CN114057859A - Antiviral target gene of coronavirus including SARS-CoV and SARS-CoV-2 and its application - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to an antiviral target gene of coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV and its application. The invention provides a combination of nucleic acids, the corresponding genes are selected from the following target genes: VPS29, VPS35, RAB7A, CCZ1, CCZ1B, C18orf8, C16orf62, CCDC22, CCDC93, COMMD2, COMMD3, COMMD3-BMI1, COMMD4, COMMD5, COMMD7, COMMD8, COMMD10, KIAA0196, KIAA1033, CCDC53, ARPC4, ACTR2, ACTR3, NPC1, NPC2, WDR81, WDR91, TFE3, SNX27, which have great potential as antiviral drug targets.

Description

Antiviral target gene of coronavirus including SARS-CoV and SARS-CoV-2 and its application

Technical Field

The invention belongs to the field of biological medicine, relates to an antiviral target gene of coronavirus, in particular to an antiviral target gene of coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV and application thereof, in particular to new application of a plurality of host genes in SARS-CoV, SARS-CoV-2, MERS-CoV, SARS-like virus and other antiviral targets of coronavirus.

Background

CRISPR-Cas9(clustered regulated short linked templates/CRI SPR-associated protein 9) is a genome-targeted modification technology. Target sites are recognized through a section of RNA, Cas9 is guided to be positioned at target genes, and DNA double-strand cutting is carried out at about 3bp upstream of PAM.

When the repair template is not available, the DSBs are reconnected through non-homologous end connection, so that insertion or mutation deletion is easy to generate, gene function is lost, and gene knock-down or knock-out is realized.

Coronaviruses have been isolated in 1965, but their knowledge is currently quite limited. Neutralizing antibodies were detected in 50% of children aged 5-9 years, and in 70% of adults, were positive. Rhinoviruses were discovered in the 50's of the 20 th century. Rhinoviruses were first found to be associated with colds, but only about 50% of colds are caused by rhinoviruses. In 1965, Tyrrell and Bynoe used the ciliated tracheal tissue of embryos to first culture coronaviruses, which were known as coronaviruses (Coronaviridae) because coronas were visualized around coronas under an electron microscope. In 1975, the committee on virus nomenclature formally named the coronaviridae.

The coronavirus particles are irregular in shape, about 60-220nm in diameter, and are wrapped by fat membrane. There are three glycoproteins on the membrane surface: spike glycoprotein (S, Spike Protein, which is the receptor binding site, cytolytic and major antigenic site); small Envelope glycoprotein (E, Envelope Protein, smaller, Envelope-bound Protein); membrane glycoproteins (M, Membrane proteins, responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope). A few classes also have hemagglutinin-esterases (HE proteins). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, has the length of 27-31kb, is the longest RNA nucleic acid chain in RNA virus, and has important structural characteristics specific to positive-strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. This structure is very similar to eukaryotic mRNA and is an important structural basis for the genomic RNA itself to function as a translation template, and the RNA-DNA-RNA transcription process is omitted. The rate of recombination between the RNA of coronaviruses and RNA is very high, and it is this high rate of recombination that causes the virus to mutate. After recombination, the RNA sequence is changed, and the amino acid sequence encoded by the nucleic acid is also changed, and the protein composed of amino acids is changed, so that the antigenicity is changed. The result of the change of antigenicity is that the original vaccine is ineffective and the immunity fails.

RNA polymerase (Viral RNA polymerase) required for RNA virus replication is not present in the mature coronavirus particles, and after entering a host cell, Viral RNA polymerase is expressed directly by using Viral genomic RNA as a translation template. This enzyme is then used to complete the transcription and synthesis of sub-genomic RNA (sub-genomic RNA), the synthesis of mRNAs of various structural proteins, and the replication of viral genomic RNA. The mature mRNA of each structural protein of coronavirus is synthesized, there is no post-transcriptional modification and cleavage process, but directly through RNA polymerase and some transcription factors, in a 'discontinuous transcription' mechanism, by recognizing specific TRS), the whole components of a mature mRNA are obtained by one-time transcription selectively from the negative strand RNA. After the replication of structural proteins and genomic RNA is completed, new coronavirus particles will be assembled (assembly) at the endoplasmic reticulum of the host cell and secreted out of the cell by the golgi apparatus, completing its life cycle.

Of the 7 human coronaviruses known, HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43 are common to the common cold and respiratory tract infections of humans and are less pathogenic and infectious. The new SARS-CoV, MERS-CoV and SARS-CoV-2 belong to beta type coronavirus, which can cause fatal respiratory system diseases, especially SARS-CoV-2 is more contagious. It has been shown that the novel coronavirus SARS-CoV-2 and HCoV-NL63, SARS-CoV all enter cells using the same cellular receptor ACE2 (angiotensin converting enzyme 2), suggesting that they have similar infection target cells and tissues. However, the pathogenicity of the three is quite obvious. Common human coronaviruses, including 229E, NL63, OC43 and HKU1, often cause mild or moderate upper respiratory tract diseases, such as the common cold. The symptoms mainly comprise rhinorrhea, headache, cough, sore throat, fever and the like, sometimes cause pneumonia, bronchitis and other lower respiratory diseases, and are common in patients with cardiopulmonary diseases, people with low immunity, infants and the elderly. MERS-CoV and SARS-CoV often cause more severe symptoms. MERS symptoms typically include fever, cough and shortness of breath, even with the development of pneumonia, with a mortality rate of about 34.4%. SARS symptoms usually include fever, chills and body aches, even with the development of pneumonia, with a mortality rate of about 9.6%, while SARS-CoV-2 is slow to develop, low fever or even asymptomatic, recurrent episodes, long latency, or can suddenly cause severe acute respiratory disease.

Disclosure of Invention

The invention aims to solve the technical problem of providing a host cell gene target for resisting coronavirus infection and a related drug target combination.

Another technical problem to be solved by the invention is to provide the above-mentioned host cell gene target against coronavirus infection and related drug target combination.

Specifically, the invention provides an antiviral target gene of coronavirus, and specifically relates to an antiviral target gene of coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV and application thereof.

The experimental data of the invention show that the genes obtained by screening CRISPR-Cas9 by using a passage mutant strain SARS-CoV-2(Sdel, S protein 683-689aa deletion) comprise VPS29, VPS35, RAB7A, CCZ1, CCZ1B, C18orf8, C16orf62, CCDC22, CCDC93, COMMD2, COMMD3, COMMD3-BMI1, COMMD4, COMMD5, COMMD7, COMMD8, COMMD10, KIAA0196, KIAA 3, CCDC53, ARPC4, ACTR2, ACTR3, NPC1, NPC2, WDR81, WDR91 and TFE 3. The S protein is not mutant, i.e., SARS-CoV-2(Sfull) in the present invention, to be distinguished from the SARS-CoV-2(Sdel) mutant. SARS-CoV-2 in the present invention includes SARS-CoV-2(Sfull) and SARS-CoV-2 (Sdel). Through SARS-CoV-2 virus infection verification, the virus infection can be obviously reduced by knocking out the corresponding gene. In addition, it is verified that the gene knockout can reduce SARS-CoV-2 infection by using SARS-CoV-2 pseudovirus. Using SARS-CoV pseudovirus verification, the gene knockout can reduce SARS-CoV infection. Further mechanism research shows that the membrane transport of ACE2 is down regulated after partial gene (COMMD3, CCDC22, VPS29, VPS35 and CCDC53) knockout, so that the combination of virus and a cell surface receptor ACE2 is influenced, and virus infection is reduced. The present invention has been completed on this basis.

The invention provides a nucleic acid combination, wherein the nucleic acid comprises DNA or RNA.

The gene corresponding to the nucleic acid is selected from the following target genes or open reading frames thereof: VPS29, VPS35, RAB7A, CCZ1, CCZ1B, C18orf8, C16orf62, CCDC22, CCDC93, COMMD2, COMMD3, COMMD3-BMI1, COMMD4, COMMD5, COMMD7, COMMD8, COMMD10, KIAA0196, KIAA1033, CCDC53, ARPC4, ACTR2, ACTR3, NPC1, NPC2, WDR81, WDR91, TFE 3.

In the present invention, an Open Reading Frame (ORF) is a sequence in a DNA sequence that has the potential to encode a protein, typically a polypeptide sequence corresponding to the expression product of the gene, beginning with a start codon and ending with a stop codon.

Preferably, the target gene or active fragment thereof comprises the coding sequence of COMMD3, CCDC22, VPS29, VPS35, CCDC 53.

The active fragment can be a nucleic acid fragment closely related to coronavirus infection besides a coding sequence of a gene expression product.

The invention provides a combination of polypeptides, wherein the polypeptides correspond to the following genes or gene fragments thereof:

VPS29，VPS35，RAB7A，CCZ1，CCZ1B，C18orf8，C16orf62，CCDC22，CCDC93，COMMD2，COMMD3，COMMD3-BMI1，COMMD4，COMMD5，COMMD7，COMMD8，COMMD10，KIAA0196，KIAA1033，CCDC53，ARPC4，ACTR2，ACTR3，NPC1，NPC2，WDR81，WDR91，TFE3。

the present invention also provides a detection kit comprising a detection reagent comprising the following target genes or active fragments thereof:

COMMD3, CCDC22, VPS29, VPS35 or CCDC 53.

The detection reagent includes, but is not limited to, a nucleic acid, an amino acid, a compound, or a conjugate that recognizes the target gene.

On the other hand, the invention provides the application of the nucleic acid combination in preparing the medicine for preventing and treating the coronavirus.

The nucleic acid can be used as a drug target for preventing and treating coronavirus.

Wherein, the medicine for preventing and treating the coronavirus can be a medicine for inhibiting the invasion, replication or budding of the coronavirus.

Preferably, the nucleic acid combination is used for screening or determining the activity of a drug candidate for controlling coronavirus. Or the nucleic acid combination is used for preparing active ingredients of the medicine for preventing and treating the coronavirus.

In the present invention, the term "coronavirus" includes viruses of the genus coronavirus such as SARS-CoV, SARS-CoV-2, MERS-CoV and the like.

In the present invention, the term "host cell" is a eukaryotic cell, such as A549, Calu-3, Vero, etc.

In the present invention, the term "list gene" means VPS29, VPS35, RAB7A, CCZ1, CCZ1B, C18orf8, C16orf62, CCDC22, CCDC93, COMMD2, COMMD3, COMMD3-BMI1, COMMD4, COMMD5, COMMD7, COMMD8, COMMD10, KIAA0196, KIAA1033, CCDC53, ARPC4, ACTR2, ACTR3, NPC1, NPC2, WDR81, WDR91, TFE3, SNX 27.

The list genes of the invention can be used as targets for screening substances or methods for inhibiting the activity of the list genes. The candidate compound may be a nucleic acid, an amino acid, or a small molecule compound. The target gene may be a protein of the list gene, an mRNA of the list gene, an expression product of the list gene, or a specific fragment of the three genes, in addition to the list gene. The "specific fragment" refers to a fragment that can distinguish the tabulated gene of the present invention from other genes.

The invention also provides the application of the genes in preparing the anti-coronavirus infection medicines, and the list genes are medicine targets for screening the anti-coronavirus infection medicines.

Wherein, the anti-coronavirus drug is a drug for inhibiting the invasion, replication, budding and other processes of coronavirus. The drug targeting form comprises but is not limited to nucleic acid, amino acid, compound, various carriers and various presentation modes.

The invention also provides a plurality of drug combinations for resisting coronavirus drug infection, wherein the drug combinations are based on a plurality of antiviral targets and comprise but not limited to drug types and presentation modes. These combinations of drugs can reduce coronavirus infection or alleviate pathological symptoms.

By using the list gene of the present invention, substances such as receptors, inhibitors or antagonists, etc. interacting therewith can be screened out by various conventional screening methods. These substances which interact with or inhibit the activity of the genes listed may be nucleic acids, amino acids, compounds, etc.

The invention obtains a plurality of genes, and obviously reduces virus infection after the genes are proved to be knocked down, so the genes can be used as targets for designing antiviral drugs. Thus, the invention provides multiple antiviral drug targets. The invention uses CRISPR-Cas9 technology to obtain the gene which can be used for SARS-CoV, SARS-CoV-2, MERS-CoV, SARS-like virus and other coronavirus antivirus medicine target point design. The infection capacity of coronavirus on the cells with the gene knockdown expression is remarkably inhibited, so that the genes have great potential as antiviral drug targets.

Drawings

Fig. 1 shows a list of genes screened by CRISPR-Cas 9.

FIG. 2 shows the effect on SARS-CoV-2(Sdel) infected cells after gene knockdown.

FIG. 3 shows the effect on SARS-CoV-2(Sfull) infected cells after gene knock-down.

FIG. 4 shows the effect on SARS-CoV-2(Sdel) pseudovirus infected cells after gene knockdown.

FIG. 5 shows the effect on SARS-CoV pseudovirus infected cells after gene knockdown.

FIG. 6 shows the effect on MERS-CoV pseudovirus infected cells after gene knockdown.

FIG. 7 shows the effect on SARS-CoV-2 binding and internalization following corresponding gene knockdown.

FIG. 8 shows the effect on SARS-CoV-2 infection following knockdown of other transporters.

FIG. 9 shows the effect on the cell membrane localization of ACE2 after knockdown of expression of the corresponding gene.

Figure 10 shows that a decrease in localization on ACE2 cell membranes affects binding to S1 protein or ACE2 antibody.

FIG. 11 shows the growth curve of SARS-CoV-2 on CCDC53 knockdown Calu-3.

FIG. 12 shows the effect of NPC inhibitory drugs Imipramine and U18666A on SARS-CoV infection

Detailed Description

EXAMPLE 1 Listing validation of viral infection efficiency on Gene knockdown cells

1.1 obtaining of Listed Gene knockdown cells

(1) And (3) designing and synthesizing sgRNA of the target list genes, connecting the sgRNA to plentiCRISPRV2-Cas9-puromycin plasmid, obtaining the correct plasmid through sequencing, co-transfecting 293T with pMD2.G and psPAX2, and harvesting lentivirus after 48 h.

(2) Adding lentivirus of the tabulated genes into A549-ACE2 cells, and adding puromycin drug sieve after 24 hours. And 4 days later, the medicine sieving is finished. Cells with a reduced list of genes were obtained.

(3) Adding lentiviruses of the genes in the list into A549-ACE2-CD26 cells, and adding hygromycin drug sieves after 24 hours. And about 6 days, finishing the medicine sieving. Cells with a reduced list of genes were obtained.

1.2 verification of Virus infection efficiency

(1) Inoculating the cells with the list genes knocked down into a 96-well plate 12h in advance, and adding corresponding viruses when the cell density reaches 80-90% to perform virus infection verification.

(2) After 24h of virus addition, the supernatant was removed and stained after virus inactivation.

(3) And (5) photographing, analyzing and counting by using a high content instrument to obtain the infection efficiency of the virus.

1.3 verification of infection efficiency by pseudovirus

(1) Inoculating the cells with the list gene knocked down into a 96-well plate 12h in advance, adding pseudovirus (containing luciferase reporter gene) when the cell density reaches 80-90%, and carrying out virus infection verification

(2) After 72h of virus addition, an equal volume of cell lysate mixture (containing luciferase substrate) was added and the fluorescence intensity values of each well were measured.

The results are shown in FIGS. 2, 3, 4, 5, 6, and compared to the control group (ctrl-sgRNA does not target a specific gene), the SARS-CoV-2(Sdel) infectivity was significantly reduced after the knockdown of the list genes, and a consistent phenotype was also seen on SARS-CoV pseudoviruses. In addition, partial list gene knockdown also significantly affected infection with SARS-CoV-2 (Sfull). In addition, MERS-CoV pseudovirus infected cells can be obviously influenced after gene knockdown. In conclusion, the list gene obtained by screening the CRISPR-Cas9 can be used as a target point for designing medicines for resisting coronavirus such as SARS-CoV, SARS-CoV-2, MRES-CoV and the like.

Example 2 Listing the Effect of Gene knockout on ACE2 expression

(1) Obtaining monoclonal cells: and diluting the list of the cells with the knocked-down genes to enable 0.5 cell per well, and selecting appropriate cells after two weeks to perform Western blotting verification to obtain a target gene knocked-down cell line.

(2) Virus binding experiments: inoculating the list gene knockout cells into a 24-well plate 24h in advance, placing on ice for 10min, adding precooled SARS-CoV-2, and incubating for 45 min. Washed 5 times with PBS, cells were lysed with TRIzol and harvested. Virus internalization assay: on the basis of the virus binding experiment, after rinsing with PBS for 5 times, 2% of culture medium is added, the mixture is incubated at 37 ℃ for 45min, the mixture is placed on ice and then washed with PBS once, proteinase K is added, the mixture is incubated for 45min, the PBS is rinsed for 3 times, and TRIzol is added to lyse cells and collect the cells. Cell lysis was used for RNA extraction, qRT-PCR to detect copy amount of N protein, GAPDH was used as internal control.

(3) Inoculating cells with list gene knockout to a 24-hole plate 24h in advance, placing on ice, adding biotin to mark cell surface membrane proteins, after 30 min, rinsing for 3 times by PBS, adding cell lysate (containing protease inhibitor) to crack cells, obtaining the cell membrane proteins by a biotin-streptavidin method, and detecting the expression condition of ACE2 on the cell membrane by Western blotting.

(4) The expression of ACE2 on the surface of the list of gene knocked out cells was tested using anti-ACE 2 antibodies. The binding ability of the antigen antibody is utilized, and then the corresponding antibody containing the fluorescent group is combined with the Fc end of the anti-ACE 2 antibody. The binding between cell surface ACE2 and anti-ACE 2 antibody was detected by flow.

(5) The binding ability of ACE2 to coronavirus spike protein on the surface of the knockdown cell was further verified. The binding condition of ACE2 and S1 on the cell surface is detected by flow by utilizing the binding capacity of S1 and ACE2 and then combining the corresponding antibody containing a fluorescent group with S1 protein.

The results are shown in FIGS. 7, 8, 9 and 10, and the binding and internalization of SARS-CoV-2 are significantly inhibited in COMMD3, VPS29 and CCDC53 knockout cells. SARS-CoV-2 infection was significantly inhibited on SNX27 knockdown cell lines. The expression level of ACE2 on the cell membrane surface of knockout cells of COMMD3, CCDC22, VPS29, VPS35 and CCDC53 is remarkably reduced (P <0.05), and the combination level of the ACE2 with S1 protein and anti-ACE 2 antibody is reduced, thereby disclosing the mechanism of part of list genes in anti-coronavirus (particularly SARS-CoV and SARS-CoV-2) infection.

Example 3 Effect of CCDC53 knockdown on the SARS-CoV-2 growth Curve

(1) Cells were seeded 24h in advance into 96-well plates, SARS-CoV-2 was added, and cell culture supernatants were harvested every 24 h.

(2) Vero cells are inoculated 24h in advance, and culture supernatant containing viruses which is diluted in proportion is added and incubated for 2 h. Methyl cellulose was added. After 48h, fix with 4% paraformaldehyde for 1 h. The membrane was ruptured with 0.2% Triton X-100 for 1 h. Primary antibody against SARS-CoV-2N was added and left overnight at 4 ℃. And (4) incubating the mixture for 2h at room temperature by using an HRP-labeled secondary antibody, adding a substrate for color development, and calculating the number of spots.

As a result, as shown in FIG. 11, the proliferation of SARS-CoV-2 was significantly inhibited on CAUu-3 knocked down by CCDC53 (P < 0.05).

EXAMPLE 4 Effect of NPC-inhibiting drugs on SARS-CoV-2 infected host

4.1 in vitro experiments

(1) Cells were seeded into 96-well plates and 10, 20, 40 μ M Imipramine was added 12h, 2h before and 2h after virus infection. 24h after virus infection, the supernatant was removed, fixed and inactivated, immunofluorescent staining was performed for the viral nucleocapsid protein, and the proportion of cells with fluorescence in each well to total cells was calculated.

(2) Cells were seeded into 96-well plates and 5, 10, 20 μ M U18666A was added 12h, 2h before and 2h after virus infection. 24h after virus infection, the supernatant was removed, fixed and inactivated, immunofluorescent staining was performed for the viral nucleocapsid protein, and the proportion of cells with fluorescence in each well to total cells was calculated.

(3) Cells were seeded into 96-well plates and Imipramine and U18666A of different solubilities as described above were added and Cell-Titer Glo assay kit was used to measure Cell viability after 48 h.

4.2 in vivo experiments

(1) The hamsters were challenged 24h after intraperitoneal injection into different hamsters at doses of 2mg/kg U18666A and 20mg/kg Imipramine. The injection of the same dose of U18666A or Imipramine was then continued daily.

(2) The hamsters of the experimental group and the control group were continuously observed and the body weight was measured.

As shown in FIG. 12, in vitro experiments show that Imipramine can inhibit SARS-CoV-2 infection, especially SARS-CoV-2(Sdel), and the phenotype is more obvious. U18666A also inhibited SARS-CoV-2 infection, with U18666A added 12h earlier being the most pronounced SARS-CoV-2(Sfull) phenotype. In addition, the addition of U18666A before and after SARS-CoV-2(Sdel) infection can significantly affect the virus infection. The in vivo experiment results show that the weight of the hamsters injected with U18666A is maintained stably, and the difference is significant compared with the control group, which indicates that U18666A has a certain protective effect in inhibiting the hamsters infected with SARS-CoV-2.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (9)

1. A combination of nucleic acids, wherein said nucleic acids comprise DNA or RNA; the gene corresponding to the nucleic acid is selected from the following target genes or open reading frames thereof:

VPS29，VPS35，RAB7A，CCZ1，CCZ1B，C18orf8，C16orf62，CCDC22，CCDC93，COMMD2，COMMD3，COMMD3-BMI1，COMMD4，COMMD5，COMMD7，COMMD8，COMMD10，KIAA0196，KIAA1033，CCDC53，ARPC4，ACTR2，ACTR3，NPC1，NPC2，WDR81，WDR91，TFE3,SNX27。

2. the nucleic acid combination of claim 1, wherein the target gene comprises:

COMMD3,CCDC22,VPS29,VPS35,CCDC53。

3. a combination of polypeptides which are the expression products of the following genes or gene fragments thereof:

VPS29，VPS35，RAB7A，CCZ1，CCZ1B，C18orf8，C16orf62，CCDC22，CCDC93，COMMD2，COMMD3，COMMD3-BMI1，COMMD4，COMMD5，COMMD7，COMMD8，COMMD10，KIAA0196，KIAA1033，CCDC53，ARPC4，ACTR2，ACTR3，NPC1，NPC2，WDR81，WDR91，TFE3,SNX27。

4. a detection kit comprising a detection reagent comprising the following target genes or active fragments thereof:

COMMD3, CCDC22, VPS29, VPS35 or CCDC 53.

5. The test kit of claim 4, wherein the detection reagent comprises but is not limited to a nucleic acid, an amino acid, a compound, or a conjugate that recognizes the target gene.

6. Use of the nucleic acid combination according to claim 1 for the preparation of a medicament for the control of coronaviruses.

7. The use of claim 6, wherein the nucleic acid is a drug target in a medicament for the control of coronaviruses.

8. The use of claim 6, wherein the agent for controlling coronavirus is an agent that inhibits coronavirus entry, replication or budding.

9. Use of the nucleic acid combination of claim 1 for the preparation of a candidate pharmaceutical active agent for screening or assaying for control of coronavirus.

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