CN111363017B - Influenza A virus nucleoprotein S69 mutant sequence, mutant and application thereof - Google Patents

Abstract

The invention discloses a mutant sequence of influenza A virus nucleoprotein S69 as well as a mutant and application thereof. The invention provides an influenza virus nucleoprotein mutant, which is a protein obtained by mutating serine S at the 69 th site of an influenza virus nucleoprotein amino acid sequence into glutamic acid E or alanine A and keeping other amino acid residues unchanged. The present invention relates to the effects of NP S69E or S69A on the virulence of viruses in cells and mice, viral polymerase activity, transcription and replication of viral RNA, NP autopolymerization, NP interaction with viral polymerase proteins, NP binding to RNA, NP intracellular localization, etc. The present invention relates to the provision of candidate strains of influenza vaccines.

Description

Influenza A virus nucleoprotein S69 mutant sequence, mutant and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an influenza A virus nucleoprotein S69 mutant sequence, a mutant and application thereof.

Background

Influenza A Virus (IAV) is the main pathogen causing influenza epidemics and sometimes causes severe respiratory diseases, and because the host has not developed immunity against the newly mutated virus, viral epidemics are often accompanied by significant morbidity and mortality in humans and animals. Influenza a viruses are enveloped viruses belonging to the orthomyxoviridae family, with a genome consisting of 8 negative-strand RNA segments. The viral ribonucleoprotein (vRNP) complex is the core component of the virus, is composed mainly of three polymerase proteins (PB1, PB2, PA), Nucleoprotein (NP) and viral rna (vrna), and plays an important role in the transcription and replication of influenza viruses. The characteristic of high variability of influenza viruses makes the current influenza vaccines unable to effectively realize the preventive protection effect, so that the research of more suitable universal vaccines is very important.

NP is encoded by the RNA segment of item 5 and is a protein that is present in high abundance in the virion. During influenza infection, subcellular localization, oligomerization, interaction with other components of vRNP, etc. are important factors in regulating viral polymerase activity and viral replication capacity. The NP has a tail loop structure, and the loop is inserted into a binding groove of an adjacent NP molecular body to mediate the oligomerization of the NP and participate in the formation of a vRNP complex. NP can interact with the viral polymerase proteins PB1, PB2, but not bind to PA. And NP has high RNA binding activity and RNA binding non-sequence specificity. NP can interact with nuclear import protein (import-alpha) in host cells, mediate NP to enter nucleus, further promote vRNP to enter nucleus, and play a certain role in vRNP nuclear export.

During viral infection of eukaryotic cells, phosphorylation modifications often occur at serine, threonine or tyrosine, and phosphorylation is a process by which phosphate groups on ATP are transferred to the acceptor protein. Phosphorylation and dephosphorylation play an important role in the viral replication cycle by regulating viral protein function. Some phosphorylation can activate protein activity and correspondingly dephosphorylate inhibitory protein activity, or conversely dephosphorylate "on" protein activity and phosphorylate "off" protein activity.

Disclosure of Invention

It is an object of the present invention to provide influenza virus nucleoprotein mutants.

The influenza virus nucleoprotein mutant provided by the invention is a protein obtained by mutating serine S at the 69 th site of an influenza virus nucleoprotein amino acid sequence into glutamic acid E or alanine A and keeping other amino acid residues unchanged.

In the influenza virus nucleoprotein mutant, the influenza virus nucleoprotein is derived from influenza A virus.

In the mutant, the amino acid sequence of the influenza virus nucleoprotein is a sequence 1 in a sequence table.

The nucleotides encoding the above mutants are also within the scope of the present invention.

Recombinant vectors containing the above nucleotides are also within the scope of the present invention.

The application of the above mutant or the above nucleotide or the above recombinant vector in any one of the following 1) to 6) is also within the scope of the present invention: the mutant is a protein obtained by mutating serine S at the 69 th site of an amino acid sequence of the nucleoprotein of the influenza virus into alanine E and keeping other amino acid residues unchanged:

1) preparing an influenza virus having reduced viral virulence;

2) preparing an influenza virus having reduced viral polymerase activity;

3) preparing an influenza virus with reduced viral RNA transcription and/or reduced viral RNA replication;

4) preparing an influenza virus with reduced binding of NP to PB 2;

5) preparing influenza virus with reduced NP nuclear entry;

6) influenza viruses are prepared that inhibit the interaction of NP with import-alpha 3.

Or, the use of the above-mentioned mutant or the above-mentioned nucleotide or the above-mentioned recombinant vector in a) or b) as follows: the mutant is a protein obtained by mutating serine S at the 69 th site of an amino acid sequence of the nucleoprotein of the influenza virus into alanine A and keeping other amino acid residues unchanged:

a) preparing an influenza virus having reduced viral virulence;

b) influenza viruses are produced that have reduced viral RNA transcription and/or increased viral RNA replication.

It is another object of the present invention to provide a recombinant influenza virus.

The recombinant influenza virus provided by the invention contains nucleotide for expressing the mutant.

Or, the application of the recombinant influenza virus in preparing influenza vaccine is also the protection scope of the invention;

alternatively, the present invention also provides an influenza vaccine (candidate strain) comprising the above-described mutant or the above-described nucleotide or the above-described recombinant vector or the above-described recombinant virus.

The application of the substance which can prevent the 69 th S site of the influenza virus nucleoprotein from phosphorylation or dephosphorylation in the preparation of the product for reducing the pathogenicity of the influenza virus to be detected is also within the protection scope of the invention;

or the application of the substance which can lead the 69 th S of the influenza virus nucleoprotein to be phosphorylated in the preparation of products for reducing the pathogenicity of the influenza virus to be detected is also within the protection scope of the invention;

or the application of the substance which can phosphorylate the 69 th S of the influenza virus nucleoprotein in the preparation of the product for reducing the activity of the influenza virus polymerase to be detected is also within the protection scope of the invention;

or the application of the substance which can phosphorylate the 69 th site S of the influenza virus nucleoprotein in preparing the product for reducing the RNA transcription and/or replication activity of the influenza virus to be detected is also the protection scope of the invention;

or the application of the substance which can lead the 69 th S of the influenza virus nucleoprotein to be phosphorylated in the preparation of products for changing the RNA transcription and/or replication of the influenza virus to be detected is also within the protection scope of the invention;

or the application of the substance which can not lead the 69 th site S of the influenza virus nucleoprotein to generate phosphorylation or dephosphorylation in the preparation of products for reducing the RNA transcription of the influenza virus to be detected and/or improving the RNA replication of the influenza virus to be detected is also within the protection range of the invention;

or the application of the substance which can not lead the 69 th S of the influenza virus nucleoprotein to generate phosphorylation or dephosphorylation in the preparation of products for changing the RNA transcription and/or replication of the influenza virus to be detected is also within the protection scope of the invention;

or the application of the substance which can phosphorylate the 69 th site S of the influenza virus nucleoprotein in preparing the nuclear product for reducing the nucleoprotein of the influenza virus to be detected is also within the protection scope of the invention;

or the application of the substance which can phosphorylate the 69 th S of the influenza virus nucleoprotein in the preparation of products for reducing the interaction between NP and PB2 of the influenza virus to be detected is also within the protection scope of the invention;

or the substance which can lead the 69 th S of the influenza virus nucleoprotein to be phosphorylated in the preparation of products for reducing the interaction between NP and import in-alpha 3 of the influenza virus to be detected is also within the protection scope of the invention.

The substance for phosphorylating or dephosphorylating the 69 th serine of the influenza nucleoprotein comprises a substance obtained by mutating the 69 th serine of the amino acid sequence of the influenza nucleoprotein into glutamic acid E or alanine A;

the substance for mutating the 69 th serine S of the influenza virus nucleoprotein amino acid sequence into the glutamic acid E or the alanine A is specifically a primer for point mutating a plasmid carrying the influenza virus nucleoprotein into a plasmid carrying the influenza virus nucleoprotein mutant;

the specific examples of the above primers are as follows:

S69E up：5’-gagagaatggtgctcgaggcttttgacgagaggaggaataaat-3’

S69E down：5’-cctctcgtcaaaagcctcgagcaccattctctctattgttaag-3’

S69Aup：5’-gagagaatggtgctcgctgcttttgacgagaggaggaataaat-3’

S69A down：5’-cctctcgtcaaaagcagcgagcaccattctctctattgttaag-3’

the 3 rd object of the present invention is to provide a method for reducing the virulence of influenza virus.

The method provided by the invention is 1) or 2):

1) the method comprises the following steps: (ii) non-phosphorylation or dephosphorylation, or sustained phosphorylation of S at position 69 of said influenza virus nucleoprotein;

2) the method comprises the following steps: the 69 th S of the influenza virus nucleoprotein is mutated into A or E.

Dephosphorylating S at 69 th site of influenza virus nucleoprotein to mutate S to A;

(ii) persistent phosphorylation of influenza virus nucleoprotein at position 69S to mutate S to E;

according to the invention, an unreported phosphorylation site, namely serine 69 (S69), on the NP of the influenza A virus is identified by using a mass spectrometry experiment, bioinformatics analysis shows that the NP S69 is highly conserved in different subtype strains of the influenza A virus, and functional analysis shows that S69 is positioned in a binding domain of the NP and PB2 and is also positioned in a binding domain of the NP and RNA. The NP S69 is mutated into glutamic acid E or alanine A, a plasmid containing NP S69 continuous phosphorylation (E is consistent with the phosphorylated S structure and the charged condition) and continuous dephosphorylation (A is consistent with the S structure) is constructed by point mutation, a reverse genetic system rescues the virus, the detection is carried out at the protein expression, cell level and gene level of the virus, the NP S69E mutation is found to reduce the activity of virus polymerase, inhibit the transcription and replication of virus RNA, and the S69A mutation inhibits the transcription of the virus RNA, so that the virus RNA replication is increased, the virus RNA replication is carried out on the cell, but the virus pathogenicity is reduced. The replication capacity and the pathogenicity of the S69A mutant virus are detected by using a mouse model, and the replication capacity of the mutant virus is maintained and the pathogenicity is reduced. Further, the influence of the NP S69 mutation on the interaction between NP and a virus polymerase protein, the combination between NP and RNA and the autopolymerization of NP is detected, the influence of the NP S69 mutation on the intracellular localization of NP is also detected, the phenomenon that an S69A group is basically consistent with a Wild Type (WT) phenomenon is found, the interaction between NP and PB2 is weakened by S69E, and the nuclear entry of NP is inhibited by S69E.

The above results show that:

(1) the NP S69A mutant maintained replicative capacity on cells and virus virulence was reduced.

(2) The NP S69A mutant maintained replication competence in animals and virus virulence was reduced.

(3) NP S69A enhances viral RNA replication, reduces viral RNA transcription; NP S69E reduces viral polymerase activity, reducing viral RNA transcription and viral RNA replication.

(4) The influence of the NP S69A mutation on the interaction between NP and viral polymerase protein, the binding between NP and RNA, and the autopolymerization of NP is basically consistent with the wild-type phenomenon, and the S69E mutation weakens the interaction between NP and PB 2.

(5) The effect of the NP S69A mutation on NP intracellular localization, similar to wild type, the S69E mutation suppressed NP nuclear entry.

Drawings

FIG. 1 shows that position S69 of NP is identified as a phosphorylation site on NP of influenza A virus. (A) Arrows mark phosphorylated NP (pnp) and NP bands. A/WSN/1933(H1N1) virus infected 293T cells at an MOI of 3, lysed 8 hours after infection, incubated with NP antibody followed by protein G agar beads, phosphatase (ALP) -treated or non-ALP-treated groups as controls, electrophoresed to separate alkaline phosphatase-sensitive protein bands from NP bands, stained, excised and mass-analyzed for pNP bands. (B) The sample-carrying protein was identified as NP in influenza A virus A/WSN/1933(H1N1) strain, and the phosphorylation site on the identified NP was marked with orange color. (C) A549 cells were infected with WT WSN or S69A virus (MOI of 1), lysed 8 hours after infection, incubated with NP antibody followed by protein G agarose beads, and detected with NP antibody and phosphoserine antibody against a group of uninfected A549 cells.

FIG. 2 is a structural formula of the NP-mutated amino acid and a crystal structure of the NP monomer; wherein, the structural formula of the (A) mutation amino acid and the crystal structure of the (B) NP monomer are marked with orange yellow at the position of S69.

FIG. 3 shows the rescue and detection of NP-S69 mutant virus. (A) 293T cells were transfected with a 12 plasmid reverse genetics system carrying NP WT or NP-S69E or NP-S69A mutants, the cells were pooled 6 hours after transfection and harvested 72 hours after transfection to examine protein expression of NP and M1. (B) Cell supernatants were collected 72 hours post transfection, blinded one generation on MDCK cells, and harvested for protein expression of NP and M1. (C) WSN WT and S69A viruses infected A549 cells with MOI of 0.001 respectively, cell supernatants were harvested at different time points, virus titers were detected, and virus multi-step growth curves were plotted.

FIG. 4 shows the effect of NP-S69A on viral pathogenicity in mice. (A) Dripping the rescued and harvested WSN WT or S69A virus into nose to infect mice, setting different titers, setting 8 viruses in each group, and observing the weight change of the mice every 14 days after infection; (B) observing the mortality of the mice; (C-E) infection of mice 10⁴PFU WSN WT or S69A virus, 3 in each group, lungs were taken 1, 3, 5 days after infection, lung index and pneumovirus titer were examined, and lung disease changes were observed.

FIG. 5 shows the effect of NP-S69E or S69A on viral polymerase activity. (A) The expression plasmids for NP WT or NP-S69E or NP-S69A were co-transfected with PB1, PB2, PA and vNS-luc expression plasmids into 293T cells and assayed 33 hours after transfection using the luciferase assay. And detecting NP and beta-actin protein expression quantity (B)12 plasmid reverse genetic system transfects 293T cell, cell lysis is carried out 48 hours after transfection, total RNA is extracted, reverse transcription is carried out, and the level of mRNA, vRNA and cRNA of M1 is detected by using real-time quantitative PCR method.

FIG. 6 shows the effect of NP-S69E or S69A on NP autopolymerization. FLAG-NP (WT or mutant) and MYC-NP (WT or mutant) were co-transfected into 293T cells, harvested 28 hours after transfection, subjected to co-IP experiments, enriched with FLAG-Beads, and assayed for MYC antibodies.

FIG. 7 is a graph of the effect of NP-S69E or S69A on the interaction of NP with PB 2. FLAG-PB2 and MYC-NP (WT or mutant) were co-transfected into 293T cells, cells were harvested 28 hours after transfection, and co-IP experiments were performed, enriched with FLAG-Beads, and MYC antibody was detected.

FIG. 8 is a graph of the effect of NP-S69E or S69A on NP interaction with PB 1. FLAG-PB1 and MYC-NP (WT or mutant) were co-transfected into 293T cells, cells were harvested 28 hours after transfection, and co-IP experiments were performed, enriched with FLAG-Beads, and MYC antibody was detected.

FIG. 9 shows the effect of NP-S69E or S69A on the interaction of NP and RNA. Polyuridylic acid-Agarose and prokaryotic expression purified HIS-NP (WT or mutant) protein were combined for half an hour for co-IP assay and detected with NP antibody.

FIG. 10 shows intracellular localization of NP-S69E or S69A. (A) The 293T cells were transfected with NP WT or mutants, cells were fixed at various time points after transfection, and the NP localization was detected using IFAs, red for NP and blue for nucleus. (B) The number of different fields is 100 cells, and different groups of localization conditions are recorded respectively, wherein N represents that the cells are mainly localized in a nucleus, N + C represents that the nucleus and cytoplasm are distributed, and C represents that the cells are mainly distributed in the cytoplasm.

FIG. 11 shows the nuclear/cytoplasmic distribution of NP-S69E or S69A. 293T cells were transfected with NP WT or NP-S69E or NP-S69A, respectively, cells were harvested at different time points (11, 24, 35 hours) after transfection, the nuclei and cytoplasm were separated, and protein expression in the nuclei or cytoplasm was detected using the western blotting method using NP, Lamin B1, HSP70 antibodies, respectively.

FIG. 12 shows the binding of NP-S69E or S69A to importin-alpha. FLAG-importin-alpha and MYC-NP WT or MYC-NP-S69E or MYC-S69A were co-transfected into 293T cells, the cells were harvested 25 hours after transfection, and co-IP experiments were performed, enriched with FLAG-Beads, and MYC antibody detection was performed.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The cells, strains and virus strains used in the following examples are as follows:

MDCK cells (Cobioer, catalog No. CBP60561), 293T cells (Shanghai Bo Guo Biotech Co., Ltd., product No. BG0021), A549 cells (Sigma-Aldrich, product No. 86012804), Escherichia coli Top10, and Escherichia coli BL 21. The influenza virus strain used in the experiment is A/WSN/1933(H1N1) and is obtained by rescuing 12 plasmid reverse genetic packaging system, propagated in MDCK cells and stored at-80 ℃.

The experimental reagents in the following examples are as follows:

sodium chloride (NaCl), HEPES, EDTA, Triton X-100, phosphatase inhibitor (phosSTOP), MnCl₂Trissylfonyl Phenyl Chloromethyl Ketone (TPCK) treated pancreatin, Cycloheximide (Cycloheximide), FLAG M2agar beads, protein G agar beads, Polyuridylic acid-agar, murine anti-FLAG antibody were purchased from Sigma-Aldrich (USA);

the preparation of murine anti-M1 monoclonal antibody is described in the following documents: koestler, T.P., Rieman, D., Muirhead, K., Greig, R.G., Poste, G., Identification and characterization of a monoclonal antibody to an anti expressed on activated macrophages, proceedings of the National Academy of science of the United States of America,1984,81: 4505-;

rabbit anti-NP polyclonal antibody (preparation method: 2 month old female rabbits were immunized with 250mg of purified His-pET30a-NP protein, 2 weeks apart, 3 booster immunizations with 150mg of protein, serum was collected), and the preparation method of polyclonal antibody is described in the following documents: liu, X, Sun, L, Yu, M, Wang, Z, Xu, C, Xue, Q, Zhang, K, Ye, X, Kitamura, Y, Liu, W, Cyclophilin A intermediates with influenza A viruses M1protein and antigens the early stage of the viral replication, cellular microbiology,2009,11: 730-;

protease inhibitors (cocktail) and pancreatin (for digestion) were purchased from Roche corporation (usa);

murine anti-beta-actin antibodies, murine anti-GAPDH antibodies, murine anti-p-Ser antibodies, murine anti-c-MYC antibodies, murine anti-Lamin B1(A11) antibodies, murine anti-HSP 70(3A3) antibodies, rabbit anti-MYC antibodies were purchased from Santa Cruz Biotechnology, Inc. (England and Ireland);

RNase inhibitor was purchased from Promega (USA);

bovine Serum Albumin (BSA) was purchased from Bovogen Biologicals (Australia);

acrylamide-pendant phosphate-tag (Phos-tag Acrylamide) was purchased from Wako corporation (Japan);

lipofectamine2000 and TRIzol were purchased from Invitrogen corporation (usa);

paraformaldehyde (paraforarmaldehyde) was purchased from national pharmaceutical group chemical agents limited (china);

4',6-diamidino-2-phenylindole (DAPI) was purchased from Thermo Fisher Scientific (USA);

DMEM cell culture medium and Fetal Bovine Serum (FBS) were purchased from Gibco (usa);

cell culture dishes were purchased from Corning corporation (usa);

pfu DNA polymerase and dNTP mix were purchased from Takara, Japan;

Ni Sepharose^TM6Fast Flow was purchased from GE Healthcare Bio-Sciences (Sweden);

glycerol was purchased from Beijing chemical plant (China);

all secondary antibodies were purchased from Baihui Zhongyuan Biotech company (China).

pHH21 and pcDNA4/TO belong TO the reverse genetic packaging system of influenza virus, pHH21 vector with RNA polymerase I promoter and terminator, for the expression of 8 strands of viral RNA segments of influenza virus. pcDNA4/TO (Invitrigen, Cat. No. V103020) carries the CMV/TO promoter, and is used TO initiate expression of viral proteins in the viral ribonucleoprotein complex of the viral rescue process, including PB1, PB2, PA and NP.

The experimental apparatus in the following examples is as follows:

4 ℃ centrifuge, CO₂Cell culture boxes, water baths and autoclaves are products of Thermo corporation;

the biological safety cabinet is a product of Beijing Dong gang Harr instrument manufacturing company Limited;

7500Real Time PCR System is product of Applied Biosystems;

the PCR instrument and the film transfer instrument are products of Bio-Rad company;

confocal laser microscopes LSCM FV1000 and Leica SP8 are Olympus products;

-80 ℃ refrigerator, 4 ℃ refrigerator and-20 ℃ refrigerator are products of Haier group company;

the ultrasonic cell crusher is a SCIENTZ-IID product.

Example 1 determination of NP phosphorylation site of WSN Strain of influenza Virus

Determination of NP phosphorylation site of influenza virus WSN strain

293T cells were infected with influenza A/WSN/1933(H1N1) (strain was rescued from the 12 plasmid reverse genetics system and propagated in MDCK cells; MOI 1), and 8 hours after infection, cells were lysed with lysates (150mM NaCl, 20mM HEPES [ pH7.4 ], 1mM EDTA, 10% glycerol, 1% Triton X-100, pH adjusted to 7.4) containing protease inhibitor (cocktail) (Roche, 50ml lysate plus 1 plate) and phosphatase inhibitor (PhosSTOP) (Sigma-Aldrich, 50ml lysate plus 1 plate) at 4 ℃ for 30 minutes and centrifuged at 12000rpm for 10 minutes; the supernatant of the cell lysate was collected. To the above supernatant was added rabbit anti-NP polyclonal antibody, incubated at 4 ℃ for 4 hours, enriched with protein G agar beads (Sigma-Aldrich), and the protein was collected.

Alkaline phosphatase (ALP) treatment group: 293T cells were infected with influenza a/WSN/1933(H1N1) strain (MOI ═ 1) 8 hours post-infection; the cells were lysed with a lysis solution (150mM NaCl, 20mM HEPES [ pH7.4 ], 1mM EDTA, 10% glycerol, 1% TritonX-100, pH adjusted to 7.4) containing protease inhibitor (cocktail) and phosphatase inhibitor (PhosSTOP) at 4 ℃ for 30 minutes, and centrifuged at 12000rpm for 10 minutes; the cell lysate supernatant was collected. To the supernatant was added rabbit anti-NP polyclonal antibody, incubated at 4 ℃ for 4 hours, enriched with protein G agar beads, treated with alkaline phosphatase ALP (NEB, M0371V, concentration 1unit/mL), and collected at 37 ℃ for 1 hour.

Control group: in contrast to the ALP treatment group, no ALP treatment was added.

The collected protein is 15% Mn²⁺-Phos-tag polyacrylamide gel (containing 50. mu.M Phos-tag acrylamide and 0.1mM MnCl)₂) SDS-PAGE. The PAGE gel was stained using an ultrafast staining solution of ExBlue protein.

The results are shown in FIG. 1A (IAV refers to influenza A virus, type A virus), and the arrow marks phosphorylated NP: (^pNP) and NP bands; the NP band was identified without the ALP group (lane 3 of FIG. 1A), and had a slower migration rate than normal NPs; while no such band was present in the ALP treated group (lane 3 of FIG. 1A); this band is sensitive to ALP and is considered to be a phosphorylated NP band. The cut-off band was sent to a mass spectrometry laboratory (LCQ Deca XP Plus, Thermo Fisher Scientific, USA) for mass spectrometry identification, which proved that the band was NP of A/WSN/1933(H1N1) strain, and the 69 th S (serine) of the NP amino acid sequence was a phosphorylatable site (FIG. 1B), the NP amino acid sequence was SEQ ID NO: 1, and the coding gene of NP protein was SEQ ID NO: 2.

Simulating continuous phosphorylation to change the 69 th S of NP to E, and marking the protein as NP-S69E protein, wherein the amino acid sequence of the protein is that the 69 th serine of sequence 1 is mutated into glutamic acid (as shown in figure 2A); the nucleotide sequence of the coding gene of the NP-S69E protein is that the tct at the 205 th-207 th position of the sequence 2 is replaced by gag.

Simulating continuous dephosphorylation, namely, mutating the 69 th S of NP into A, and recording the result as NP-S69A protein, wherein the amino acid sequence of the protein is that the 69 th serine of sequence 1 is mutated into alanine (as shown in figure 2A); the nucleotide sequence of the coding gene of the NP-S69A protein is that tct at the 205 th-207 th position of the sequence 2 is replaced by gct.

Secondly, determining the phosphorylation of NP with S69 using phosphorylated antibodies

1. Rescue of WSN WT virus, NP S69E mutant virus, and NP S69A mutant virus

1) Virus rescue

293T cells (Shanghai Bogu Biotech Co., Ltd., product number BG0021) were plated in a 60mm dish and transfected when 90% confluency was reached within 24 hours; the cells were replaced with Opti-MEM medium before transfection, and the reverse genetics system 12 plasmid (pHH21-WSN-PB2, pHH21-WSN-PB1, pHH21-WSN-PA, pHH21-WSN-HA, pHH21-WSN-NP or pHH21-WSN-NP-S69E or pHH21-WSN-NP-S69A, pHH21-WSN-NA, pHH21-WSN-M, pHH21-WSN-NS, pcDNA4/TO-PB2, pcDNA4/TO-PB1, pcDNA4/TO-PA, pcDNA4/TO-NP or pcDNA4/TO-NP-S69E or pcDNA4/TO-NP-S69A, each 1. mu.g) was mixed; the procedure was as per Lipofectamine2000 instructions; and (3) carrying out transfection for 6 hours, changing the transfection solution into DMEM containing pancreatin (the final concentration is 2 mu g/mL) treated by TPCK, culturing for 72-96 hours, and then harvesting cell supernatant, namely the virus solution saved.

Among the above-mentioned 12 plasmids, pHH21-WSN-PB2, pHH21-WSN-PB1, pHH21-WSN-PA, pHH21-WSN-HA, pHH21-WSN-NP, pHH21-WSN-NA, pHH21-WSN-M, pHH21-WSN-NS, pcDNA4/TO-PB2, pcDNA4/TO-PB1, pcDNA4/TO-PA, and pcDNA4/TO-NP are described in "Neumann G, et al," Generation of flowable A viruses from bound cDNAs, Proc Natl Acad Sci U S A,1999,96(16):9345-50 ", respectively.

pHH21-WSN-NP was a recombinant plasmid obtained by cloning the gene encoding the NP protein (SEQ ID NO: 2) into pHH21 vector (a gift from professor Yoshiro Kawaoka, university of Wisconsin, Madison, USA, described in "Neumann G, et al, Generation of influenza A viruses from cloned cDNAs, Proc Natl Acad Sci U S.A.1999; 96(16) 9345-50") among BsmBI cleavage sites.

pHH21-WSN-NP-S69E is a recombinant plasmid obtained by cloning the coding gene of NP-S69E protein into BsmBI enzyme cutting sites of pHH21 vector.

pHH21-WSN-NP-S69A is a recombinant plasmid obtained by cloning the coding gene of NP-S69A protein into BsmBI enzyme cutting sites of pHH21 vector.

pcDNA4/TO-NP was a recombinant plasmid obtained by cloning the gene coding for the NP protein (SEQ ID NO: 2) between the KpnI and EcoRI cleavage sites of pcDNA4/TO vector (Invitrigen, catalog No. V103020).

pcDNA4/TO-NP-S69E is a recombinant plasmid obtained by cloning the gene coding for NP-S69E protein into the KpnI and EcoRI cleavage sites of pcDNA4/TO vector.

pcDNA4/TO-NP-S69A is a recombinant plasmid obtained by cloning the gene coding for NP-S69A protein into the KpnI and EcoRI cleavage sites of pcDNA4/TO vector.

The above pHH21 and pcDNA4/TO carrying the full-length NP-S69E or NP-S69A can be prepared as follows: pHH21 and pcDNA4/TO vectors carrying full-length NP (A/WSN/1933(H1N1)) were subjected TO point mutation (S69E and S69A), and each point mutation cloning was performed by a point mutation Kit (Newpage Site-Directed Mutagenesis Kit). The point mutation primers are:

S69E up：5’-gagagaatggtgctcgaggcttttgacgagaggaggaataaat-3’

S69E down：5’-cctctcgtcaaaagcctcgagcaccattctctctattgttaag-3’

S69A up：5’-gagagaatggtgctcgctgcttttgacgagaggaggaataaat-3’

S69A down：5’-cctctcgtcaaaagcagcgagcaccattctctctattgttaag-3’

plasmid specific point mutation sequence identification (Senhua large gene sequencing) DNAstar software MegAlign function and original sequence alignment was used.

2) Detecting the viral titer

Preparing cells: taking well-grown MDCK cells (Cobioer, catalog number CBP60561), digesting by conventional method, diluting with DMEM containing 2% FBS to prepare cell suspension, counting cells, and adjusting cell concentration to 1 × 10⁵one/mL of suspension was then added to a 96-well cell culture plate at 100 μ L/well. 5% CO at 37 ℃₂The culture was carried out in an incubator overnight or for 8-12 hours to allow a monolayer to be filled.

And (3) virus dilution: the WSN WT virus, NP-S69A mutant virus and NP-S69E mutant virus obtained in 1 were diluted in 10-fold gradient, and 6 to 8 gradients were selected for the test using DMEM containing TPCK-pancreatin (final concentration: 2. mu.g/mL) as the diluent. And (3) subpackaging the diluent, adding the virus to be detected, uniformly mixing, and diluting one by one according to a gradient.

Infection with virus: after the cells in the 96-well plate grow to be full of a monolayer, discarding the original culture solution; two arrays of 96-well plates were set up with 100. mu.L of each dilution as negative controls. Adding 8-10 multiple wells of virus diluent from low concentration, 100 μ L each, and using 10 wells^-1-10^-8Serial graded dilutions of virus sequentially infect cells in 96-well plates. Placing at 37 ℃ with 5% CO₂Culturing under the condition.

Cytopathic well number was observed and recorded: the end point was judged as 72 hours.

Calculating the virus titer according to a Reed-muench method,

(1) calculating the number of pathological wells (A) and the number of negative wells (B) of each virus dilution cell;

(2) calculating the accumulation numbers of the cytopathic effect and the negative holes, and accumulating the accumulation numbers of the cytopathic effect holes from bottom to top (C); accumulating the negative hole accumulation number from top to bottom (D);

(3) calculate the percentage of cytopathic wells: a ratio (E) ═ C)/(C) + (D) ], (F) × 100;

(4) calculating distance ratio

Distance ratio (percentage higher than 50% -50)/(percentage higher than 50% -percentage lower than 50%)

(5) End result

TCID₅₀Per 100 μ L ═ negative log of virus dilution + distance ratio higher than 50%

The results of the detection of WSN WT virus, NP S69A mutant virus, and NP S69E mutant virus are shown in table 1 below:

TABLE 1

As can be seen from table 1, WSN WT virus, NP S69A mutant virus had corresponding viral titers; whereas the NP S69E mutant virus was not detected (n.d).

2. Identification of whether NP S69 is phosphorylated during WSN virus infection

WSN WT virus rescued in 1 above and NP-S69A mutant virus (MOI ═ 1) were infected with A549 cells (Sigma-Aldrich, product No. 86012804), and 8 hours after infection, the cells were lysed with a lysate (150mM NaCl, 20mM HEPES [ pH7.4 ], 1mM EDTA, 10% glycerol, 1% TritonX-100, pH adjusted to 7.4) containing protease inhibitor (cocktail) and phosphatase inhibitor (PhosSTOP) at 4 ℃ for 30 minutes, and centrifuged at 12000rpm for 10 minutes; the supernatant of the cell lysate was collected. The supernatant was added with rabbit anti-NP polyclonal antibody and incubated at 4 ℃ for 4 hours, and then protein G agarose beads were used for enrichment to collect the protein. Uninfected cells were also used as controls.

The obtained protein was subjected to SDS-PAGE and western blotting experiments.

The results are shown in fig. 1C, and it can be seen that the amounts of Input (original protein expression as control) detected by rabbit anti-NP polyclonal antibody and S69A and WT protein after enrichment are respectively consistent, the S69A protein band detected by serine phosphorylation antibody is obviously reduced compared with WT band, and it is determined that S69 on NP is phosphorylation modified during WSN virus infection.

3. Conservation and functional analysis of NP S69 site

The conservative analysis of Influenza Virus strains is that the complete sequences of 2389 strain H1N1, 108 strain H2N2, 2106 strain H3N2, 353 strain H4N6, 804 strain H5N1, 119 strain H7N7, 185 strain H7N9, 831 strain H9N2 and 56 strain H11N2 are aligned using Influenza Virus Sequence Database of NCBI (https:// www.ncbi.nlm.nih.gov/genes/FLU/Database/nph-select.cgigo% 04dat abase). Multiple sequence alignment showed that the NP S69 site is highly conserved among different subtypes of influenza a virus (table 2).

Subsequent observation of the surface accessibility of the S69 site to the NP crystal structure revealed that S69 (indicated by arrows) was exposed on the NP monomer surface (see FIG. 2B), and that the three-dimensional (3D) crystal structure of the NP was achieved using PyMOL

And (4) designing a software program. And S69 PB2 binding on NP andthe RNA binding domain, suggesting that it may be a functional serine, suggests that NP S69 phosphorylation or dephosphorylation may play a role in viral replication.

TABLE 2 conservation of NP S69 in different subtypes of influenza A virus

Example 2 Effect of NP S69 phosphorylation and dephosphorylation on viral virulence

Effect of NP S69 phosphorylation or dephosphorylation on viral replication at cellular level

1. Rescue of WSN WT and NP S69 mutant viruses

293T cells were plated in 60mm dishes and transfected when 90% confluence was achieved within 24 hours; the cells were replaced with Opti-MEM medium before transfection, and the reverse genetics system 12 plasmid (pHH21-WSN-PB2, pHH21-WSN-PB1, pHH21-WSN-PA, pHH21-WSN-HA, pHH21-WSN-NP or pHH21-WSN-NP-S69 or pHH21-WSN-NP-S69E, pHH21-WSN-NA, pHH21-WSN-M, pHH21-WSN-NS, pcDNA4/TO-PB2, pcDNA4/TO-PB1, pcDNA4/TO-PA, pcDNA4/TO-NP or pcDNA4/TO-NP-S69 or pcDNA4/TO-NP-S E, each 1. mu.g) was mixed; the procedure was as per Lipofectamine2000 instructions; the solution is changed after 6 hours of transfection, DMEM containing pancreatin (the final concentration is 2 mug/mL) treated by TPCK is changed, and the DMEM is cultured for 72 to 96 hours;

the WSN WT virus cell supernatant, NP S69A mutant virus cell supernatant and NP S69E mutant virus cell supernatant were collected and the cells were collected separately.

2、TCID₅₀The experiment detects the titer of the rescued virus

Preparing cells: taking well-grown MDCK cells (Cobioer, catalog number CBP60561), digesting by conventional method, diluting with DMEM containing 2% FBS to prepare cell suspension, counting cells, and adjusting cell concentration to 1 × 10⁵one/mL of suspension was then added to a 96-well cell culture plate at 100 μ L/well. 5% CO at 37 ℃₂The culture was carried out in an incubator overnight or for 8-12 hours to allow a monolayer to be filled.

And (3) virus dilution: the supernatant of the WSN WT virus cell rescued in the step 1, the supernatant of the NP S69A mutant virus cell and the supernatant of the NP S69E mutant virus cell are respectively diluted by 10 times of gradient, and 6-8 gradients are selected for testing. The virus diluent is DMEM containing TPCK-pancreatin, and is first packed into diluent, then added with the virus to be tested, mixed and diluted one by one.

Infection with virus: after the cells in the 96-well plate grow to be full of a monolayer, discarding the original culture solution; two rows of 96-well plates were provided with 100. mu.L of each dilution as negative controls. Adding 8-10 multiple wells of virus diluent from low concentration, 100 μ L each, and using 10 wells^-1-10^-8Serial graded dilutions of virus sequentially infect cells in 96-well plates. Placing at 37 ℃ with 5% CO₂Culturing under the condition.

Cytopathic well number was observed and recorded: the end point was judged as 72 hours.

Calculating the virus titer according to the calculation of a Reed-muench method,

(1) calculating the number of pathological wells (A) and the number of negative wells (B) of each virus dilution cell;

(2) calculating the accumulation numbers of the cytopathic effect and the negative holes, and accumulating the accumulation numbers of the cytopathic effect holes from bottom to top (C); accumulating the negative hole accumulation number from top to bottom (D);

(3) calculate the percentage of cytopathic wells: a ratio (E) ═ C)/[ (C) + (D) ], (F) × 100;

(4) calculating distance ratio

Distance ratio (percentage higher than 50% -50)/(percentage higher than 50% -percentage lower than 50%)

(5) End result

TCID₅₀Per 100 μ L ═ negative log of virus dilution + distance ratio higher than 50%

The detection results of the WSN WT virome cell supernatant, the NP S69A mutant virome cell supernatant and the NP S69E mutant virome cell supernatant are shown in table 3, and it can be seen that the WSN WT group and the NP S69A mutant group have corresponding virus titers, which indicates that the NP S69A mutant group maintains the virus replication ability; the NP S69E mutation group was not detected (N.D), indicating that NP S69E did not package infectious virus.

TABLE 3

3. Monitoring of expression of the viral proteins NP and M1

The expression of the major viral proteins NP and M1 in the cells collected in the step 1 (shown in FIG. 3A) was determined by the western blotting method, and the supernatant of the collected cells was rescued to infect MDCK cells (first generation), and the expression of the major proteins NP and M1 in the harvested cells was determined by the western blotting method (shown in FIG. 3B), specifically as follows:

the cells were lysed with a lysis solution containing protease inhibitor (Cocktail) (formulation 150mM NaCl, 20mM HEPES, 1mM EDTA, 1% Triton X-100, 10% glycerol, adjusted to pH7.4) at 4 ℃ for 30 minutes; centrifuging at 12000rpm for 10 minutes and collecting supernatant; adding loading buffer, boiling the sample at 99 ℃ for 10 minutes, and separating protein by SDS-PAGE; transferred to PVDF (Immobilon-P) membranes (Millipore Corporation, USA); the blocking solution (TBST solution containing 5% skimmed milk and 1% BSA) is slowly shaken at 4 ℃ overnight or at normal temperature for 2 hours; incubating at normal temperature for 1-2 hours by using a mouse anti-M1 antibody, a mouse anti-beta-actin antibody and a rabbit anti-NP antibody; the secondary antibody is correspondingly added with Horse Radish Peroxidase (HRP) labeled anti-mouse or anti-rabbit antibody; enhanced chemiluminescent detection reagent (TIANGEN Biotech, China) imaging.

The results are shown in fig. 3A and fig. 3B, and it can be seen that the S69A mutant obtained by rescue is similar to the WT group NP in expression, the S69A M1protein expression level is slightly weaker than that of WT, and the first generation NP and M1 are normally expressed after the S69A mutant is rescued; the S69E mutant has normal NP expression, extremely low M1 expression level, no NP and M1 expression after generation transmission, and probably because the NP S69 site is continuously phosphorylated to block the transcription of virus genes or inhibit the expression of virus proteins, the S69E group rescues the virus which can not be continuously infected.

4. Multistep growth curve of virus

The WSN WT virus and WSN NP-S69A mutant virus with the measured titers were infected with a549 cells (MOI ═ 0.001) at 12, 24, 36, 48, 60, and 72 hours, respectivelyCollecting cell supernatant, and using TCID₅₀The experiment detects the virus titer at different time points (the detection method is the same as the previous method), and a multistep growth curve of the virus is drawn on A549 cells.

As shown in fig. 3C, it can be seen that the NP-S69A mutant virus was able to replicate continuously on cells, and compared to WT, the S69 continuous dephosphorylation (NP S69A) virus titer of NP was decreased, indicating that the NP S69A group decreased the virulence of the virus on cells.

Second, the influence of NP S69A on virus pathogenicity in animal body

Different titers (10) obtained from one of the above were assigned to groups of 8 mice per group (BALB/c mice: purchased from Experimental animals technologies, Inc., Wei Tony Hua, Beijing)²、10³And 10⁴PFU) WSN WT virus and NP-S69A mutant virus were nasally inoculated into BALB/c mice. The control group was mice that were inhaled with sterilized PBS. The mice in each group were observed daily for body weight and mortality.

The change in body weight of the mice was observed daily for 14 days after the inoculation, and the results are shown in FIG. 4A, 10⁴The body weight of mice in the PFU S69A mutant virus infection group is slightly reduced; PBS group and 10²、10³PFU S69A mutant virus-infected groups showed no significant weight loss.

Mice were observed daily for mortality 14 days post inoculation, and the results are shown in FIG. 4B, 10⁴The mortality rate of PFU WT WSN-infected mice was 100%, and the S69A mutant was not lethal to each dose group.

Let 10⁴PFU titer WSN WT or S69A mutant virus infected mice dissected groups of 3 mice each, used for 1, 3, 5 days after infection to take mice lungs, observe lung lesion status, test lung index (wet lung weight/body weight x 100) and lung virus titer. As shown in fig. 4C and 4D, 10⁴Lung dissection of mice infected with PFU virus, and the S69A mutant virus-infected mice were found to have reduced lung lesions compared to WSN WT-infected mice (fig. 4C); the S69A mutant virus group showed a slight decrease in pulmonary index from WT mice 5 days post-infection (fig. 4D), and pneumovirus titers were greater than 1 log reduction from WT groups at each time point of detection post-infection for the S69A mutant virus group₁₀Order of PFU/mL (FIG. 4E).

The above results demonstrate that the virus (NP S69A) reduces the virulence of the virus in animals and that the virus replicates in the animals.

Mechanism research of influence of NP S69 phosphorylation and dephosphorylation on pathogenicity of influenza A virus

1. Effect of NP S69 phosphorylation and dephosphorylation on viral polymerase Activity

NP is one of the major components of the vRNP complex, which plays an important role in transcription and replication of the viral genome, and mutations at conserved amino acid sites in NP lead to reduced viral polymerase activity. The following experiments were thus carried out:

1) vRNP luciferase detection

293T cells were plated in 24-well plates and transfected into 293T cells simultaneously with 0.1. mu.g of pcDNA4/TO-PB1, pcDNA4/TO-PB2, pcDNA4/TO-PA, pcDNA4/TO-NP WT or pcDNA4/TO-NP-S69A or pcDNA4/TO-NP-S69E, pcDNA-beta-gal (Promega, Madison, Wis.) and 0.25. mu.g of pHH21-NS-Luc (Promega, Madison, Wis.) expression plasmids (described in Li, Z., Watanabe, T., Hatta, M., Watanabe, S., Nanbo, A., Ozawa, M., Kakugawa, S., Shimojima, Yamamada, S., Neuman, S., J.4102, J., P.4153, Joulo, Joule, 4102, J.; the above plasmid was also transfected, without NP, as a control; after transfection of cells at 37 ℃ were incubated for 33 hours; using 5 × Cell lysis buffer (Sigma-Aldrich, USA), it was diluted to 1 × Cell lysis buffer; cracking at 4 ℃ for 1 hour; centrifuging at 12000rpm for 10 min at 4 ℃; mu.L of Z buffer (21.5g Na) using beta-gal chromogenic substrate₂HPO₄·12H₂O、6.2g NaH₂PO₄·2H₂O、0.75g KCl、0.246g MgSO₄·7H₂O, adjusting to pH 7.0) +200 μ L ONPG (prepared with Z buffer, concentration 4mg/mL), and mixing; adding 20 mu L of cell lysate supernatant into the chromogenic substrate mixed solution, uniformly mixing, and standing at 37 ℃ for no more than 30 minutes until light yellow appears; 100 μ L of each sample was added to a 96-well plate, 4 replicates for each sample. Determining the OD 450; add 50. mu.L luciferase substrate to each EP tube; the fluorescence was rapidly measured by adding 10. mu.L of lysis supernatant to each tube. Each timeThe tubes were kept for the same reaction time; dividing the value measured by beta-gal by the fluorescence value of luciferase to obtain the corresponding enzyme activity value.

The cell lysate is detected by a western blotting detection method as follows:

the cells were lysed with protease inhibitor (Cocktail) containing lysis buffer (formulation 150mM NaCl, 20mM HEPES, 1mM EDTA, 1% Triton X-100, 10% glycerol, adjusted to pH7.4) for 30 minutes at 4 ℃; centrifuging at 12000rpm for 10 minutes and collecting supernatant; adding loading buffer, boiling the sample at 99 ℃ for 10 minutes, and separating protein by SDS-PAGE; transferred to PVDF (Immobilon-P) membrane (Millipore Corporation, USA); blocking solution (TBST solution containing 5% skimmed milk and 1% BSA) is slowly shaken overnight at 4 ℃ or at room temperature for 2 hours; incubating for 1-2 hours at normal temperature by using corresponding antibodies; the secondary antibody is correspondingly added with Horse Radish Peroxidase (HRP) labeled anti-mouse or anti-rabbit antibody; enhanced chemiluminescent detection reagent (TIANGEN Biotech, China) imaging.

As shown in FIG. 5A, the polymerase activity of the virus in the S69E group was lower than that of the WT group by 50%, and the polymerase activity in the S69A group was slightly higher than that of the WT group.

2) Viral RNA transcription and replication assays

293T cells were plated in 60mm dishes and transfected when 90% confluence was achieved within 24 hours; the cells were replaced with Opti-MEM medium before transfection, and the reverse genetics system 12 plasmid (pHH21-WSN-PB2, pHH21-WSN-PB1, pHH21-WSN-PA, pHH21-WSN-HA, pHH21-WSN-NP or pHH21-WSN-NP-S69A or pHH21-WSN-NP-S69E, pHH21-WSN-NA, pHH21-WSN-M, pHH21-WSN-NS, pcDNA4/TO-PB2, pcDNA4/TO-PB1, pcDNA4/TO-PA, pcDNA4/TO-NP or pcDNA4/TO-NP-S69A or pcDNA4/TO-NP-S69E, each 1. mu.g) was mixed; the procedure was as described in Lipofectamine 2000; the transfection was carried out by changing the medium to DMEM containing TPCK-treated pancreatin (final concentration: 2. mu.g/mL) at 6 hours, and the cells were collected at 50 hours after the transfection.

Extracting cellular RNA using different primers mRNA, oligo (dT); vRNA, 5 'AGCAAAAGCAGG-3'; cRNA, 5 'AGTAGAAACAAGG-3' for reverse transcription. Then, real-time quantitative PCR is carried out by using upstream and downstream primers 5'-TCTGATCCTCTCGTCATTGCAGCAA-3' and 5'-AATGACCATCGTCAACATCCACAGC-3' of the M1 gene; housekeeping gene GAPDH as controlThe upstream and downstream primers are 5'-GGTGGTCTCCTCTGACTTCAACA-3' and 5'-GTTGCTGTAGCCAAATTCGTTGT-3'; 40 cycles using the program 95 ℃ 30 seconds, 95 ℃ 5 seconds and 60 ℃ 31 seconds; for data 2^-ΔΔCTCalculating a method; 3 replicate wells were made for each sample.

As a result, as shown in FIG. 5B, it can be seen that mRNA, vRNA and cRNA were decreased in the S69E group to a different extent than in the WT group; mRNA was decreased in S69A group compared to WT and vRNA and cRNA were increased compared to WT group.

The above results indicate that the mutation NP S69E inhibits the transcription and replication of viral RNA, and the mutation NP S69A inhibits the transcription of viral RNA, enhancing viral RNA replication. NP S69E has also been shown to reduce the transcriptional activity and reduce the replication activity of viral RNA polymerase; NP S69A reduces the transcription activity of viral RNA polymerase and increases the replication activity of viral RNA polymerase.

2. Effect of NP S69 phosphorylation or dephosphorylation on NP oligomerization

NP is the major component of the vRNP complex, NP oligomerization is the basis for vRNP formation, NP interacts with components of the vRNP complex other than PA, and NP S69E affects viral polymerase activity, and NP 69 site is in the domain where NP binds to PB2 and NP binds to RNA, and NP S69E or S69A mutation is examined for NP autopolymerization considering that NP S69 phosphorylation or dephosphorylation may have an effect on NP oligomerization or NP binding to PB2, PB1 or RNA, as follows:

the plasmid FLAG-NP WT was a recombinant plasmid obtained by cloning the NP-encoding gene (SEQ ID NO: 2) into the expression vector pcDNA3-FLAG (addgene, cat. No. 20011) between KpnI and EcoRI cleavage sites.

The plasmid FLAG-NP S69A is a recombinant plasmid obtained by cloning a coding gene of NP-S69A into an expression vector pcDNA3-FLAG between KpnI and EcoRI enzyme cutting sites.

The plasmid FLAG-NP S69E is a recombinant plasmid obtained by cloning a coding gene of NP-S69E into an expression vector pcDNA3-FLAG between KpnI and EcoRI enzyme cutting sites.

The plasmid MYC-NP WT was a recombinant plasmid obtained by cloning the NP-encoding gene (SEQ ID NO: 2) between EcoRI and KpnI cleavage sites of pCMV-MYC (Clontech, Cat. No. 635689).

The plasmid MYC-NP S69A is a recombinant plasmid obtained by cloning a coding gene of NP-S69A between EcoRI and KpnI enzyme cutting sites of pCMV-MYC.

The plasmid MYC-NP S69E is a recombinant plasmid obtained by cloning a coding gene of NP-S69E between EcoRI and KpnI enzyme cutting sites of pCMV-MYC).

The above Flag-pcDNA3.0 and pCMV-MYC vectors carrying full-length NP-S69E or NP-S69A can be prepared as follows:

the Flag-pcDNA3.0 and pCMV-MYC vectors carrying full-length NP (A/WSN/1933(H1N1)) were subjected to point mutation (S69E, S69A), and each point mutation cloning was performed by a point mutation Kit (Newpen Site-Directed Mutagenesis Kit). The point mutation primers are:

S69E up：5’-gagagaatggtgctcgaggcttttgacgagaggaggaataaat-3’

S69E down：5’-cctctcgtcaaaagcctcgagcaccattctctctattgttaag-3’

S69A up：5’-gagagaatggtgctcgctgcttttgacgagaggaggaataaat-3’

S69A down：5’-cctctcgtcaaaagcagcgagcaccattctctctattgttaag-3’

the 293T cells were transfected with plasmid FLAG-NP WT or plasmid FLAG-NP S69A or plasmid FLAG-NP S69E, and plasmid MYC-NP WT or plasmid MYC-NP S69A or plasmid MYC-NP S69E.

Detecting by using a co-IP experiment; the cells were lysed with cocktail containing lysis buffer (formula above) for 30 min at 4 ℃; collecting supernatant by centrifugation at 12000rpm for 10 min; adding FLAG beads, and combining at 4 ℃ for 2-4 hours; washing with washing solution (formula of 300mM NaCl, 20mM HEPES, 1mM EDTA, 1% TritonX-100, 10% glycerol, adjusted to pH7.4) for 5 times, 10 minutes each time; centrifuging at 4500rpm for 1 min at 4 deg.C, removing supernatant, collecting precipitate and small amount of supernatant, adding loading buffer, decocting at 99 deg.C for 10 min, centrifuging at 12000rpm for 1 min, and detecting supernatant by SDS-PAGE and western blotting.

The results are shown in fig. 6, and the detected S69E or S69A mutant NP protein bands are consistent with NP WT, suggesting that S69 phosphorylation or dephosphorylation does not affect NP autopolymerization.

3. Effect of NP S69 phosphorylation or dephosphorylation on NP interaction with viral polymerase proteins

1) Phosphorylation and dephosphorylation of S69 on NP-PB 2 interaction

Detecting the influence of S69 phosphorylation and dephosphorylation on the interaction of NP and PB2, which is as follows:

the plasmid FLAG-PB2 is a recombinant plasmid obtained by cloning a coding gene (sequence 3) of PB2 into KpnI and XhoI enzyme cutting sites of an expression vector pcDNA3-FLAG (addgene, catalog number 20011).

Plasmid FLAG-PB2 and plasmid MYC-NP WT or plasmid MYC-NP-S69A or plasmid MYC-NP-S69E were transfected into 293T cells. Detection was performed in a co-IP assay, which was repeated three times.

As shown in FIG. 7, the detected NP S69A group was consistent with the NP WT group, and the detected NP-S69E protein band was significantly lighter than that of NP-S69A and NP WT, indicating that NP S69 phosphorylation (NP S69E) inhibited NP-PB 2 interaction.

2) Phosphorylation and dephosphorylation of S69 on NP-PB 1 interaction

The influence of S69 phosphorylation and dephosphorylation on NP and PB1 interaction was detected as follows:

the plasmid FLAG-PB1 is a recombinant plasmid obtained by cloning a nucleotide sequence of a coding gene (sequence 4) of PB1 into KpnI and XhoI enzyme digestion sites of an expression vector pcDNA3-FLAG (addgene, catalog number 20011).

Plasmid FLAG-PB1 and plasmid MYC-NP WT or plasmid MYC-NP S69A or plasmid MYC-NP S69E were transfected into 293T cells. Detection was performed in a co-IP assay, which was repeated three times.

As a result, as shown in FIG. 8, the detected NP S69A group coincided with the WT group; the NP S69E protein band was slightly attenuated compared to NP S69A and WT NPs, showing that phosphorylation of S69 had a slight effect on NP-PB 1 interaction.

4. Effect of NP S69 phosphorylation or dephosphorylation on NP-RNA binding

The interaction between NP WT or NP-S69E or NP-S69A and RNA was tested, because NP and RNA were bound without sequence specificity, Polyuridylic acid-Agarose beads were selected to bind NP (purified NP and its S69 mutant from prokaryotic expression and protein harvested) and a co-IP experiment was performed as follows:

His-pET30a-NP WT was a recombinant plasmid obtained by cloning the NP-encoding gene (SEQ ID NO: 2) into the KpnI and EcoRI cleavage sites of His-pET30a vector (EMD Biosciences (Novagen), cat. No. 69909-3).

His-pET30a-S69E is a recombinant plasmid obtained by cloning the coding gene of NP S69E between the KpnI and EcoRI cleavage sites of the His-pET30a vector.

His-pET30a-S69A is a recombinant plasmid obtained by cloning the coding gene of NP S69A between the KpnI and EcoRI cleavage sites of the His-pET30a vector.

The His-pET30a vector carrying the full-length NP-S69E or NP-S69A described above can be prepared as follows:

His-pET30a vector carrying full-length NP (A/WSN/1933(H1N1)) was point mutated (S69E, S69A), and each point mutation cloning was performed by a point mutation Kit (Newpen Site-Directed Mutagenesis Kit).

The point mutation primers are:

S69E up：5’-gagagaatggtgctcgaggcttttgacgagaggaggaataaat-3’

S69E down：5’-cctctcgtcaaaagcctcgagcaccattctctctattgttaag-3’

S69A up：5’-gagagaatggtgctcgctgcttttgacgagaggaggaataaat-3’

S69A down：5’-cctctcgtcaaaagcagcgagcaccattctctctattgttaag-3’

adding 1 mu g of each of the recombinant plasmids His-pET30a-NP WT, His-pET30a-S69E and His-pET30a-S69A into BL21 competent cells respectively, and placing the cells on ice for 30 minutes; heat shock is carried out for 90 seconds at the temperature of 42 ℃, and the mixture is placed on ice for 5 minutes; coating on a flat plate with corresponding resistance by using a glass rod, and culturing for 12-15 hours in a constant-temperature incubator at 37 ℃; picking a monoclonal colony from the plate, inoculating the colony in 200mL LB liquid culture medium containing corresponding resistance (the final concentration is 100 mu g/mL), placing the colony in a shaker at 37 ℃ and culturing at 200 rpm; when the culture was cultured to OD600 between 0.6-0.8, 1mL was taken as a pre-induction control, and IPTG was added to a final concentration of 0.5mM, and induced at 37 ℃ for 3 hours or on a shaker at 16 ℃ for 8 hours;

the cells were collected, centrifuged at 4000rpm at 4 ℃ for 5 minutes, the supernatant was discarded, and a Binding buffer (50 mM NaH formulation) was used₂PO₄300mM NaCl and 10mM imidazole, adjusted to pH 8.0), the cells were resuspended, sonicated at 4 ℃ and centrifuged at 10000rpm for 15 minutes at 4 ℃, and the centrifuged supernatant was applied to a 1mL nickel column (Ni sepharose)^TM) Binding for 2 hours at 4 ℃; wash buffer (formulation 50mM NaH) was used₂PO4, 300mM NaCl, 20mM imidazole, adjusted to pH 8.0) about 1L elution nickel column; 10mL of Elution buffer (formulation 50mM NaH) was used₂PO4, 300mM NaCl, 500mM imidazole, adjusted to pH 8.0) and the protein concentration was measured by BCA method to obtain NP protein at 147.5. mu.g/mL, NP S69E at 295.8. mu.g/mL, and NP S69A at 425.3. mu.g/mL.

Equal amounts of Polyuridylic acid-Agarose (Sigma-Aldrich, Cat. No. P8563) were combined with NP WT protein, NP-S69E protein, and NP-S69A protein, respectively, at 4 ℃ for half an hour for co-IP experiments, and detected with NP antibody.

As shown in FIG. 9, it can be seen that the NP S69A group or S69E group was identical to the WT group, showing that NP-S69 phosphorylation or dephosphorylation had no effect on NP-RNA binding.

5. Effect of phosphorylation or dephosphorylation of NP S69 on intracellular localization of NP

The IFAs were used to observe cell location of NP WT, NP-S69E, and NP-S69A at various time points and to count the cells as follows:

transfecting 293T cells with the recombinant plasmid Flag-NP WT or Flag-NP-S69E or Flag-NP-S69A, washing the cells for 8, 11, 24 and 35 hours after transfection by PBS for 1 time, and fixing the cells for more than 20 minutes at room temperature by 4% paraformaldehyde; washed with PBST (PBS containing 1% Triton X-100) for 10 min; binding rabbit anti-NP antibody, binding for 1 hour at 37 ℃; blocking with 4% bovine serum albumin in PBST for 1 hour; wash with PBST for 5 × 8 min; binding to TRITC-conjugated anti-rabbit IgG or FITC-conjugated anti-mouse IgG at 37 ℃ for 1 hour; PBST wash 5 × 8 min; DAPI staining for 15 min; wash with PBST for 5 × 8 min; the sections were observed using a Leica SP8 confocal laser microscope.

The results are shown in FIG. 10, (A) NP WT or mutants were transfected into 293T cells, cells were fixed at various time points after transfection, NP localization was detected using IFAs, red for NP, and blue for nucleus. (B) Recording different groups of positioning conditions of 100 cells with different visual fields, wherein N represents that the cells are mainly positioned in cell nucleuses, N + C represents that the cell nucleuses and cytoplasms are distributed, and C represents that the cells are mainly distributed in the cytoplasms; the distribution of NP-S69E in both the nucleus and cytoplasm was observed at 8 and 11 hours after transfection using IFAs, and cell counts showed that NP-S69E was significantly less distributed in the nucleus than NP WT and NP-S69A at 11 hours after transfection, whereas NP WT and NP-S69A mutants were positioned substantially similarly, predominantly in the nucleus, and NP-S69E was observed to be less distributed in the nucleus than NP WT and NP-S69A at 24 and 36 hours after transfection.

To further confirm whether phosphorylation and dephosphorylation of S69 had an effect on intracellular localization of NP, the results were examined at various time points using a nucleoplasmic separation assay,

the recombinant plasmid Flag-NP WT or Flag-NP-S69E or Flag-NP-S69A was transfected into 293T cells, and CLB solution (formulation 10mM HEPES, 10mM NaCl, 1mM KH) containing cocktail was used₂PO₄、5mM NaHCO₃、1mM CaCl₂、0.5mM MgCl₂5mM EDTA) were collected from the cells transfected for 11, 24, 35 hours; incubating on ice for 5 min; homogenizing for 50 times with a glass homogenizer; centrifuging at 7500rpm for 5 minutes at 4 ℃; the supernatant after centrifugation is a cytoplasm component and a plasma membrane component, and is precipitated into a cell nucleus component and cell debris; resuspending the centrifuged pellet with TSE solution (10 mM Tris, 300mM sucrose, 1mM EDTA) containing 0.1% Triton X-100 and cocktail, homogenizing 30 times, and centrifuging at 4 ℃ and 5000rpm for 5 minutes; resuspending the precipitate with TSE solution, washing 2 times; the final precipitate was purified nuclear fraction, dissolved in 50 μ L TSE; the whole process is ice-bath operation.

The nuclei were controlled with Lamin B1 and the cytoplasm with HSP70 using SDS-Page and western blotting assays. Protein expression in the nucleus or cytoplasm was detected using NP, Lamin B1, HSP70 antibodies, respectively.

As shown in FIG. 11, it was found that the NP-S69A group was substantially identical to the WT group, and that at 11 hours after transfection, NP-S69E was significantly reduced in the cell nucleus expression level as compared with NP WT and NP-S69A, while NP-S69E was significantly increased in the cytoplasm expression level as compared with NP WT and NP-S69A. At both 24 and 35 hours post-transfection, NP-S69E was expressed in the nucleus in less amounts than NP WT and NP-S69A. All results showed that NP S69 phosphorylation attenuated NP nuclear entry.

The effect of phosphorylation and dephosphorylation of S69 on NP- α 3 interaction was further examined.

293T cells were transfected with plasmid FLAG-import-alpha 3 (recombinant plasmid obtained by cloning the amino acid sequence of NCBI accession No. NP-002259.1 between the KpnI and XhoI cleavage sites of expression vector pcDNA 3-FLAG) and MYC-NP (WT or S69E or S69A). Detection was performed in a co-IP assay, which was repeated three times.

As shown in FIG. 12, it can be seen that the NP-S69A group was substantially identical to the WT group, and the S69E histone band was detected to be significantly lighter than those of the S69A and WT groups, indicating that NP S69 phosphorylation (NP S69E) blocked NP from interacting with import in-. alpha.3. The above results suggest that NP-S69 phosphorylation attenuates the nuclear import of NP by inhibiting NP interaction with import- α 3.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> influenza A virus nucleoprotein S69 mutant sequence, mutant and application thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 498

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Thr Lys Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met

20 25 30

Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu

50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Val Asp Gly Lys Trp Arg Arg Glu Leu Ile Leu Tyr Asp

100 105 110

Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Thr Met Val Asp

225 230 235 240

Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Phe Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Ser Ala Val Ala Ser Gly

275 280 285

Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Arg Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Lys Val

340 345 350

Val Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Glu Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg

385 390 395 400

Ala Ser Ser Gly Gln Ile Ser Ile Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Asp Arg Pro Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Leu Met

435 440 445

Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Ser Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr

485 490 495

Asp Asn

<210> 2

<211> 1497

<212> DNA

<213> Artificial sequence

<400> 2

atggcgacca aaggcaccaa acgatcttac gaacagatgg agactgatgg agaacgccag 60

aatgccactg aaatcagagc atctgtcgga aaaatgattg atggaattgg acgattctac 120

atccaaatgt gcaccgaact taaactcagt gattatgagg gacggctgat tcagaacagc 180

ttaacaatag agagaatggt gctctctgct tttgacgaga ggaggaataa atatctagaa 240

gaacatccca gtgcggggaa agatcctaag aaaactggag gacctatata caggagagta 300

gatggaaagt ggaggagaga actcatcctt tatgacaaag aagaaataag acgaatctgg 360

cgccaagcta ataatggtga cgatgcaacg gctggtctga ctcacatgat gatctggcac 420

tccaatttga atgatgcaac ttaccagagg acaagagctc ttgttcgcac aggaatggat 480

cccaggatgt gctcactgat gcagggttca accctcccta ggaggtctgg ggccgcaggt 540

gctgcagtca aaggagttgg aacaatggtg atggaattga tcagaatgat caaacgtggg 600

atcaatgatc ggaacttctg gaggggtgag aatggacgga gaacaaggat tgcttatgaa 660

agaatgtgca acattctcaa agggaaattt caaacagctg cacaaagaac aatggtggat 720

caagtgagag agagccggaa tccaggaaat gctgagttcg aagatctcat ctttttagca 780

cggtctgcac tcatattgag agggtcagtt gctcacaagt cctgcctgcc tgcctgtgtg 840

tatggatctg ccgtagccag tggatacgac tttgaaagag agggatactc tctagtcgga 900

atagaccctt tcagactgct tcaaaacagc caagtataca gcctaatcag accaaatgag 960

aatccagcac acaagagtca actggtgtgg atggcatgcc attctgctgc atttgaagat 1020

ctaagagtat caagcttcat cagagggacg aaagtggtcc caagagggaa gctttccact 1080

agaggagttc aaattgcttc caatgaaaac atggagacta tggaatcaag tacccttgaa 1140

ctgagaagca gatactgggc cataaggacc agaagtggag ggaacaccaa tcaacagagg 1200

gcttcctcgg gccaaatcag catacaacct acgttctcag tacagagaaa tctccctttt 1260

gacagaccaa ccattatggc agcattcact gggaatacag aggggagaac atctgacatg 1320

agaaccgaaa tcataaggct gatggaaagt gcaagaccag aagatgtgtc tttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcaacgagcc cgatcgtgcc ctcctttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtacga caattaa 1497

<210> 3

<211> 2280

<212> DNA

<213> Artificial sequence

<400> 3

atggaaagaa taaaagaact aaggaatcta atgtcgcagt ctcgcactcg cgagatactc 60

acaaaaacca ccgtggacca tatggccata atcaagaagt acacatcagg aagacaggag 120

aagaacccag cacttaggat gaaatggatg atggcaatga aatatccaat tacagcagac 180

aagaggataa cggaaatgat tcctgagaga aatgagcagg gacaaacttt atggagtaaa 240

atgaatgacg ccggatcaga ccgagtgatg gtatcacctc tggctgtgac atggtggaat 300

aggaatggac cagtgacaag tacagttcat tatccaaaaa tctacaaaac ttattttgaa 360

aaagtcgaaa ggttaaaaca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420

atacgtcgaa gagttgacat aaatcctggt catgcagatc tcagtgccaa agaggcacag 480

gatgtaatca tggaagttgt tttccctaac gaagtgggag ccaggatact aacatcggaa 540

tcgcaactaa cgacaaccaa agagaagaaa gaagaactcc agggttgcaa aatttctcct 600

ctgatggtgg catacatgtt ggagagagaa ctggtccgca aaacgagatt cctcccagtg 660

gctggtggaa caagcagtgt gtacattgaa gtgttgcatt tgacccaagg aacatgctgg 720

gaacagatgt acactccagg aggggaggcg aggaatgatg atgttgatca aagcttaatt 780

attgctgcta gaaacatagt aagaagagcc acagtatcag cagatccact agcatcttta 840

ttggagatgt gccacagcac gcagattggt ggagtaagga tggtaaacat ccttaggcag 900

aacccaacag aagagcaagc cgtggatatt tgcaaggctg caatgggact gagaattagc 960

tcatccttca gttttggtgg attcacattt aagagaacaa gcggatcatc agtcaagaga 1020

gaggaagagg tgcttacggg caatcttcag acattgaaga taagagtgca tgagggatat 1080

gaagagttca caatggttgg gagaagagca acagctatac tcagaaaagc aaccaggaga 1140

ttgattcagc tgatagtgag tgggagagac gaacagtcga ttgccgaagc aataattgtg 1200

gccatggtat tttcacaaga ggattgtatg ataaaagcag ttagaggtga cctgaatttc 1260

gtcaataggg cgaatcagcg attgaatccc atgcaccaac ttttgagaca ttttcagaag 1320

gatgcaaagg tgctctttca aaattgggga attgaatcca tcgacaatgt gatgggaatg 1380

atcgggatat tgcccgacat gactccaagc accgagatgt caatgagagg agtgagaatc 1440

agcaaaatgg gggtagatga gtattccagc gcggagaaga tagtggtgag cattgaccgt 1500

tttttgagag ttagggacca acgtgggaat gtactactgt ctcccgagga ggtcagtgaa 1560

acacagggaa cagagaaact gacaataact tactcatcgt caatgatgtg ggagattaat 1620

ggtcctgaat cagtgttggt caatacctat cagtggatca tcagaaactg ggaaactgtt 1680

aaaattcagt ggtcccagaa tcctacaatg ctgtacaata aaatggaatt tgagccattt 1740

cagtctttag ttccaaaggc cgttagaggc caatacagtg ggtttgtgag aactctgttc 1800

caacaaatga gggatgtgct tgggacattt gataccgctc agataataaa acttcttccc 1860

ttcgcagccg ctccaccaaa gcaaagtgga atgcagttct cctcattgac tataaatgtg 1920

aggggatcag gaatgagaat acttgtaagg ggcaattctc cagtattcaa ctacaacaag 1980

accactaaaa gactcacagt tctcggaaag gatgctggcc ctttaactga agacccagat 2040

gaaggcacag ctggagttga gtccgcagtt ctgagaggat tcctcattct gggcaaagaa 2100

gacaggagat atggaccagc attaagcata aatgaactga gcaaccttgc gaaaggagag 2160

aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg gaaacggaac 2220

tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280

<210> 4

<211> 2274

<212> DNA

<213> Artificial sequence

<400> 4

atggatgtca atccgacttt acttttctta aaagtgccag cacaaaatgc tataagcaca 60

actttccctt atactggaga ccctccttac agccatggga caggaacagg atacaccatg 120

gatactgtca acaggacaca tcagtactca gaaaggggaa gatggacaac aaacaccgaa 180

actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt 240

ggttatgccc aaacagattg tgtattggaa gcaatggcct tccttgagga atcccatcct 300

ggtatctttg agacctcgtg tcttgaaacg atggaggttg ttcagcaaac acgagtggac 360

aagctgacac aaggccgaca gacctatgac tggactctaa ataggaacca gcctgctgca 420

acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgaatcc 480

ggaaggctca tagacttcct taaggatgta atggagtcaa tgaacaaaga agaaatggag 540

atcacaactc attttcagag aaagagacga gtgagagaca atatgactaa gaaaatggtg 600

acacagagaa caataggtaa aaggaagcag agattgaaca aaaggagtta tctaattagg 660

gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca 720

attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actagcaagg 780

agtatatgtg agaaacttga acaatcagga ttgccagttg gaggcaatga gaagaaagca 840

aagttggcaa atgttgtaag gaagatgatg accaattctc aggacactga aatttctttc 900

accatcactg gagataacac caaatggaac gaaaatcaga accctcggat gtttttggcc 960

atgatcacat atataaccag aaatcagccc gaatggttca gaaatgttct aagtattgct 1020

ccaataatgt tctcaaacaa aatggcgaga ctgggaaagg ggtacatgtt tgagagcaag 1080

agtattaaaa ttagaactca aatacctgca gaaatgctag caagcatcga tttgaaatac 1140

ttcaatgatt caactagaaa gaagattgaa aaaatccggc cgctcttaat agatgggact 1200

gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagtac tgtattaggc 1260

gtctccatcc tgaatcttgg acaaaagaga cacaccaaga ctacttactg gtgggatggt 1320

cttcaatctt ctgatgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa 1380

gccggagtca acaggtttta tcgaacctgt aagctacttg gaattaatat gagcaagaaa 1440

aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatggg 1500

tttgttgcca atttcagcat ggagcttccc agctttgggg tgtctgggat caacgagtct 1560

gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt 1620

ccagcaaccg ctcaaatggc ccttcagctg ttcatcaaag attacaggta cacgtaccgg 1680

tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg 1740

gagcaaaccc attccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac 1800

attagaaatc tccacattcc tgaagtctgc ttgaaatggg aattaatgga tgaggattac 1860

caggggcgtt tatgcaaccc actgaaccca tttgtcaacc ataaagacat tgaatcagtg 1920

aacaatgcag tgataatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt 1980

gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagccaaaga 2040

ggaatacttg aagatgaaca aatgtaccaa aagtgctgca acttatttga aaaattcttc 2100

cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc 2160

agagcccgaa ttgatgcacg aattgatttc gaatctggaa ggataaagaa agaggagttc 2220

actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274

Claims (10)

1. An influenza virus nucleoprotein mutant, which is a protein obtained by mutating serine S at the 69 th site of an influenza virus nucleoprotein amino acid sequence into glutamic acid E or alanine A and keeping other amino acid residues unchanged;

the influenza virus nucleoprotein is derived from influenza A virus.

2. The mutant according to claim 1, characterized in that: the amino acid sequence of the influenza virus nucleoprotein is a sequence 1 in a sequence table.

3. A nucleotide encoding the mutant of claim 1 or 2.

4. A recombinant vector comprising the nucleotide of claim 3.

5. Use of the mutant of claim 1 or 2 or the nucleotide of claim 3 or the recombinant vector of claim 4 in any one of the following 1) -6): the mutant is a protein obtained by mutating serine S at the 69 th site of an amino acid sequence of nucleoprotein of influenza virus into glutamic acid E and keeping other amino acid residues unchanged:

1) preparing an influenza virus having reduced viral virulence;

2) preparing an influenza virus having reduced viral polymerase activity;

3) preparing an influenza virus with reduced viral RNA transcription and/or reduced viral RNA replication;

4) preparing an influenza virus with reduced NP binding to PB 2;

5) preparing an influenza virus with reduced NP nuclear entry;

6) preparing an influenza virus that inhibits the interaction of NP with import-alpha 3;

the influenza virus nucleoprotein is derived from influenza A virus;

the influenza virus is influenza A virus.

6. Use of the mutant of claim 1 or 2 or the nucleotide of claim 3 or the recombinant vector of claim 4 in a) or b) as follows: the mutant is a protein obtained by mutating serine S at the 69 th site of an amino acid sequence of nucleoprotein of influenza virus into alanine A and remaining amino acid residues are unchanged:

a) preparing an influenza virus having reduced viral virulence;

b) preparing an influenza virus with reduced viral RNA transcription and/or increased viral RNA replication;

the influenza virus nucleoprotein is derived from influenza A virus;

the influenza virus is influenza A virus.

7. A recombinant influenza virus containing nucleotides expressing the mutant of claim 1 or 2;

the influenza virus is influenza A virus.

8. Use of the recombinant influenza virus of claim 7 in the preparation of an influenza vaccine.

9. An influenza vaccine comprising the mutant of claim 1 or 2 or the nucleotide of claim 3 or the recombinant vector of claim 4 or the recombinant virus of claim 7.

10. A method of reducing the pathogenicity of an influenza virus, 1) or 2) as follows:

1) the method comprises the following steps: (ii) non-phosphorylation or dephosphorylation, or sustained phosphorylation of S at position 69 of said influenza virus nucleoprotein;

2) the method comprises the following steps: mutating the 69 th S of the influenza virus nucleoprotein to A or E;

the influenza virus nucleoprotein is derived from influenza A virus;

the influenza virus is influenza A virus.

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