CN102675429A - Virus capping system polypeptide inhibitor for controlling coronavirus - Google Patents
Virus capping system polypeptide inhibitor for controlling coronavirus Download PDFInfo
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Abstract
The invention relates to a virus capping system polypeptide inhibitor for controlling coronavirus and a method for inhibiting the activity of the coronavirus capping system by utilizing the polypeptide inhibitor. The polypeptide inhibitor comprises the polypeptide with the following amino acid sequence: 1) an amino acid sequence shown in SEQ ID No.1, 2) a sequence which has equivalent functions, is derived from the sequence shown in SEQ ID No.1 and is obtained by replacing, adding and/or deleting one or more amino groups in the amino acid sequence shown in SEQ ID No.1, or 3) a truncated sequence of the sequence shown in SEQ ID No.1, wherein the truncated sequence at least comprises GGASCCLYCRCH. The inhibitor has extremely high specificity, overcomes the defect that the traditional virus capping system inhibitors have low specificity and simultaneously overcomes the defect that the present nonspecific treatment drugs have high side effects and are easy to cause sequelae as a guide drug for controlling coronavirus.
Description
Technical field
The invention belongs to molecular biology, virusology, biological chemistry, albumen (enzyme) structure function research and biological medicine technology field.Particularly relating to a kind of can be used for suppresses adding the cap rhetorical function and duplicating the peptide inhibitor of transcribing of coronavirus such as SARS-CoV.
Background technology
Coronavirus (Coronavirus) is one type of infected person, poultry, domestic animal, and then causes the pathogenic RNA virus of respiratory tract, alimentary canal inflammation, diarrhoea.Wherein infect SARS (the Severe Acute Respiratory Syndromes that causes by sars coronavirus (SARS-CoV); Be called for short SARS); Be a kind of new respiratory infectious disease, with heating, dry cough, uncomfortable in chest be cardinal symptom, it is depleted that the respiratory system of rapid progress appears in severe patient; Infectivity is extremely strong, disease progression is quick (Ziebuhr, 2003).2003, during the outburst of this virus world wide, lethality rate reached 10%, especially in the grownup especially up to 50%, caused serious social fear and financial loss.Still do not have effective vaccine and specificity therapeutic method at present, and the extensive existence of coronavirus and high recombination fraction be prone to the New Development wild-type virus takes place, thereby be one of main health threat of facing of the whole world.
, mainly be divided three classes at present to the treat-ment of sars coronavirus: 1. immune-regulating factor, like Interferon, rabbit, ribavirin etc.; 2. stop the poisoning intrusion cellular elements, like the proteic monoclonal antibody of S, to the polypeptide of the crucial integration region of S albumen etc.; 3. suppress the virus replication molecule, like the varial polymerases specific inhibitor, broad spectrum proteinase inhibitor etc.In these methods, immune-regulating factor and broad spectrum proteinase inhibitor all are non-specificity therapeutic methods, and spinoff is bigger, and the patient of healing stays sequela easily, and result of treatment exists bigger individual difference (Groneberg et al., 2005; Tong, 2009).
The terminal cap sequence of viral RNA 5 ' has stable virus RNA, guide RNA location, initial viral protein translation, the critical functions such as immunity identification of escaping, be that virus replication is transcribed necessary (Banerjee, 1980; Daffis et al., 2010; Furuichi and Shatkin, 2000; Lewis and Izaurralde, 1997; Muthukrishnan et al., 1975; Schwer et al., 1998; Shatkin, 1976; Shimotohno et al., 1977; Zust et al., 2011).Therefore, how effectively to suppress virus and add the cap system, become a frontier (Dong et al., 2008 of exploitation antiviral; Shuman, 2001; Shuman, 2002), there has been the inhibition virus of some wide spectrums to add the medicine of cap system at present, like (Bouvet et al., 2010) such as AdoHcy, Sinfungin, ATA.But these nonspecific inhibition medicines impact host's the cap system that adds itself easily, and bigger spinoff is arranged, and also fail to get into actual operational phase.
The fundamental research of sars coronavirus has obtained certain progress at present; The mechanism of duplicating and transcribing there has been certain understanding; And each proteic 26S Proteasome Structure and Function also there are some deep researchs, wherein the study on the synthesis of coronavirus rna gene group 5 ' end cap minor structure have been obtained the progress of mutagenicity.Discover recently; Sars coronavirus Nonstructural Protein nsp16 is after interacting with another Nonstructural Protein of virus nsp10; Has 2 '-O-methyltransgerase (2 '-O-MTase) activity; Participate in sars coronavirus RNA 5 ' terminal Cap-1 type cap synthetic (Bouvet et al., 2010; Lugari et al., 2010; Pan et al., 2008), and the independent Nonstructural Protein nsp16 of sars coronavirus almost detects less than activity, has only FCV (FCoV) Nonstructural Protein nsp16 can detect 2 ' extremely low-O-MTase active (Decroly et al., 2008).Coronavirus Nonstructural Protein nsp10 is as the stimulating factor of nsp16, and itself does not have 2 '-O-MTase function, but nsp10 can bind nucleic acid and participate in duplicating and transcribing of RNA, brings into play important regulatory function (Donaldson et al., 2007a; Donaldson et al., 2007b; Matthes et al., 2006), and the nsp10 structure has obtained resolving (Joseph et al., 2006; Su et al., 2006).Owing to after two Nonstructural Protein nsp16 of SARS-CoV interact with nsp10, just have 2 '-O-MTase activity, so be a good potential drug target spot.Yet up to the present, also less than relevant report about this action target spot.
Summary of the invention
The objective of the invention is to be to provide a kind of virus that can be used for the coronavirus control efficiently to add cap system peptide inhibitor.
To achieve these goals; The present invention is a design basis with sars coronavirus Nonstructural Protein nsp10 and nsp16 compound crystal structure (Fig. 1); Combine sars coronavirus Nonstructural Protein nsp16 with synthetic polypeptide competition; Prevention nsp10 combines with nsp16's, and (2 '-O-MTase) activity stops sars coronavirus RNA to be processed to Cap-1 type cap thereby suppress sars coronavirus nsp16 methyltransgerase.The result shows; In institute's design of series polypeptide; Sequence (K29) shown in the SEQ ID No.1 and truncated sequence SEQ ID No.2 (K12) thereof have the nsp10 of prevention and the effect of nsp16 bonded; And (2 '-O-MTase) active effect finally suppresses duplicating of coronavirus to present inhibition sars coronavirus nsp16 methyltransgerase.Those skilled in the art can design modifier or analogue based on SEQ ID No.1 according to aforesaid method, in order to stop nsp10 and the effect of nsp16 bonded.For example, can the last amino acids of sequence shown in the SEQ ID No.1 be lacked, or the 26th Lys is replaced with Arg.
In view of the above, the present invention at first provides a peptide species, and its aminoacid sequence is: the 1) aminoacid sequence shown in the SEQ ID No.1; Or, 2) aminoacid sequence shown in the SEQ ID No.1 through replacement, add and/or lack that one or several amino obtains have same function by sequence deutero-sequence shown in the SEQ ID No.1; Or 3) truncated sequence of sequence shown in the SEQ ID No.1, this truncated sequence comprises GGASCCLYCRCH at least.Preferred said truncated sequence is GGASCCLYCRCH.
The present invention also provides a kind of peptide inhibitor that is used to suppress coronavirus, and it comprises above-mentioned polypeptide.
The present invention also provides a kind of coronavirus antiviral, and it comprises above-mentioned polypeptide.
Aforementioned polypeptides can perhaps obtain through engineered means vivoexpression through artificial directly synthetic.Therefore the present invention also provides the gene of coding aforementioned polypeptides, contains said expression carrier, and the host cell that contains said expression vector.Described polypeptide, gene, expression vector or host cell all can directly or indirectly prepare the anti-coronavirus medicine.Therefore, the present invention also comprises their application in the preparation anti-coronavirus.
The present invention also provides a kind of and suppresses coronavirus and add cap and modify or duplicate the method for transcribing, and comprises using described polypeptide to suppress combining of nsp10 and nsp16.
The present invention provides peptide inhibitor; The interaction that suppresses between two Nonstructural Proteins of virus through polypeptide suppresses the activity that coronavirus adds the cap system; And host itself is not exerted an influence and spinoff, make this polypeptide suppress to have very high specificity and application prospect.
Description of drawings
Fig. 1 shows the crystalline structure of nsp10 and nsp16 mixture cocrystallization.(A) nsp16-nsp10 complex body crystalline structure.Wherein nsp10 is green (zone shown in the arrow), and nsp16 is light blue (shown in the arrow with exterior domain), and both all show with the ribbon pattern.(B) nsp10 shows that with the ribbon pattern nsp16 shows with the surface charge pattern.(C) nsp10 and nsp16 all show with the surface charge pattern.1-positive surface charge district, 2-surface negative charge district.
Fig. 2 shows that results of interaction takes place for nsp10 total length and different truncated mutant and nsp16.The Nsp10 total length has 139 amino acid."+" expression has interaction, and "-" expression does not interact.
Fig. 3 shows nsp10 total length and the different truncated mutant activation effect to nsp16.(A) assay of the total length nsp14 of purifying and nsp16.(B) the total length nsp10 of purifying and truncated mutant assay thereof.(C) total length nsp10 and truncated mutant thereof activate the detected result of nsp16 effect.(D) total length nsp10 and truncated mutant thereof influence the binding ability of nsp16 and substrate SAM.
Fig. 4 shows the polypeptide principle of design.(A) effect of nsp10 albumen each several part, red (arrow 1) is the activation structure territory, and green (arrow 2) is the binding domains that combines with nsp16, and pink (arrow 3) is the activation structure territory, and blue (arrow 4) is exposed to the part of protein surface for binding domains.(B) nsp10 crystalline structure exterior view, its Smalt (arrow 5) is for being exposed to the binding domains of nsp10 protein surface.(C) position view of five polypeptide difference mimic nsp10.
Fig. 5 shows that synthetic peptide inhibitor K29 relies on effect to the metering that is combined with that suppresses sars coronavirus Nonstructural Protein nsp16 and substrate SAM.Final concentration can effectively suppress combining of nsp16 and substrate SAM greater than the K29 of 100 μ M.But control group K8 does not just have this to suppress effect.
Fig. 6 shows the inhibition effect of synthetic five peptide species suppressor factor to sars coronavirus nsp16 methyltransgerase (being the capping enzyme system).
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment only to be used to the present invention is described and be not used in the scope of restriction requirement of the present invention protection.
The design and the checking of embodiment 1 peptide inhibitor
1, sars coronavirus Nonstructural Protein nsp10 and the structural analysis of nsp16 compound crystal and inhibition principle
Shown in Figure 1 is the crystalline structure figure of nsp10 and nsp16 mixture cocrystallization.Among Figure 1A, that green is nsp10, and nattier blue is nsp16, can find out, both interact through protein surface, form a complex body.Research according to before us shows, nsp10 promoted combining of nsp16 and substrate (viral RNA) and with the combining of substrate SAM, thereby 2 '-O-MTase methyl transferase activity of activated nsp16.What Figure 1B showed is the surface charge distribution plan of nsp16, and wherein blue region is positively charged surface (routine zone as shown in arrow 1), and red area is electronegative surface (routine zone as shown in arrow 2).Because viral RNA is electronegative, therefore can be incorporated into the positively charged RNA in nsp16 surface and combine (zone shown in the arrow 1 of Figure 1B) in the ditch.Nsp10 is with after nsp16 combines, and expanded the area (zone shown in the arrow 1 of Fig. 1 C) that viral RNA combines ditch, and what make viral RNA and nsp16-nsp10 complex body combines closelyr, causes the active enhancing of nsp16.According to the above principle; If know the specifying information of nsp10 and nsp16 bonding surface; Can design special polypeptide competition and be attached to the surface of nsp16, thereby stop the combination of nsp10, reach the purpose of the 2 '-O-MTase methyl transferase activity that suppresses nsp16.
2, yeast two-hybrid is identified nsp10 and the interactional Minimum Area of nsp16
The complete nsp16 of construction expression on the yeast two-hybrid system pGBKT7 of clontech company carrier, and called after BD16, the nsp10 that construction expression is complete on the pGADT7 carrier and the nsp10 of various truncated mutants, and called after AD10; AD Δ N41, AD Δ N64, AD Δ N90; AD Δ C19, AD Δ C32, AD Δ C53; AD Δ N55& Δ C32, AD Δ N64& Δ C32, concrete design of primers is seen table 1.Method according to the clontech yeast two-hybrid system; In the AH109 yeast strain; Carry out interaction zone checking, the result is as shown in Figure 2, with the minimum interaction zone of nsp16 be the 65-107 amino acids AD10 Δ N64& Δ C32 (aa65-107) of nsp10.
3, checking nsp10 different zones is to the activation capability of nsp16
In order to verify whether minimum interaction zone nsp10 Δ N64& Δ C32 (aa 65-107) has the ability that activates nsp16, the different lengths truncated mutant clone with sars coronavirus Nonstructural Protein nsp14, nsp16, nsp10 and nsp10 is building up on the protein expression vector pET30a respectively, respectively called after nsp14; Nsp16, nsp10, nsp10 Δ N41; Nsp10 Δ N64; Nsp10 Δ C32, nsp10 Δ C19, and expression and purification goes out albumen (Fig. 3 A and 3B).Concrete design of primers is seen table 1.Through a series of its protein functions of biochemical test test; Though find that at last the various truncated mutants of nsp10 have all weakened the binding ability (Fig. 3 D) of nsp16 and substrate SAM, nsp10 Δ N64 and nsp10 Δ C32 can not activate methyltransgerase (2 '-O-MTase) active (Fig. 3 C) of nsp16.Prove that minimum interaction zone nsp10 Δ N64& Δ C32 (aa 65-107) can't activate nsp16, need bigger regional nsp10 Δ N41& Δ C19 (aa 42-120) to accomplish and combine and the active effect of activation nsp16.
Table 1 recombinant clone design of primers
4, the principle of design of polypeptide inhibition is with synthetic
Because minimum interaction zone nsp10 Δ N64& Δ C32 (aa 65-107) can combine nsp16; But can not activate the activity of nsp16; Therefore designing the polypeptide simulation should zone (the aa 65-107 of nsp10) specific combination nsp16; Cause nsp16 to combine, suppress the active purpose of nsp16 thereby play with the nsp10 of normal function.Zone green among Fig. 4 A is the minimum interaction zone nsp10 Δ N64& Δ C32 (aa 65-107) of nsp10 and nsp16, and the zone that wherein is distributed in the nsp10 protein surface is aa 68-96 (blue portion is represented, like Fig. 4 B).Be peptide inhibitor mimic best region here, so difference simulation polypeptide K12 and K29 (Fig. 4 C).At the non-surf zone (green area outside Fig. 4 A Smalt part coverage, promptly this zone is buried in active site of protein) of minimum interaction zone, difference simulation polypeptide K10 and K8 (Fig. 4 C).Simultaneously at active region (the aa 42-64 of nsp10) (red area among Fig. 4 A), the polypeptide K20 (Fig. 4 C) that simulation should the zone is to test its influence to active region.Article five, the design information of polypeptide is following:
The design information of five polypeptide of table 2
| The polypeptide title | Aminoacid sequence | The zone of simulation nsp10 |
| K8 | PTTCANDP | 100-107 |
| K10 | DLKGKYVQIP | 91-100 |
| K12 | GGASCCLYCRCH | 69-80 |
| K20 | NCVKMLCTHTGTGQAITVTP | 40-59 |
| K29 | FGGASCCLYCRCHIDHPNPKGFCDLKGKY | 68-96 |
Article five, polypeptide all obtains through the synthetic mode.Experiment through early stage is found, in the synthetic process, introduces the amination of N end, and after the C end acetylation modification, the solvability of polypeptide increases.Therefore five synthetic polypeptide all pass through N end amination and C end acetylation modification synthesizes.Prove that through various system testings polypeptide K12 and K29 have the obvious suppression effect, other control group polypeptide then do not have effect (embodiment 2 ~ 4), meet principle of design and requirement.
Embodiment 2: peptide inhibitor K29 suppresses sars coronavirus Nonstructural Protein nsp16 and combines with substrate SAM
The reagent preparation:
10 * reaction buffer: 400mM Tris-HCl (pH 7.5), 20mM MgCl
2, 20mMDTT.
Isotropic substance
3H substrate: S-adenosyl [methyl-3H] methionine (67.3Ci/mmol, 0.5 μ Ci/ μ l).
Show toughener: Enlightening buffer (PerkinElmer).
Implementation step:
1. [40mM Tris-HCl (pH 7.5), 2mM MgCl in the reaction system of 25 μ l
2, 2mM DTT] and good sars coronavirus Nonstructural Protein nsp16 (0.5 μ M) and the nsp10 (4 μ M) (Chen et al., 2011) of adding purifying.
2. in reaction, add the peptide inhibitor K29 (0 μ M, 10 μ M, 100 μ M, 400 μ M) or the control group polypeptide K8 (0 μ M, 10 μ M, 100 μ M, 500 μ M) of different concns, mix respectively.
3. in each reaction system, add 2 μ Ci S-adenosyl [methyl-3H] methionine (67.3Ci/mmol, 0.5 μ Ci/ μ l), mix respectively.
4. reaction system is positioned on ice, carried out UV-crosslinked in 30 minutes with the 254nm UV-irradiation.
5. the protein substrate mixture in the reaction system is through the SDS-PAGE electrophoresis detection.
6. steeped the SDS-PAGE film 30 minutes with showing that toughener Enlightening buffer (PerkinElmer) invades, and make public a week with the X-mating plate at-70 degree.
7. towards X-ray sheet developing, show and suppress effect.
Result of implementation: as showing among Fig. 5, peptide inhibitor K29 can effectively suppress combining of sars coronavirus Nonstructural Protein nsp16 and substrate SAM, and becomes metering to rely on effect.Then unrestraint effect of control group polypeptide K8.
Embodiment 3: peptide inhibitor K29 and K12 suppress the methyl transferase activity (viral RNA adds the cap system activity) of sars coronavirus Nonstructural Protein nsp16
The reagent preparation:
10 * reaction buffer: 400mM Tris-HCl (pH 7.5), 20mM MgCl
2, 20mMDTT, 400units RNase inhibitor, 0.1mM SAM.
Isotropic substance
3H substrate: S-adenosyl [methyl-3H] methionine (67.3Ci/mmol, 0.5 μ Ci/ μ l)
Viral RNA substrate: m7GpppA-RNA (deriving from sars coronavirus RNA)
RNA purifying filler: the DEAE-Sephadex G250 that activation is good
Implementation step:
1. [40mM Tris-HCl (pH 7.5), 2mM MgCl in the reaction system of 30 μ l
2, 2mM DTT, 40units RNase inhibitor, 0.01mM SAM] and good sars coronavirus Nonstructural Protein nsp16 (3.3 μ M) and the nsp10 (14 μ M) of adding purifying.
2. the peptide inhibitor K29 or the K12 that in reaction, add different concns (0-320 μ M) respectively, and other control group polypeptide K8, K10, K20 mixes respectively.
3. in each reaction system, add 2 μ Ci S-adenosyl [methyl-3H] methionine (67.3Ci/mmol, 0.5 μ Ci/ μ l), mix respectively.
4. in each reaction system, add 3 μ g m7GpppA-RNA (sars coronavirus RNA) substrates.
5.37 degree was hatched 1.5 hours.
6. make purification column, the viral RNA behind the purification reaction with RNA purifying filler (the DEAE-Sephadex G250 that activation is good).
7. use liquid scintillation counter, detect
3The isotopic content of H counts out the inhibition effect.
Result of implementation: as showing among Fig. 6, peptide inhibitor K29 and K12 all can effectively suppress the methyl transferase activity (viral RNA adds the cap system activity) of sars coronavirus Nonstructural Protein nsp16, and become metering to rely on effect.Control group polypeptide K8, K10, then unrestraint effect of K20.
Embodiment 4: peptide inhibitor K29 and K12 suppress coronavirus MHV duplicating in the 17Cl-1 cell
MHV (MHV) belongs to the coronaviridae member with sars coronavirus, and it adds the cap system and has identical attribute.Therefore peptide inhibitor also can be used for the control of other coronavirus.With MHV is example.Synthetic new peptide inhibitor K29, K12, and before peptide inhibitor, introduce leading peptide and modify, get into intracellular efficient so that improve peptide inhibitor.
Implementation step:
1. press the normal cell cultural method and cultivate 17Cl-1 cell to 80% saturated level.
2. with MHV virus infection 17Cl-1 cell 2 hours.
3. remove virus infection liquid, add the normal cell nutrient solution, and the adding final concentration is peptide inhibitor K29 and the K12 of 200 μ M in cell culture fluid.
Result of implementation:, add the cell of peptide inhibitor K29 and K12, under the MHV virus infection through paired observation; Observed 72 hours continuously; Cytopathic effect (CPE) do not occur, and cytopathic effect has appearred in control group, viral number continues to increase; Explain that using K29 and K12 polypeptide can suppress coronavirus duplicates, and is well protected cell.
Embodiment 5: the vivoexpression of peptide inhibitor K29
Respectively peptide inhibitor K29 encoding sox is cloned on protein expression vector pET30 (a) and the pGEX-6P-1.The K29 gene order is shown in SEQ ID No.38, and directly synthetic by Synesis Company through bridging PCR, the polypeptide K29 of reorganization has guiding peptide and 6 * His label.Transform BL21 (DE3), ordinary method is cultivated and abduction delivering, obtains recombinant polypeptide suppressor factor K29 through protein expression and purification system (Ni-NTA Superflow Cartridges) by specification method purifying.The SDS-PAGE electrophoresis detection is consistent with expection.Adopt the method for embodiment 4 to carry out the inhibition of the duplicating experiment of sars coronavirus in the 17Cl-1 cell, the result shows it and directly synthetic polypeptide result is consistent.
Pertinent literature:
Banerjee,A.K.(1980).5′-terminal cap structure in eucaryotic messenger ribonucleic acids.Microbiol Rev 44,175-205.
Bouvet,M.,Debarnot,C.,Imbert,I.,Selisko,B.,Snijder,E.J.,Canard,B.,and Decroly,E.(2010).In vitro reconstitution of SARS-coronavirus mRNA cap methylation.PLoS Pathog 6,e1000863.
Chen,Y.,Su,C.,Ke,M.,Jin,X.,Xu,L.,Zhang,Z.,Wu,A.,Sun,Y.,Yang,Z.,Tien,P.,Ahola,T.,Liang,Y.,Liu,X.and Guo,D.(2011)Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp 16/nsp 10 Protein Complex.PLoS Pathog7(10),e1002294.
Daffis,S.,Szretter，K.J.,Schriewer,J.,Li,J.,Youn,S.,Errett,J.,Lin,T.Y.,Schneller,S.,Zust,R.,Dong,H.,et al.(2010).2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members.Nature 468,452-456.
Decroly，E.,Imbert,I.,Coutard,B.,Bouvet,M.,Selisko,B.,Alvarez,K.,Gorbalenya,A.E.,Snijder,E.J.,and Canard,B.(2008).Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing(nucleoside-2′O)-methyltransferase activity.J Virol 82,8071-8084.
Donaldson,E.F.,Graham,R.L.,Sims,A.C.,Denison,M.R.,and Baric,R.S.(2007a).Analysis of murine hepatitis virus strain A59 temperature-sensitive mutant TS-LA6 suggests that nsp 10 plays a critical role in polyprotein processing.J Virol 81,7086-7098.
Donaldson,E.F.,Sims,A.C.,Graham,R.L.,Denison,M.R.,and Baric,R.S.(2007b).Murine hepatitis virus replicase protein nsp 10 is a critical regulator of viral RNA synthesis.J Virol 81,6356-6368.
Dong,H.,Zhang,B.,and Shi,P.Y.(2008).Flavivirus methyltransferase:a novel antiviral target.Antiviral Res 80,1-10.
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Lewis,J.D.,and Izaurralde,E.(1997).The role of the cap structure in RNA processing and nuclear export.Eur J Biochem 247,461-469.
Lugari,A.,Betzi,S.,Decroly,E.,Bonnaud,E.,Hermant,A.,Guillemot,J.C.,Debarnot,C.,Borg,J.P.,Bouvet,M.,Canard,B.,et al.(2010).Molecular mapping of the RNA Cap2′-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.J Biol Chem 285,33230-33241.
Matthes,N.,Mesters,J.R.,Coutard,B.,Canard,B.,Snijder,E.J.,Moll,R.,and Hilgenfeld,R.(2006).The non-structural protein Nsp 10 of mouse hepatitis virus binds zinc ions and nucleic acids.FEBS Lett 580,4143-4149.
Muthukrishnan,S.,Both,G.W.,Furuichi,Y.,and Shatkin,A.J.(1975).5′-Terminal7-methylguanosine in eukaryotic mRNA is required for translation.Nature 255,33-37.Pan,J.,Peng,X.,Gao,Y.,Li,Z.,Lu,X.,Chen,Y.,Ishaq,M.,Liu,D.,Dediego,M.L.,Enjuanes,L.,and Guo,D.(2008).Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.PLoS One 3,e3299.
Schwer,B.,Mao,X.,and Shuman,S.(1998).Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme.Nucleic Acids Res 26,2050-2057.
Shatkin,A.J.(1976).Capping ofeucaryotic mRNAs.Cell 9,645-653.
Shimotohno,K.,Kodama,Y.,Hashimoto,J.,and Miura,K.I.(1977).Importance of 5′-terminal blocking structure to stabilize mRNA in eukaryotic protein synthesis.Proc Natl Acad Sci U S A 74,2734-2738.
Shuman,S.(2001).The mRNA capping apparatus as drug target and guide to eukaryotic phylogeny.Cold Spring Harb Symp Quant Biol 66,301-312.
Shuman,S.(2002).What messenger RNA capping tells us about eukaryotic evolution.Nat Rev Mol Cell Biol 3,619-625.
Su,D.,Lou,Z.,Sun,F.,Zhai,Y.,Yang，H.,Zhang,R.,Joachimiak,A.,Zhang,X.C.,Bartlam,M.,and Rao,Z.(2006).Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10.J Virol 80,7902-7908.
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Ziebuhr,J.(2003).SARS--unprecedented global response to a newly emerging disease.Int J Med Microbiol 293,229-231.
Zust,R.,Cervantes-Barragan,L.,Habjan,M.,Maier,R.,Neuman,B.W.,Ziebuhr,J.,Szretter,K.J.,Baker,S.C.,Barchet,W.,Diamond,M.S.,et al.(2011).Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.Nat Immunol 12,137-143.
< 110>Wuhan University
< 120>a kind of virus that can be used for the coronavirus control adds cap system peptide inhibitor
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<210> 13
<211> 26
<212> DNA
< 213>artificial sequence
<400> 13
ttcgaattcc tgcatcaagg gttcgc 26
<210> 14
<211> 27
<212> DNA
< 213>artificial sequence
<400> 14
atgcatatgt gtgacttgaa aggtaag 27
<210> 15
<211> 26
<212> DNA
< 213>artificial sequence
<400> 15
ttcgaattcc tgcatcaagg gttcgc 26
<210> 16
<211> 32
<212> DNA
< 213>artificial sequence
<400> 16
atgcatatga ccatggctgg aaatgctaca ga 32
<210> 17
<211> 26
<212> DNA
< 213>artificial sequence
<400> 17
ttcgaattcc agacggtaca gactgt 26
<210> 18
<211> 33
<212> DNA
< 213>artificial sequence
<400> 18
atgcatatga ccatggctgt aacaccagaa gct 33
<210> 19
<211> 26
<212> DNA
< 213>artificial sequence
<400> 19
ttcgaattcc tgggtcatta gcacaa 26
<210> 20
<211> 32
<212> DNA
< 213>artificial sequence
<400> 20
atgcatatga ccatggctgg aaatgctaca ga 32
<210> 21
<211> 26
<212> DNA
< 213>artificial sequence
<400> 21
ttcgaattcc tgggtcatta gcacaa 26
<210> 22
<211> 32
<212> DNA
< 213>artificial sequence
<400> 22
atgcatatga ccatggctgg aaatgctaca ga 32
<210> 23
<211> 24
<212> DNA
< 213>artificial sequence
<400> 23
ttcgaattca ggatttggat ggtc 24
<210> 24
<211> 34
<212> DNA
< 213>artificial sequence
<400> 24
atgcatatga ccatgcaaga gtcctttggt ggtg 34
<210> 25
<211> 26
<212> DNA
< 213>artificial sequence
<400> 25
ttcgaattcc tgggtcatta gcacaa 26
<210> 26
<211> 51
<212> DNA
< 213>artificial sequence
<400> 26
tgccatatgg ataaaattca tcatcatcat cactgtgtga agatgttgtg t 51
<210> 27
<211> 29
<212> DNA
< 213>artificial sequence
<400> 27
ccgctcgagt tactgcatca agggttcgc 29
<210> 28
<211> 51
<212> DNA
< 213>artificial sequence
<400> 28
tgccatatgg ataaaattca tcatcatcat cacactgtaa caccagaagc t 51
<210> 29
<211> 29
<212> DNA
< 213>artificial sequence
<400> 29
ccgctcgagt tactgcatca agggttcgc 29
<210> 30
<211> 50
<212> DNA
< 213>artificial sequence
<400> 30
tgccatatgg ataaaattca tcatcatcat cacgctggaa atgctacaga 50
<210> 31
<211> 29
<212> DNA
< 213>artificial sequence
<400> 31
ccgctcgagt tacagacggt acagactgt 29
<210> 32
<211> 50
<212> DNA
< 213>artificial sequence
<400> 32
tgccatatgg ataaaattca tcatcatcat cacgctggaa atgctacaga 50
<210> 33
<211> 29
<212> DNA
< 213>artificial sequence
<400> 33
ccgctcgagt tactgggtca ttagcacaa 29
<210> 34
<211> 50
<212> DNA
< 213>artificial sequence
<400> 34
tgccatatgg ataaaattca tcatcatcat cacgctggaa atgctacaga 50
<210> 35
<211> 29
<212> DNA
< 213>artificial sequence
<400> 35
ccgctcgagt tactgcatca agggttcgc 29
<210> 36
<211> 25
<212> DNA
< 213>artificial sequence
<400> 36
gcccatggct gcaagtcaag cgtgg 25
<210> 37
<211> 31
<212> DNA
< 213>artificial sequence
<400> 37
gcctcgagtc agttgttaac aagaatatca c 31
<210> 38
<211> 87
<212> DNA
< 213>artificial sequence
<400> 38
tttggtggtg cttcatgttg tctgtattgt agatgccaca ttgaccatcc aaatcctaaa 60
ggattctgtg acttgaaagg taagtac 87
Claims (10)
1. a peptide species, its aminoacid sequence is: the 1) aminoacid sequence shown in the SEQ ID No.1; Or, 2) aminoacid sequence shown in the SEQ ID No.1 through replacement, add and/or lack that one or several amino obtains have same function by sequence deutero-sequence shown in the SEQ ID No.1; Or 3) truncated sequence of sequence shown in the SEQ ID No.1, this truncated sequence comprises GGASCCLYCRCH at least.
2. the truncated sequence polypeptide according to claim 1 is characterized in that, wherein said 3) is GGASCCLYCRCH.
3. peptide inhibitor that is used to suppress coronavirus, it comprises claim 1 or 2 described polypeptide.
4. coronavirus antiviral, it comprises claim 1 or 2 described polypeptide.
5. the gene of coding claim 1 or 2 said polypeptide.
6. contain the said expression carrier of claim 5.
7. the host cell that contains the said expression vector of claim 6.
8. claim 1 or 2 described polypeptide, the described gene of claim 5, the described expression vector of claim 6 or the described host cell of claim 7 application in preparation anti-coronavirus medicine.
9. one kind is suppressed coronavirus and adds cap and modify or duplicate the method for transcribing, and comprises using claim 1 or 2 described polypeptide to suppress combining of nsp10 and nsp16.
10. the method for preparing claim 1 or 2 said polypeptide, it is artificial directly synthetic or obtain through vivoexpression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101819675A CN102675429A (en) | 2012-06-05 | 2012-06-05 | Virus capping system polypeptide inhibitor for controlling coronavirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101819675A CN102675429A (en) | 2012-06-05 | 2012-06-05 | Virus capping system polypeptide inhibitor for controlling coronavirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
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Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101819675A Pending CN102675429A (en) | 2012-06-05 | 2012-06-05 | Virus capping system polypeptide inhibitor for controlling coronavirus |
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| Country | Link |
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| CN (1) | CN102675429A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555599A (en) * | 2013-11-05 | 2014-02-05 | 武汉大学 | High-throughput screening method for anti-coronavirus medicine |
| CN107245095A (en) * | 2017-06-15 | 2017-10-13 | 武汉大学 | Peptide inhibitor for suppressing ten kinds of coronavirus |
| CN116854787A (en) * | 2023-09-05 | 2023-10-10 | 四川大学 | Cas7-11 protein inhibitor and application thereof |
-
2012
- 2012-06-05 CN CN2012101819675A patent/CN102675429A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| KE M ET AL.: "Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2-O-methyltransferase activity of nsp10/nsp16 complex", 《VIRUS RES.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555599A (en) * | 2013-11-05 | 2014-02-05 | 武汉大学 | High-throughput screening method for anti-coronavirus medicine |
| CN107245095A (en) * | 2017-06-15 | 2017-10-13 | 武汉大学 | Peptide inhibitor for suppressing ten kinds of coronavirus |
| CN107245095B (en) * | 2017-06-15 | 2020-05-26 | 武汉大学 | Polypeptide inhibitor for inhibiting ten kinds of coronavirus |
| CN116854787A (en) * | 2023-09-05 | 2023-10-10 | 四川大学 | Cas7-11 protein inhibitor and application thereof |
| CN116854787B (en) * | 2023-09-05 | 2023-11-07 | 四川大学 | Cas7-11 protein inhibitor and application thereof |
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Application publication date: 20120919 |