gentleness, and 2mM EDTA is in the damping fluid of pH8.0.The solution that obtains is concentrated and uses the desalination once more of Econo-Pac 10DG post by freeze-drying.Then with the freeze-drying once more of purified RNA oligomer ,-20 ℃ of preservations.The similar 16-nt that contains same sequence has justice and the antisense DNA chain can be from Genosys Biotechnologies, the Inc purchase.Absorption (A260) according to the 260nm place utilizes following molar extinction coefficient ( ₂₆₀, at 20 ℃ M ^-1Cm ^-1): (+) RNA, 151 530; (-) RNA, 165 530; (+) DNA, 147 300; (-) DNA, 161 440; DsRNA, 262 580; RNA/DNA, 260 060; DNA/RNA, 273 330; DsRNA, 275 080 calculate nucleic acid samples concentration.The optical extinction coefficient of strand calculates (Cantor et al. by 20 ℃ of monomers and dimeric optical extinction coefficient, (1965) J.Mol.Bio.13,65-77), suppose that molar absorptivity is that nearest-neighbor characteristic and oligonucleotide are strand (Hung et al. at 20 ℃, (1994), Nucleic Acids Res.22,4326-4334).Double-stranded molar extinction coefficient is from 20 and 90 ℃ A ₂₆₀Value utilizes following expression formula to calculate: ε (260,20 °)=[A, _{260,20 °)}/ A, _{260,90 °)}] * ε _{(260,90 °, calc)}, at this, ε _{(260,90 °, calc)}The molar extinction coefficient that obtains from the summation of strand when to be that supposition is double-stranded dissolve under 90 ℃ temperature fully.

Embodiment 3 polyacrylamide gels displacement binding analysis: the RNA of synthetic strand 16-nt and DNA oligonucleotide utilize T4 polynucleotide kinase [γ at its 5 ' end ³²P] ATP mark and usefulness sex change urea-PAGE purifying.About 1: 1 of mol ratio strand (ss) is had adopted RNA (or DNA) and sense-rna (or DNA) at 50mM Tris, and 100mM NaCl is in the damping fluid of pH8.0.Solution is annealed two strands at 2 minutes slow then cool to room temperature of 90 ℃ of heating.With ultimate density is binding buffer liquid (the 50mM Tris-glycine that 0.4 μ M NS1A (1-73) joins 20 μ l respectively, 8% glycerine, the 1mM dithiothreitol (DTT), the tRNA of 50 nanograms/microlitre, the RNasin of 40 units, pH8.0) (dsRNA (RR) in four two strandss (ds) nucleic acid in, RNA-DNA (RD) and DNA-RNA (DR) crossbred, and dsDNA (DD), 10,000cpm, final concentration approximate 1 nmole).With reaction mixture ice bath 30 minutes.The protein-nucleic acid mixture is at 4 ℃, and 15% non-sex change PAGE was 150 volts of voltage electrophoresis 6 hours, and at 50mM Tris-boric acid, 1mMEDTA dissolves from free two strands or strand oligomer in the damping fluid of pH8.0.

Embodiment 4 gel permeation chromatography analyses: micromole's solution 10mM potassiumphosphate of the duplex of 4 16-nt (RR, RD, DR and DD), 100mM KCl, 50 μ M EDTA, pH7.0 buffer preparation and as above-mentioned method annealing.Utilize never these two strandss of purifying in annealed or the superfluous strand of Superdex-75HR 10/30 gel-filtration column (Pharmacia) then, then two strands is regulated concentration to 4 μ M.Then each double-strandednucleic acid being joined and making two strands and albumen molar ratio among the 1.5mMNS1A (1-73) (monomeric concentration) is 1: 1.On Superdex-75HR 10/30 post (Pharmacia), carry out gel permeation chromatography.Calibrate this post with four standard proteins: albumin (67kDa), ovalbumin (43kDa), chymotrypsinogen A (25kDa) and ribonuclease A (13.7kDa).At the 10mM potassiumphosphate, 100mM KCl, 50 μ M EDTA carry out chromatography at 20 ℃ in the pH7.0 damping fluid, and flow velocity is 0.5 milliliter of per minute.With the albumen-double-stranded sample upper prop of 1: 1 mol ratio, by their A ₂₆₀Detect the existence of the nucleic acid that flows out part; Owing to compare the ε of NS1A (1-73) with nucleic acid double chain ₂₆₀Relatively little, can ignore the contribution that it absorbs UV.

The purifying of embodiment 5NS1A (1-73)-DSRNA mixture: NS1A (1-73) dimer and the dsRNA mixture part of collecting mol ratio corresponding to first peak in the gel permeation chromatography and be 1: 1, (Amicon Inc.) is concentrated to below 1 milliliter with the Centricon thickener.Then this concentrating sample is gone up once more and collected main outflow part on the same gel-filtration column once more.The concentration of the NS1A of purifying (1-73)-dsRNA mixture absorbs by the UV that measures the 260nm place to be determined.By with 4 μ M sample pipetting volumes, 100 μ l, utilize the analysis mode gel-filtration to preparation immediately and purity and stability a month later sample detection mixture of preparation.

Embodiment 6 sedimentation equilibriums: utilize a Beckman XL-I instrument to carry out the sedimentation equilibrium experiment at 25 ℃.Behavior for these free components of independent assessment, utilize the charcoal-Epon chamber in 12 millimeters paths, Beckman 8-road to carry out the short column chromatography of NS1A (1-73) and dsRNA under 30K to 48K rpm speed, last sample concentration is respectively every milliliter of every milliliter of 0.2-2 milligram and 0.2-0.6 milligram.Utilize the Rayleigh interference optical system to obtain data.For study NS1A (1-73) dimer and dsRNA in conjunction with behavior, the long column chromatography is carried out to the sample with the mixture of gel permeation chromatography purifying in the charcoal-Epon chamber that utilizes Beckman 6-road (1.2 centimetres of paths) under 16K to 38K rpm speed.Utilize UV to absorb optical system at 260nm, last sample concentration obtains these data when being 0.3,0.5 and 0.6 absorbance units.For guaranteeing the sample balance, short column totally 4 hours every 0.5 hour, long column was measured every 1-6 hour totally in 28 hours.Utilize WINMACH (by Yphantis, D.A. and Larry, the J exploitation, by National Analytical Ultracentrifugation Facility at TheUniversity of Connecticut supply and marketing) program calculating, when the difference that separates twice scanning room of one hour is disturbed in the scope of optics at 0.005-0.008 for Rayleigh, or for absorbancy in the 0.005OD, determine to have set up balance.

Utilize the WINNL106 program, carry out data analysis according to the Windows95 version of primary nonlinear least square program NONLIN (Johnson et al., 1981).These data or be each data centralization at specific other match of branch of going up sample concentration and speed, or be at the different last sample concentration and/or the match of velocity joint in the combination of several data sets.Overall fit is meant by the binding constant InK that utilizes all data sets and handle as General Parameters and obtains together.That causes for fear of departing from Beer ' s law is complicated, unless indicate the absorption data OD different with the baseline district≤1.0 cutoff value editors.

Little partially specific volume v of NS1A (1-73) _NS1With solvent density ρ, utilize sednterp program (Laue et al., 1992) to be respectively 0.7356 and 1.01156 as calculated at 25 ℃.Little specific volume v of dsRNA _RNA, be defined as 0.5716 (seeing conclusion for details) by the sedimentation equilibrium experiment of dsRNA sample.NS1A (the 1-73)-little specific volume v of dsRNA mixture _Mixture, suppose that 1: 1 stoichiometry utilizes Cohn and Edsall (Cohn﹠amp; Edsall, 1943) method is calculated as 0.672.

The calculating of embodiment 7 dissociation constants: 1: 1 NS1A (1-73)-dsRNA complex dissociation constant is that the amount according to free NS1A (1-73) albumen and free dsRNA in the original solution is that equimolar hypothesis is calculated.If when the composite sample of using in these are measured through the gel-filtration purifying was actually 1: 1 stoichiometry, this hypothesis was effective.In this case, the amount correspondence of free dsRNA and free NS1A (1-73) dissociated amount from 1: 1 mixture.In addition only be 3% because the molecular weight of NS1A (1-73) dimer and dsRNA reduces the difference of (defining among the Eq.2 below), with two freely macromole be used as and between sedimentation period, have same hydrodynamic force type of process.The concentration of the ith type of idealized system is distributed and can be expressed as during sedimentation equilibrium

$C_i\left(r\right)=C_i{\left(r^1\right)e}^{{\mathrm{\&sigma;}}_i({\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2-{\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2)}----(E_q.1)$

(Johnson et al., 1981) are C (r) wherein _iBe the weight concentration of ith component when radius is r, r ' is a reference position in the post solution.i in the above-mentioned equation is the molecular weight (Yphantis﹠amp that reduces; Waugh, 1956):

σ _i＝M _i(1- viρ)ω ²/RT. (Eq.2)

Mi among the Eq.2 and vi are the molecular weight of ith kind and little partially specific volume, and R is a gas law constant, and T is an absolute temperature, and ω is a circular frequency.Concentration is used weight concentration scale (every milliliter of milligram) expression usually, yet, use volumetric molar concentration m in this example, mi=C _I/ M _iMore convenient.

Based on the principle of mass conservation (Van Holde﹠amp; Baldwin, 1958), dsRNA can be expressed from the next

Quality m ^oBe meant the concentration of original solution, m (r) when referring to sedimentation equilibrium radius be the concentration of r.Subscript " RNA, t ", " RNA, free " and " RNA, x " refer to dsRNA respectively, the total amount of the dsRNA in free dsRNA and NS1A (1-73)-dsRNA mixture; T _mAnd r _bBe meant the radius value of meniscus and bottom in the solution post respectively.In order to simplify following result, r ' is set at r _mThe position.Comprehensive equation 3 obtains then:

M (r wherein _b) _{RNA, free}And m (r _b) _{RNA, x}Refer to concentration respectively at the free dsRNA and the dsRNA in NS1A (1-73)-dsRNA mixture of solution column bottom.Same equation also can be represented NS1A (1-73) albumen.At m ^o _RNAEqual m ^o _NS1The time obtain equation:

Utilization is for specific protein: RNA mixture σ _RNA≈ σ _NS1The fact, Eq.5 is presented at reference position m (r ') _{RNA, free}=m (r ') _{NS1, free}, like this, m when any radius r (r) _RNA,Free=m (r) _{NS1, free}

Finally, the absorption of radius r can be expressed as during sedimentation equilibrium:

$A_{260}\left(r\right)=E_{x}m(r^1{)}_{\mathrm{RNA}}{e}^{\mathrm{\&sigma;RNA}({\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2-{\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2)}+(1/E_x)K_a[E_{x}m(r^1{)}_{\mathrm{RNA}}{e}^{\mathrm{\&sigma;RNA}({\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2-{\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A20038010864300621.png"\; state="\{\{state\}\}">r</figure-callout>}}^2/2)}{]}^2----(E_q.6)$

In the superincumbent equation, E _x=(ε _RNA+ ε _NS1) l, wherein ε is an optical extinction coefficient, l is an optical range.Ka is the binding constant that the volumetric molar concentration scale is represented, at m _RNA=m _NS1Condition under with function m _xAnd m _RNAYuan shows (Eq.7).

Ka＝mx/mRNA2 (Eq.7)

Like this, the articulated system of NS1A (1-73) and dsRNA is reduced to the simple system of two components between sedimentation period.It is easy to use fitting parameter K ₂=K _a/ E _xCarry out match, the dissociation constant of NS1A (1-73) and dsRNA mixture, K with ideal monomer-dimer oneself bonded NONLIN model _dFrom following equation, calculate:

K _D＝1/(E _xE ₂). (Eq.8)

Embodiment 8NMR spectroscopy: all NMR data all are to collect in the Varian in 4 roads INOVA500 and 600NMR spectrometer system at 20 ℃.Service routine VNMR (Varian Associates), NMRCompass (Molecular Simulations, Inc.), and AUTOASSIGN (Zimmerman et al., (1997) J.Mol.Biol.269 592-610) carry out the processing and the analysis of data.Chemical shift of proton is with reference to interior 2,2-dimethyl-2-silicon pentane-5-sulfonic acid; ¹³C and ¹⁵The chemical shift indirect reference of N is used magnetogyric ratio separately, ¹³C: ¹⁵N (0.251449530) and ¹³N: ¹⁵H (0.101329118) (Wishart et al., (1995) J.Biomol.NMR 6,135-140).

Embodiment 9NS1A (1-73) sequence-specific location: be used for localized free ¹³C, ¹⁵N-NS1A (1-73) NMR sample prepares in the NMR test tube that the Shigemi susceptibility matches, and wherein has 270 μ l to contain 50mM ammonium acetate and 1mM sodiumazide, the 95%H of pH 6.0 ₂O/5%D ₂Dimer protein concentration is 1.0-1.25mM in the O solution.By ¹³C, ¹⁵The proteic three resonance NMR spectrum of N-enrichment are analyzed automatically, and (Zimmerman etal., (1997) J.Mol.Biol.269 592-610) determines skeleton to utilize computer program AUTOASSIGN ¹H, ¹³C, ¹⁵N and ¹³C The resonance location.The input of AUTOASSIGN comprises and comes from two dimension ¹H- ¹⁵N HSQC and three-dimensional HNCO spectrographic peak lists come from the peak lists that 3 inner residues [HNCA, CBCANH, and HA (CA) NH] and 3 mutual residues [CA (CO) NH, CBCA (CO) NH and HA (CA) be NH (CO)] are tested simultaneously in addition.The details of these pulse sequences and parameters optimization is commented (Montelione et al., (1999), Berliner, L.J. elsewhere, and Krishna, N.R., Eds, Vol.17, pp 81-130, Kluwer Academic/Plenum Publishers, New York).The used peak lists of AUTOASSIGN is to utilize NMRCompass to produce by automatic peak value collection, and edit is to remove tangible noise peak and artificial spectrum then.Then by manual analyzing three-dimensional HCC (CO) NH TOCSY (Montelione et al., (1992) J.Am.Chem.Soc.114,10974-10975), HCCH-COSY (Ikruaet al., (1991) J.Biomol.NMR 1,299-304) and ¹⁵The TOCSY that N-edits (Fesik et al., (1988) J.Magn.Reson.78,588-593) experiment and two-dimentional TOCSY spectrum, the mixing time of record is 32,53 and 75ms (Celda and Montelione (1993) J.Magn.Reson.Ser.B 101 189-193) obtains side chain resonance location (except the aromatic nucleus side chain ¹³Beyond the C location).

Embodiment 10NMR chemical shift disturbance experiments: purifying and preparation as mentioned above ¹⁵The NS1A of N-enrichment (1-73).Use 250 μ l earlier ¹⁵The NS1A of N-enrichment (1-73), the 0.1mM dimer, in the 50mM ammonium acetate, 1mM sodiumazide, 5%D ₂O, pH6.0 solution collect floating preteins ¹H ^N- ¹⁵N HSQC spectrum.16-nt have justice and sense-rna chain with 1: 1 molar ratio at the 200mM ammonium acetate, anneal in the solution of pH7.0, freeze-drying 3 times, with the dissolving of same NMR sample buffer, final RNA two strands concentration is 10mM.Come titration free with this highly spissated dsRNA solution then ¹⁵The NS1A of N-enrichment (1-73) NMR sample, the preparation ratio is the albumen-dsRNA sample of 2: 1,1: 1,1: 1.5 and 1: 2 than (dsRNA) for (dimer protein).In order to prevent the precipitation of NS1A (1-73), sample solution is to prepare by the free protein solution is slowly joined among the spissated dsRNA.Free ¹⁵The HSQC spectrum of the NS1A of N-enrichment (1-73) is that 80 scanning and 200 * 2048 mixture data points obtain with every increment, changes 1024 * 2048 points over to after the zeroing of t1 dimension.Utilize 320 scanning of every increment to collect the HSQC spectrum of dsRNA volumetry experiments of measuring with same numerical analysis.

Embodiment 11CD measures: the Aviv model 62-DS spectropolarimeter that utilizes the cell that is equipped with 1 centimetre of path length is at 20 ℃, and the 200-350 nanosection writes down CD spectrum.From 1.1 milliliters, the sample in the above-mentioned phosphate buffered saline buffer of 4 μ M obtains four nucleic acid double chains (RR, RD, DR, CD spectrum DD).Each two strands and 1.5mM NS1A (1-73) (monomer concentration) are in conjunction with forming albumen and mol ratio of 1: 1 of two strands then.Under same condition, collect the CD spectrum of these albumen-double-stranded mixture, suppose that the total double-stranded concentration of each sample remains 4 μ M.Also obtained the CD spectrum of NS1A (the 1-73)-dsRNA mixture of 1.1 milliliters free NS1A (1-73) of 4 μ M concentration in identical phosphate buffered saline buffer and column purification.The summation that utilization comes from free NS1A (1-73) and each double-strandednucleic acid CD data separately obtains the CD spectrum of the albumen-double-stranded mixture of calculating.CD spectrum is stated as ε _L-ε _R, unit is M ^-1Cm ^-1Every mole of Nucleotide.

Embodiment 12 is by purifying and the sign of gel permeation chromatography to NS1A (1-73)-DSRNA mixture: above-mentioned four NS1A (1-73)-nucleic acid double chain mixture further utilizes the analysis mode gel permeation chromatography to analyze the formation of mixture.NS1A (1-73)-dsRNA mixture shows two main peaks in the tomographic map of 260 nanometer detection (Fig. 2 A), and the mixture that contains dsDNA and RNA/DNA only by wash-out be a peak (Fig. 2 B, C, D).Because the chromatography elutriant detects the absorption in 260 nanometers, the state of these tomographic map reflection nucleic acid in these samples.In the example of dsRNA (Fig. 2 A), quicker and slow elution peak is respectively corresponding to NS1A (1-73)-dsRNA mixture and unconjugated dsRNA two strands.For the elution time of elution peak faster and corresponding molecular weight (～26kDa) with 1: 1 stoichiometry (the albumen dimer is to dsRNA) mixture be consistent.About 70% RNA and albumen werte under chromatography condition, have been used in the mixture component.Do not observe the peak that forms corresponding to mixture in other sample.These results further are NS1A (1-73) and dsRNA, rather than combine evidence is provided with dsDNA or RNA/DNA crossbred specificity in this research.Permanent stability (Fig. 3) at follow-up experiment (for example, sedimentation equilibrium and CD) and evaluating combined thing are also used preparation type gel permeation chromatography purifying NS1A (1-73)-dsRNA mixture before.The NS1A of fresh purifying (1-73)-dsRNA mixture chromatograph once more produces one unimodal (Fig. 3 A) corresponding to relatively stable and pure mixture.Yet,, show that mixture can irreversible slowly decomposition when long-time 4 ℃ of increases (Fig. 3 B) of observing free dsRNA after depositing one month.

Embodiment 13 sedimentation equilibriums: free NS1A (1-73) and DSRNA: use sedimentation equilibrium to determine the stoichiometry that mixture forms between NS1A (1-73) and 16-bp dsRNA two strands and the constant that dissociates.At first, utilize a plurality of application of sample concentration and many rotating speeds that NS1A (1-73) albumen of purifying and the dsRNA sample of purifying are carried out short column balance chromatography.NS1A in solution (1-73) albumen exists with dimeric forms, and molecular weight is every mole of 16,851 gram, and signal (No data) does not significantly dissociate.In some instances, NS1A (1-73) sample that is used for the experiment of these sedimentation equilibriums comprises the existence of big non-special aggregate.The total amount of each sample collection bodily form formula changes to some extent, and separates from the dimer kind down high-speed.It is the accumulation process of a sample dependence slowly that this indication is assembled.Then and dsRNA compound protein sample by the gel-filtration purifying, carry out sedimentation equilibrium afterwards and detect (see figure 3).The dsRNA sample of purifying shows as a kind of ideal one-component solution between sedimentation period.With the match of the one-component of data and NONLIN model and the molecular weight of the reduction of the estimation that obtains do not change with the change of load sample concentration and/or speed.Utilize Eqn.2 (on seeing) like this, can calculate little specific volume of dsRNA according to the molecular weight of the minimizing of estimating.The value that obtains, v _RNAThere is good consistence (Ralston, 1993) in=0.57 unit with little partially specific volume of typical DNA (0.55-0.59 unit) and RNA (0.47-0.55 unit).v _RNAValue from dsRNA than from typical R NA sample will be more approaching the fact may be owing to its double-stranded configuration.In the molecular weight that reduces, according to conservative estimation, nearly 7% error is translated into about identical error in little specific volume.In this analyzes, suppose that the formation of mixture has no significant effect dsRNA and the proteic little specific volume of NS1A (1-73).

Stoichiometry and thermodynamics that the mixture that embodiment 14 gets according to sedimentation equilibrium forms: utilize above-mentioned in preparation purifying and by analysis the gel-filtration of type determine it is NS1A (1-73)-dsRNA composite sample research NS1A (1-73) albumen of homogeneous and the combination (Fig. 3 A) of dsRNA.Determine the stoichiometry (Fig. 4 A) of mixture according to 16000 data of changeing the per minute collection.Under low like this speed, free dsRNA and NS1A (1-73) albumen has one less than 0.5 σ i value (Eqn.2).Under the condition of such lower velocity, the kind of two lower molecular weight (just, free NS1A (1-73) and free dsRNA) is redistributed no longer significantly, like this absorption curve is had the contribution of a baseline.Accordingly, utilize NONLIN (Fig. 4 A and table 3) that these data fittings are become an ideal single component model.Apparent molecular weight (the M that estimates _App) approximate 24.4kDa, with very approaching from the molecular weight of 1 of corresponding amino acid and nucleotides sequence column count: 1NS1A (1-73)-dsRNA mixture.Low relatively RMS value and residue figure at random (illustration of Fig. 4 A) show and the good match of stoichiometry in 1: 1.As the OD of data with the substrate of clipping the solution post ₂₆₀Be 0.8 when editor, the quality of this match be further improved (table 3).Every mole of molecular-weight average 26,100 gram of estimating, 1: the formula molecular weight of 1NS1A (1-73)-dsRNA mixture about 3% within.This NS1A (1-73)-dsRNA mixture that shows this purifying has 1: 1 stoichiometry.In order to estimate dissociation constant Kd, according to 1: 1 stoichiometry, the data of 3 different load sample concentration and 3 speed fitted to the balance monomer-dimer NONLIN model (Fig. 4 B) of NONLIN then.Utilize this model, when making a decision with little RMS value and residual at random curve, data have obtained good match.In order to confirm that this model of fit is correct, independent data set also respectively or the different for example single load sample concentration of combination of combined utilization in the data of 3 different speed, or different load sample concentration but in the data of a speed, or the like come match.For each match, compared different models.In all examples, the monomer-dimer model is best.An exception is the data that obtain at 16,000 rpms, and it is for single-component system and the same well match of monomer-dimer model.Also may come editing data with different cutoff value in the bottom of chamber; This just caused final fitting result the 0.8-1.5 absorbance units to clip scope independently of one another.According to the specific match of carrying out, the Kd value of utilizing Eq.8 to calculate drops in the narrow relatively scope K _d=0.4-1.4 μ M.

Table 3

The apparent molecular weight of NS1A (1-73)-dsRNA mixture

	The NONLIN match
			C
t0 a	O.D. hold back b～1.0 RMS c M app d M app/M x e	O.D. hold back～0.8 RMS c M app d M app/M x e
			0.6 0.5 0.3 associating matches	0.0061 27.5 1.02 0.0043 23.3 0.86 0.0063 24.9 0.92 0.0056 24.4 0.91	0.0051 28.8 1.07 0.0040 26.0 0.96 0.0065 24.4 0.90 0.0054 25.2 0.94

^aThe concentration of the initial solution of the absorption measurement by 260 nanometers.

^bOD greater than cutoff value ₂₆₀Data are not included in the match.

^cRoot-mean-square value with the absorbance units match.

D apparent molecular weight, unit are every mole of kilograms, are (Fig. 4 A) by the ideal solution match of data and single component is estimated.Data or at the independent match of each load sample concentration or all 3 data sets of associating match.

E is according to the apparent molecular weight (M of sedimentation equilibrium data _App) with calculate by corresponding amino acid and nucleotide sequence 1: the ratio (M of the molecular weight of 1NS1A (1-73)-dsRNA mixture _x).

Embodiment 15 free NS1A's (1-73) ¹H, ¹⁵N and ¹³The resonance of C location: determine the proteic complete basically NMR resonance of free NS1A (1-73) location, this is that to analyze the mixture of it and dsRNA by NMR needed.In a word, altogether 65/71 (92%) specified ¹⁵N- ¹The HN site utilizes AUTOASSIGN, and (Zimmerman et al., (1997) J.Mol.Biol.269 592-610) locate automatically.This automatic analysis provides 71/78H by inner residue and/or sequence connectedness ^α, 68/73C ^α, 64/71C ' and 44/68C ^βThe resonance location.The result who has confirmed these AUTOASSIGN is analyzed in the craft of the three same resonance data of then carrying out, and has also finished remaining skeletal atom and 60/68C ^βResonance location.Located and removed Met ¹NH ², Pro ³¹N and C-terminal residue Ser ⁷³C ' and Pro before residue A la ³⁰All skeleton resonance in addition.Complete non--exchange the proton of all residues and the complete side chain location (not comprising aromatic carbon) of protonated carbon have been obtained then.About commutative side-chain radical, all ArgN have also been located ^εH, GlnN ^{ε 2}H, AspN ^{δ 2}H and TrpN ^{ε 1}H resonance, but in these spectrum, do not observe ArgN ^ηThe hydroxyl proton of H or Ser and Thr.Under the condition of 20 ℃ of pH6.0, these NS1A's (1-73) ¹H, ¹³C and ¹⁵The chemical shift data of N is at BioMagResBank (http://www.bmrb.wisc.edu; Accession number 4317) deposits.

When in Fig. 5, having shown 20 ℃ of pH6.0 ¹⁵The NS1A of N enrichment (1-73) ¹H- ¹⁵N HSQC spectrum.All framework amide peaks are (except Pro ³¹And Met ¹N-terminal) marks all, as ArgN ^εH, GlnN ^{ε 2}H, AspN ^{δ 2}H and TrpN ^{ε 1}The side chain resonance of H.In a word, spectrum has showed that rational good chemical shift distributes, even the 15N-1HN intersection peak of some degeneracys is arranged.For example, Arg ³⁷And Arg ³⁸Residue is to H ^N, N, C ', C ^α, H ^αAnd C ^βIdentical chemical shift is almost arranged.

Embodiment 16 utilizes the mapping of chemical shift turbulent antigenic determinant: a series of by collecting ¹H ^N- ¹⁵The dsRNA of N HSQC spectrum and 16bp has finished together ¹⁵The titration of the NS1A of N enrichment (1-73) detects. ¹H and ¹⁵The chemical shift of N nuclear is responsive to their partial electronic environment, therefore is used as the probe of labelled protein and unmarked RNA interphase interaction.Observe and directly contact with RNA or participate in the strongest disturbance being arranged in electronic environment in conjunction with the residue of conformational change main behind the RNA.

Record contains the sample of 0.1mM NS1A (1-73) dimer concentration, and the molar ratio that reduces successively of dimer protein and dsRNA is four HSQC spectrum of 2: 1,1: 1,1: 1.5 and 1: 2 o'clock.When this ratio induced protein precipitation when above that reaches 5: 1.In the HSQC spectrum of the sample of 2: 1 ratios, ¹HN- ¹⁵The non-constant width in N intersection peak is difficult to analyze, and shows that albumen might form the more mixture of macromolecule with dsRNA.Although introduce more dsRNA, be equal to or less than 1: 1 stoichiometric spectrum and show that one group of peak is only arranged to improve susceptibility.Because the macromolecule of NS1A (1-73)-dsRNA mixture, the skeleton location of the NS1A in the new mixture (1-73) fails to finish.Yet, by comparing HSQC spectrum (Fig. 5 B of free and dsRNA bonded NS1A (1-73), the data that produce in the above-mentioned titration experiments), do not influence framework amide chemical shift in spiral 3 and 3 ' although observe the formation of mixture, the residue in nearly all spiral 2 and 2 ' shows when mixture forms ¹⁵N and ¹H displacement disturbance.In addition, spiral 1 and 1 ' also shows the chemical shift disturbance when mixture forms.In conjunction with the time ¹⁵N- ¹Describe in the three-dimensional structure of the variation of H chemical shift by the free NS1A (1-73) among Fig. 6.When forming (representing with blue-greenish colour), mixture observes all significantly corresponding to the chemical shift disturbance of NS1A (1-73) skeletal atom, or in the spiral 2 and 2 ' that contains many arginine and Methionin, or (Fig. 7 B) in the close spiral that contacts 1 and 1 ' arranged with spiral 2 and 2 '.Yet skeleton NHs residue does not experience tangible chemical shift to be changed, and shows seldom or does not have structural modification (representing with pink colour), trends towards away from tangible conjugated antigen determinant.The zone that these results have determined dsRNA bonded antigenic determinant in antiparallel-spiral 2 and 2 ' or evaluation on every side, with the research of the rite-directed mutagenesis of front be consistent (Wanget al., (1999) RNA, 5:195-205).Further show since away from the chemical shift of the amide group of conjugated antigen determinant not by the disturbance of the formation of mixture institute, the structure of all NS1A (1-73) is not because of the combination havoc of dsRNA.

Embodiment 17 circular dichroism (CD) spectrum: circular dichroism for nucleic acid and proteinic secondary structure element and completely conformational characteristic useful probe is provided.For protein, the kind (Johnson, W.C., Jr. (1990) Proteins 7:205-214) of the main reflection in the zone bone framework image of CD spectrographic 180-240 nanometer.In case formation protein-nucleic acid mixture is observed the variation of CD spectrographic more than 250 nanometers mainly be because the variation (Gray of nucleic acid secondary structure, D.M. (1996) Circular Dichroism and the ConformationAnalysis of Biomolecules, Plenum Press, New York, 469-501).The CD spectrum of the two strands of four 16bp (RR, RD, DR and DD) and they double-stranded type (Fig. 7, red vestige) separately is distinct and is distinctive (Gray and Ratliff (1975) Biopolymers 14:487-498; Wells and Yang (1974) Biochemistry 13:1317-1321; Gray et al., (1978) Nucleic AcidsRes.5:3679-3695).The feature of RR two strands is in 295 nanometers slight negative direction band to be arranged, in 210 nanometers a strong negative band is arranged, a positive dirction band is arranged near 260 nanometers, be the distinctive feature of A-type dsRNA conformation (Fig. 7 A) (Huang et al., (1994) NucleicAcids Res.25:4098-4105).The DD two strands has roughly the same forward and reverse band more than 220 nanometers, the result of intersection is the forward band (Id., Gray et al., (1992) MethodsEnzymol.211:389-406) that a typical B-DNA (Fig. 7 D) arranged in 260 nanometers.Two crossbred RD and DR show the characteristics that differ from one another significantly, however the two all roughly (Fig. 7 B, 7C) ((Hung et al., (1994), Nucleic Acids Res.22:4326-4334) between A-type dsRNA and B-type dsDNA structure; Roberts and Crothers (1992) Science 258:1463-1466; Ratmeyer et al., (1994) Biochemistry 33:5298-5304; Lesnik and Freier (1995) Biochemistry 34:10807-10815); Clark et al., (1997) Nucleic Acids Res.25:4098-4105.In addition, the intensity at the forward band of 260 nanometers seems the heteroduplex that the A feature is arranged the most responsive (Clark et al., (1997) Nucleic Acids Res.25:4098-4105).The CD spectrum (orange vestige) that in Fig. 7, has shown NS1A (1-73) when RR, RD, DR or DD two strands at equimolar amount exists.

In the example of dsRNA (Fig. 7 A), NS1A (the 1-73)-dsRNA mixture of use gel-filtration purifying is used for avoiding the interference (seeing Fig. 2 and 3) of free dsRNA existence.The spectrum (blue trace) that has also shown the free NS1A (1-73) in each situation.NS1A (1-73) has mainly occupied CD spectrographic 200-240 nanosection (Qian et al., (1995) RNA 1:948-956), and the structural information of nucleic acid double chain mainly occupies the zone of 250-320 nanometer.Above-mentioned gel Displacement Analysis and gel-filtration data presentation have only dsRNA substrate and NS1A (1-73) to form mixture.Yet, the same with shown in Fig. 8 A, the mixture of formation (yellow vestige) fails to cause the significant CD spectrum change that the 250-320 nanosection is the most responsive to the nucleotide double conformation.These digital proofs RNA two strands is the main A-type conformation that keeps it in albumen-dsRNA mixture.In addition, the CD spectrum (yellow) of dsRNA-NS1A (1-73) and the simple free NS1A (1-73) of adding and free dsRNA use the spectrum (green) of computer calculates also closely similar in the 200-240 nanosection, show that NS1A (1-73) skeleton structure does not change because of the formation of mixture widely yet.Although NS1A (1-73) is not and other double-stranded combination, also each RD, DR and DD are mixed with the NS1A (1-73) of equimolar amount and the CD spectrum that obtains (Fig. 7 B, C, D) in contrast.The CD spectrum of detected mixture was not to be consistent with independent two strands and proteic spectrographic summation when these data acknowledgements changed when the structure of these molecules.

I has been determined in interaction between the dsRNA of the 16bp that is formed by the proteic N-terminal of the NS1 of influenza A virus and two synthetic oligonucleotide) NS1A (1-73) and dsRNA combination, and discord dsDNA or the combination of corresponding xenogenesis two strands; Ii) NS1A (1-73)-dsRNA mixture show 1: 1 stoichiometry and approximate 1 μ mole dissociation constant; Iii) the antiparallel spiral 2 and 2 ' that symmetry is relevant plays important effect in conjunction with target dsRNA; Iv) the skeleton structure of the structure of dsRNA and NS1A (1-73) is not changing in the binding molecule accordingly significantly than them in their mixtures form.Comprehensively, this information provides important biophysics evidence for the model of the hypothesis of setting up this new dsRNA binding motif and the mixture between double-stranded RNA.In addition, this information has determined that the mixture between NS1A (1-73) and 16bp dsRNA is a suitable reagent that will carry out three-dimensional structural analysis future, that is to say that it is 1: 1 mixture of a homogeneous.

Embodiment 18NS1A (1-73): the biophysics feature of DSRNA mixture: gel displacement polyacrylamide gel electrophoresis, gel permeation chromatography and CD spectropolarimetry show all that NS1A (1-73) is specific and combine with dsRNA that homotactic dsRAN and heteroduplex are not had detectable affinity.A large amount of spectral evidence in the document comprises that the definite dsDNA of nucleus magnetic resonance, X ray, CD and Raman spectral investigation is the folding B-type conformational characteristic of C2 '-internal sugar, it is the A-type structure of feature with C3 '-internal sugar that dsRNA adopts one, and the DNA/RNA crossbred shows middle conformation (Huang.et al., (1994) Nucleic Acids Res.22:4326-4334 between A-and B-type conformation; Lesnik and Freier (1995) Biochemistry 34:10807-10815; Dickerson et al., (1982) Science 216:75-85; Chou et al., (1989) Biochemistry28:2435-2443; Lane et al., (1991) Biochem.J.279:269-81; Arnottet al., (1968) Nature 220:561-564; Egli et al., (1993) Biochemistry 25:41-50; Gyi et al., (1996), Biochemistry35:12538-12548; Nishizaki et al., (1996) Biochemistry35:4016-4025; Salazar et al., (1996) Biochemistry35:8126-8135; Rice and Gao (1997) Biochemistry 36:399-411; Hashem etc., (1998) Biochemistry 37:61-72; Gray et al., (1995) MethodsEnzymol.246:19-34).

In addition, topologizing of the two strands of standard is different, and the A-type is wide with one, and shallow ditch is a feature, and the B-type is narrow with one, and dark major groove is a feature.Because NS1A (1-73) only clearly combines with dsRNA, and does not have sequence-specific, clearly this albumen is to distinguish these nucleic acid spirals (A-type conformation just) according to double-stranded conformational to a great extent.Yet, do not get rid of molecule and carry out recognition process yet according to the existence of 2 '-OH base of each chain of two strands.These results are NS1A albumen and NS1A (1-73) and another target RNA of total length, the specific stem in a spliceosome small nuclear rna s-prominent, and the combination of U6 snRNA provides explanation (Qianet al., (1994) J.Virol.68:2433-2441; Wang and Krug, (1996) Virology 223:41-50).The belemnoid of supposing this U6 snRNA has formed an A-type structure that resembles dsRNA in solution, allow NS1A (1-73) and U6 snRNA so that NS1A (1-73) and 16-bp dsRNA fragment are the similar fashion formation mixture of feature herein.

Aforesaid sedimentation equilibrium experiment confirm NS1A (1-73) dimer and dsRNA two strands were with 1: 1 mode combination, and dissociation constant Kd is about 1 μ M.What is interesting is that about 30% dsRNA is to be (Fig. 2 A) that does not form mixture in 1: 1 the molecular sieve experiment in dimer and double-stranded mol ratio, detects more free dsRNA (Fig. 1) in gel mobility shift assay.The part of finding unconjugated dsRNA is changed to another prepared product from a NS1A (1-73) prepared product, does not observe in the gel permeation chromatography of the composite sample of fresh purifying not in conjunction with dsRNA (Fig. 3 A).In addition, observe slowly to dissociate when being compounded in long-term storage (Fig. 3 B).Therefore, infer that NS1A (1-73) has shown irreversible slowly self-polymerization under the condition of using in these research.This hypothesis also obtains to support by the macromole that utilizes laser light scattering to observe in the sedimentation equilibrium experiment as detection method.In addition, in the gel permeation chromatography of some free NS1A (1-73) samples, observed a leading peak before NS1A (1-73) dimer wash-out, showing may be the peak of polymkeric substance.Yet, with the NS1A (1-73) of purifying-when the dsRNA mixture is gone up sample once more to gel-filtration column, do not observe excessive free dsRNA.Sample shows as close composite with the Kd value of μ M scope, is consistent with what estimate in the sedimentation equilibrium experiment.The mixture itself that forms to a certain extent, provides the purifying mechanism of NS1A (1-73) dimer-active dsRNA mixture of " inactive substance " isolating active that exists from sample.Therefore, no matter the character of pollutent, aggregation and/or underproof sample, these factors do not influence based on the sedimentation equilibrium of the NS1A that utilizes purifying (1-73)-dsRNA mixture tests stoichiometry and the estimation of dissociating constant.In addition, the mixture of gel-purified shows as closely, and the real example of the mixture of homogeneous shows that these mixtures should meet the structural analysis of X-radiocrystallgraphy or nucleus magnetic resonance.

Embodiment 19NS1A (1-73): DSRNA affinity and stoichiometric alternately estimation are compared: the NS1A (1-73) that utilizes the gel displacement to measure of front: the estimation of dsRNA affinity has reported that the scope of apparent dissociation constant (KD) value is 20-200nM (Qian et al., 1995; Wang et al., 1999).These researchs all are that the longer dsRNA substrate with different sequences carries out in measuring with the above-mentioned biophysics of ratio in a small amount.In the work in early days, the stoichiometry of observing NS1A (1-73): dsRNA combination (according to the gel shift value) depends on the length of dsRNA substrate, and in conjunction with being (Wang et al., 1999) that narrow and do.Reported similar total length NS 1A narrow do in conjunction with the result (Lu et al., (1995) Virology214,222-228).Mixture between NS1A that describes among the application (1-73) and 16-bp dsRNA duplex molecule be when the RNA-of a plurality of NS1A in conjunction with the territory along long dsRNA in conjunction with the time the interactional part completely that takes place model, think with esoteric the same.Observed in the present invention 1: 1 stoichiometry of applicant has been got rid of interaction and other the cooperation that may the occur influence between possible protein-protein in the multiple bonded pattern of a bigger system.In the NS1A protein binding during to bigger dsRNAs, have under the situation in many possible non-specific combination sites, by the configuration entropy effect adjust apparent affinity (Wang et al., (1999) RNA5,195-205).For example, Wang et al. (1999) has reported that NS1A (1-73) is higher 10 times than the affinity of the dsRNA substrate of similar 55-bp to the affinity of the dsRNA substrate of 140-bp.Because this several reasons, the segmental affinity costant of simple 1: 1 NS1A (1-73) dimer of reporting among the application and 16-bp dsRNA is lower than the apparent affinity of the bigger cooperation system of reporting before.Yet, the mixture model of describing in this work has only been caught the part of complete a plurality of entire infrastructure information in conjunction with cooperation system, the mixture of describing in this work is well characterized, be easy to produce, be more suitable for the interactional detailed structural research of protein-dsRNA of following NS1A-RNA molecular recognition process.

RNA binding site among the embodiment 20NS1A (1-73): L-Ala scanning mutation research (the Wang et al. that in NS1A, carries out recently, 1999) disclose i has been determined in the combination of bigger dsRNA fragment and U6snRNA) in conjunction with target, albumen must be dimer and ii) R only ³⁸Be in conjunction with the sin qua non of RNA institute, even K ⁴¹Also play a significant effect.The RNA conjugated antigen determinant support of the NS1A (1-73) that determines by the chemical shift disturbance of above-mentioned 15N-1H HSQC resonance and expanded these accidental datas.The chemical shift of all the framework amide base resonance in spiral 2 and 2 ' is in case combine and will change with dsRNA.The one or more basic side chain that is exposed to solvent of residue comprises Arg in this and the spiral 2 and 2 ' ³⁸And Lys ⁴¹The model that (Fig. 6 B) participates in the direct contact of dsRNA is consistent.Also may be exposed to the basic side chain Arg of solvent ³⁷And Arg ⁴⁴, also have the Arg of part embedding ³⁵And Arg ⁴⁶Side chain (participates in intramolecularly and intermolecular salt bridge (Chien et al., (1997), NatureStruct.Biol.4:891-895; Liu et al., (1997) NatureStruct.Biol.4:896-89917) also direct and dsRNA interaction.In addition, the chemical shift noisy data has been got rid of participation (the Chien et al. of the potential RNA binding site on spiral 3 and 3 ' of hypothesis, (1997)), because most of skeleton 1HN, the 15N atom of residue do not show any chemical shift on the 3rd spiral when mixture forms, show the conjugated antigen determinant away from spiral 3 and 3 ', and all cage constructions of NS1A (1-73) are not influenced by the RNA bonded.The difference of the chemical shift of some residues on the spiral 1 and 1 ' in protein core district is owing to the variation of RNA interaction inductive self environment.Generally speaking, these nuclear magnetic resonance datas show that the folded conformation of six coiled strands of NS1A (1-73) is complete when combining with dsRNA.The conclusion that NS1A (1-73) and dsRNA did not all demonstrate a large amount of skeleton structure changes when this conclusion and the mixture that obtains from CD research formed has good consistence.

The three-dimensional model of embodiment 21NS1A (1-73)-DSRNA mixture: the analysis of all data of NS1A (1-73)-dsRNA mixture has disclosed the new constitutional features of the nonspecific dsRNA combined function of encoding shown herein.The binding site of NS1A (1-73) is made up of the antiparallel spiral 2 and 2 ' that is rich in the arginine surface.The model of supposing and our cumulative NS1A (1-73) are consistent to the knowledge of the binding characteristic of dsRNA, and the mating surface of the structure of proteic symmetric form crosses the ditch (Fig. 8) of the A-type RNA of standard.In the model of this hypothesis, the side chain of antiparallel spiral 2 and 2 ' outside arginine and Methionin and the phosphoric acid ester skeleton of the big furrow rim of antiparallel formation interact with a kind of symmetric pattern, simultaneously the base formation hydrogen bond in the surface ion between spiral 2 and 2 ' pair and the ditch.Remarkable similar space (about 16.5 ) between NS1A (1-73) spiral 2 and 2 ' and cross distance (about 16.8 ) between the phosphoric acid ester of ditch and increased voucher for the model on the ditch that NS1A (1-73) ' is sitting in ' A-type RNA needs the A-type to carry out correct stop.In addition, the interaction of these albumen-RNA seldom or not needs specific sequence, and it is consistent (Hatada and Fukuda (1992) J.Gen.Virol.73,3325-3329 that this point also lacks distinctive particular sequence with the interaction of NS1A and dsRNA; Lu et al., (1995) Virology 214,222-228; Qian et al., (1995) RNA 1,948-956).

Embodiment 22 and other albumen: the comparison of DSRNA mixture: in the background that puts it into known RNA-protein-interacting, the NS1A of the supposition that the application advocates (1-73): the dsRNA model has constituted a model that new albumen-dsRNA mixture forms.The alpha-helix peptide of arginine enrichment, for example come from HIV-1 Rev albumen known by the interaction between specific major groove be attached to ds RNA (Battiste et al., (1996), Science273:1547-1551.).Yet the major groove in the A-type two strands of standard is too narrow too dark even can not hold a single alpha-helix.As a result, Rev-albumen-RNA mixture causes the distortion of serious nucleic acid Id structure in conjunction with the spiral of arginine enrichment.Therefore, the similar interaction between the major groove of NS1A (1-73) spiral 2/2 ' and its target dsRNA can be got rid of, and albumen and nucleic acid kept the conformation of their free state when former because mixture formed.Most of albumen in conjunction with dsRNA typically contain the ubiquitous ca.70 amino acid that surpasses a copy, α ₁-β ₁-β ₂-β ₃-α ₂Module is called as dsRNA in conjunction with territory (dsRBD) (Fierro-Monti﹠amp; Matthews, 2000).The X-ray crystal structure that comes from the mixture of the dsRBD of the conjugated protein A of Africa xenopus RNA-and dsRNA discloses two alpha-helixs of two interchain and adds that a ring forms the interaction (Ryter﹠amp of a face of two ditches of 16bpwindow-of leap of collective and intervenient major groove-two strands; Schultz, 1998).In fact the contact between all these albumen-RNA all comprises 2 ' hydroxylic moiety in the ditch and the non-bridging oxygen in the phosphodiester backbone.Also reported the similar viewpoint (Ramos et al., 2000) in the nucleus magnetic resonance structure of the mixture between proteic dsRBD of fruit bat staufen and dsRNA recently.For NS1A (1-73), the most of right and wrong of the interaction in two individual system between albumen dsRNA are sequence-specific, disturbance (Kharrat et al., 1995 of causing relatively little two strands and free protein structure; Bycroft et al., 1995; Nanduri et al., 1998).Yet different with this model is that the unspiralized region of dsRDB has formed crucial contacting with Nucleotide.Vital except comprising to Nucleotide identification, as if do not have to occur and form beyond the non-helical conformation that does not also form behind the mixture in NS1A (1-73), these dsRBM modules lack the very possible symmetrical feature that uses of NS1A (1-73) in the molecular recognition process.

Industrial usability

The present invention has applied for the application aspect the growth of control influenza virus, influenza virus chemistry and antiviral therapy.

Though, be appreciated that these examples are the explanation to principle of the present invention and application in this article by describing the present invention with reference to specific example.Therefore, be appreciated that can carry out many modifications explains the situation, and can design other arrangement and do not leave by the spirit and scope of the present invention that additional claim limits.

All publications of quoting in the specification sheets have all shown the state of the art of the those skilled in the art under the present invention.All these publications are used as a reference by this paper, and its degree just is cited as a reference by specific independent indicating as each independent publication.

Table 1: influenza A virus NS1 protein sequence

NS1A-I：1

NS1A-I：2

NS1A-I：3

NS1A-I：4

NS1A-I：5

NS1A-II：1

NS1A-II：2

NS1A-II：3

NS1A-II：4

NS1A-II：5

Table 2: influenza B virus N S2 protein sequence

NS1B I-all

NS1B II-1

NS1B II-2

Claims (42)

1. composition that comprises reaction mixture, this mixture contain influenza virus NS1 albumen or its dsRNA binding fragment and in conjunction with the mixture of described proteic dsRNA.

2. the described composition of claim 1, wherein NS1 albumen is the NS1 albumen (NS1A) of influenza A.

3. the described composition of claim 2, it contains the proteic dsRNA of described NS1A in conjunction with the territory.

4. the described composition of claim 3, wherein said dsRNA binding fragment comprises the 1-73 amino-acid residue of NS1A.

5. the described composition of claim 1, the NS1 albumen (NS1B) that wherein said NS1 albumen is influenza B.

6. the described composition of claim 5, it comprises the proteic dsRNA of described NS1B in conjunction with the territory.

7. the described composition of claim 6, wherein said dsRNA binding fragment comprises the 1-93 amino-acid residue of NS1B.

8. the described composition of claim 1, the length of wherein said dsRNA is about 16 base pairs.

9. the described composition of claim 1, wherein said dsRNA bound fraction comprises the 1-73 amino-acid residue of NS1A, and the length of wherein said dsRNA is about 16 base pairs.

10. the described composition of claim 1, wherein said dsRNA bound fraction comprises the 1-93 amino-acid residue of NS1B, and the length of wherein said dsRNA is about 16 base pairs.

11. the described composition of claim 1, it further comprises a kind of detected resisiting influenza virus and suppresses active compound.

12. the described composition of claim 1, wherein NS1 albumen or dsRNA can be detected ground mark.

13. an evaluation has the method that resisiting influenza virus suppresses active compound, it comprises:

A) preparation comprises influenza virus NS1 albumen or its dsRNA in conjunction with the territory, plant in conjunction with described proteic dsRNA or its in conjunction with the territory, and the reaction system of candidate compound; With

B) combination degree between detection NS1 albumen and dsRNA, wherein when compound existed, with respect to contrast, the combination reduction between NS1 albumen and dsRNA showed that compound has the activity of inhibition to influenza virus.

14. the described method of claim 13, wherein NS1 albumen or its dsRNA are fixed on the solid support in conjunction with the territory.

15. the described method of claim 13, wherein candidate compound prior to or join in the reaction system simultaneously with NS1 albumen and dsRNA.

16. the described method of claim 13, wherein candidate compound joins in the reaction system after adding NS1 albumen and dsRNA.

17. the described method of claim 13, it further is included in before the described detection, with detectable marker mark dsRNA, NS1 albumen or its dsRNA in conjunction with the territory.

18. comprising with NS1 albumen or its dsRNA, the described method of claim 17, wherein detectable mark combine territory bonded antibody or its fragment.

19. the described method of claim 17, wherein detectable mark comprises a kind of enzyme, and further comprises the substrate of this enzyme in the reaction system.

20. the described method of claim 17, wherein detectable mark comprises radio isotope.

21. the described method of claim 17, wherein detectable mark comprises fluorescent mark.

22. the described method of claim 13, wherein said detection is undertaken by FRET (fluorescence resonance energy transfer).

23. the described method of claim 13, wherein said detection is undertaken by the fluorescence polarization anisotropy measurement.

24. the described method of claim 13, wherein NS1 albumen or its dsRNA binding fragment are to exist with the form with the glutathione-S-transferase fusion rotein in reaction system.

25. the described method of claim 13, wherein said NS1 albumen is NS1A albumen.

26. the described method of claim 13, wherein said NS1 albumen is NS1B albumen.

27. the described method of claim 13, wherein reaction system comprises the proteic fragment of NS1, and this fragment comprises the proteic dsRNA of described NS1 in conjunction with the territory.

28. the described method of claim 27, wherein dsRNA comprises NS1A (1-73) in conjunction with the territory.

29. the described method of claim 27, wherein dsRNA comprises NS1B (1-93) in conjunction with the territory.

30. the described method of claim 13, wherein the length of dsRNA is about 16 base pairs.

31. the described method of claim 13, wherein authentication method comprises high-throughput screening method.

32. an evaluation has the method that suppresses active compound to influenza virus, it comprises:

A) preparation comprises influenza virus NS1 albumen or its dsRNA in conjunction with the territory, in conjunction with described proteic dsRNA or its reaction system in conjunction with territory and candidate compound; With

B) combination degree between detection NS1 albumen and dsRNA, wherein when compound existed, with respect to contrast, the combination reduction between NS1 albumen and dsRNA showed that compound has the activity of inhibition to influenza virus; With

C) determine b) in identify have and suppress the degree of active compound in the growth of vitro inhibition influenza virus.

33. the described method of claim 32 identifies that wherein having the method that suppresses active compound is selected from: a) chemical shift of NMR disturbance, b) gel permeation chromatography, or (c) utilize the sedimentation equilibrium of the ultracentrifuge of analysis mode to measure.

34. the described method of claim 32 further comprises d) determine c) in the compound identified in the growth of vitro inhibition influenza virus in inhuman animal, suppress the degree that influenza virus is duplicated.

35. one kind prepares and is used for the method for compositions that body is interior or the vitro inhibition influenza virus is duplicated, it comprises:

A) preparation comprise influenza virus NS1 albumen or its dsRNA in conjunction with the territory, in conjunction with described proteic dsRNA or its in conjunction with the territory, and the reaction system of candidate compound;

B) combination degree between detection NS1 albumen and dsRNA, wherein when compound existed, with respect to contrast, the combination reduction between NS1 albumen and dsRNA showed that compound has the activity of inhibition to influenza virus;

C) determine b) the middle degree of identifying with the active compound vitro inhibition influenza virus growth of inhibition;

D) determine c) in the compound identified in the growth of vitro inhibition influenza virus in inhuman animal, suppress the degree that influenza virus is duplicated; With

E) by with d) in identify in inhuman animal, suppress compound that influenza virus duplicates with effective inhibition dosage and carrier preparation with the preparation composition.

36. the described method of claim 35, it further comprises f) according to c) and d) in the result that obtains determine effective inhibition dosage of compound.

37. the described method of claim 35, wherein carrier is fit to use to animal by suction or insufflation.

38. an evaluation is as the method for the compound of the inhibitor of influenza virus, it comprises:

A) coordinator of acquisition influenza virus NS1 protein three-dimensional structure;

B) carry out rational medicinal design according to the coordinator with the described three-dimensional structure that obtains in the step a) and select the potential compound, wherein said selection is to unite with the computer simulation of NS1-dsRNA mixture to carry out;

C) the potential compound is contacted with influenza virus; With

D) activity of mensuration influenza virus, wherein when compound existed, the influenza virus specific activity did not reduce when having compound, just identified that the potential compound is the compound that suppresses influenza virus.

39. the described method of claim 38, wherein NS1 albumen is that NS1A albumen or its dsRNA are in conjunction with the territory.

40. the described method of claim 39, wherein dsRNA is NS1A (1-73) in conjunction with the territory.

41. the described method of claim 38, wherein NS1 albumen is that NS1B albumen or its dsRNA are in conjunction with the territory.

42. the described method of claim 41, wherein dsRNA is NS1B (1-93) in conjunction with the territory.

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