Novel coronavirus infection restrictive factor and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a novel coronavirus infection restrictive factor and application thereof. The invention also discloses a method for inhibiting the replication of the coronavirus, which inhibits the replication of the coronavirus by up-regulating the expression level of the VHL protein or the N-terminal active fragment of the VHL protein in the host cell. The NCBI (National Center of Biotechnology Information) login number of the VHL protein is NP000542.1, and the amino acid sequence of the active fragment at the N end of the VHL protein is MPRRAENWDEAEVGAEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR. The amino acid sequence of the active fragment at the N end of the VHL protein is shown in the description. According to the novel coronavirus infection restrictive factor and the application thereof, the replication of coronavirus can be inhibited by up-regulating the expression level of the VHL protein or the N-terminal active fragment of the VHL protein in a host cell, so that the novel coronavirus infection restrictive factor is used for preparing an antiviral pharmaceutical preparation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel coronavirus infection-restricting factor and its applications. Background Technology

[0002] Existing drugs and vaccines against novel coronavirus infection mainly target viral proteins (such as Spike protein, 3CL protease, etc.), but the virus can easily escape through mutation; host-targeted therapies mostly focus on immune-related pathways (such as RLR, TLR, cGAS-STING pathway, etc.), lacking exploration of the antiviral function of intrinsic cellular limiting factors.

[0003] The VHL gene (NCBI Gene ID: 7428; UniProt ID: P40337) is a classic tumor suppressor gene that regulates hypoxia response by ubiquitinizing and degrading HIF-1α. The VHL protein it encodes is mainly responsible for regulating cell growth and division, and initiating apoptosis (programmed cell death) upon DNA damage, thereby preventing tumor formation.

[0004] Traditional research on tumor suppressor genes (such as VHL) has focused on tumor regulation (such as the HIF-1α degradation pathway), and their antiviral functions have not been revealed; at the same time, there is a lack of high-depth proteomics methods to systematically screen the dynamic changes of host factors in viral infection. Summary of the Invention

[0005] The purpose of this invention is to provide a novel coronavirus infection-restricting factor and its application. By upregulating the expression level of VHL protein or the N-terminal active fragment of VHL protein in host cells, it can inhibit coronavirus replication, thereby being used to prepare antiviral drug formulations.

[0006] To achieve the above objectives, the present invention provides a method for inhibiting coronavirus replication by upregulating the expression level of VHL protein or the N-terminal active fragment of VHL protein in host cells, thereby inhibiting coronavirus replication.

[0007] Furthermore, the NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR (SEQ ID NO.1).

[0008] Furthermore, the coronavirus is SARS-CoV-2.

[0009] To achieve the above objectives, the present invention also provides a coronavirus inhibitory factor, which is a VHL protein or an N-terminal active fragment of a VHL protein or a formulation containing an N-terminal active fragment of a VHL protein. The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0010] Furthermore, the coronavirus is SARS-CoV-2.

[0011] To achieve the above objectives, the present invention also provides the application of the aforementioned coronavirus inhibitory factor in the preparation of a formulation for inhibiting the replication of SARS-CoV-2 virus.

[0012] To achieve the above objectives, the present invention also provides the application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in the preparation of antiviral drugs, wherein the NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0013] To achieve the above objectives, the present invention also provides the application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in screening antiviral drugs, wherein the NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0014] The advantages and positive effects of the novel coronavirus infection-restricting factor and its application described in this invention are as follows: 1. This invention reveals for the first time the antiviral function of the tumor suppressor gene VHL, breaking through the existing scope of host target research; and its mechanism of action is unique, VHL specifically inhibits the synthesis of viral negative strand RNA (a key step in viral replication) without affecting the early life cycle, which is different from many identified host restriction factors.

[0015] 2. VHL is constitutively highly expressed in target organ tissues (such as lungs and intestines) infected with the novel coronavirus. It does not require interferon or other innate immune pathway activators for activation. Antiviral therapy based on VHL can effectively avoid the immune side effects during interferon antiviral treatment.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 These are the fluorescence microscopy observation results of the novel coronavirus infection model in this embodiment of the invention; Figure 2 This is a heatmap of tumor suppressor gene expression in the infected group versus the control group in an embodiment of the present invention; Figure 3 This illustrates the cytopathic effect and GFP expression in the embodiments of the present invention. Figure 4 The results of qPCR detection of total viral RNA in this embodiment of the invention; Figure 5 The results of qPCR detection of viral subgenomic RNA in this embodiment of the invention; Figure 6 This illustrates the effect of the Δ1-60aa mutant on subgenomic RNA synthesis in this embodiment of the invention. Figure 7 This refers to the HIF-1α degradation ability of the Δ1-60aa mutant in the embodiments of the present invention; Figure 8 This invention provides an example of VHL analysis of the viral life cycle. In example A, knocking down VHL in HEK293T-ACE2-N cells does not affect the expression of total intracellular ACE2 protein. In example B, knocking down VHL in HEK293T-ACE2-N cells does not affect the expression of cell surface ACE2 protein. In examples C and D, knocking down VHL in HEK293T-ACE2-N cells does not affect VSV-G or Spike-mediated pseudovirus invasion (indicating that VHL does not restrict the invasion steps of novel coronavirus infection). Example E is a schematic diagram of the experimental steps for detecting negative-strand RNA of novel coronavirus infection. Example F shows that knocking down VHL in HEK293T-ACE2-N cells promotes the synthesis of negative-strand RNA at an early time point of novel coronavirus infection, indicating that VHL does not affect the early steps of the viral life cycle, but only inhibits the negative-strand RNA replication stage. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0021] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0022] This invention provides a method for inhibiting coronavirus replication by upregulating the expression level of VHL protein or the N-terminal active fragment of VHL protein in host cells.

[0023] Furthermore, the NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0024] Furthermore, the coronavirus is SARS-CoV-2.

[0025] The present invention also provides a coronavirus inhibitory factor, which is a VHL protein or an N-terminal active fragment of a VHL protein or a formulation containing an N-terminal active fragment of a VHL protein; The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0026] Furthermore, the coronavirus is SARS-CoV-2.

[0027] The present invention also provides the application of the above-mentioned coronavirus inhibitory factor in the preparation of a formulation that inhibits the replication of SARS-CoV-2 virus.

[0028] The present invention also provides the application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in the preparation of antiviral drugs, wherein the NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0029] This invention also provides the application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in screening antiviral drugs. The NCBI accession number of the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

[0030] This invention reveals for the first time the antiviral function of the tumor suppressor gene VHL, breaking through the existing scope of host target research. Furthermore, its mechanism of action is unique: VHL specifically inhibits viral negative-strand RNA synthesis (a key step in viral replication) without affecting the early life cycle, unlike many identified host-restricting factors. VHL exhibits constitutive high expression in target organs and tissues infected with the novel coronavirus (such as the lungs and intestines), and does not require stimulation by interferon or other innate immune pathway activators. VHL-dependent antiviral therapy can effectively avoid the immune side effects associated with interferon antiviral treatment.

[0031] The following examples provide a detailed explanation.

[0032] In this embodiment of the invention, HEK293T cells were derived from ATCC CRL-3216 (human colon adenocarcinoma cell line Caco-2), and then a stable HEK293T-ACE-N cell line expressing ACE2 and SARS-CoV-2-infected N protein was constructed via PLVX-puro vector lentiviral transduction. Caco-2-ACE2-N cells were obtained using the same method.

[0033] Example 1. Constructing a novel coronavirus infection model by infecting host cells: Human intestinal epithelial cells (Caco-2) were infected with the SARS-CoV-2-GFP strain (Wuhan-Hu-1, MN908947) at an MOI of 0.1. Infection status was confirmed 24 hours later by observing GFP using a cell fluorescence microscope. Figure 1The SARS-CoV-2-GFP strain originated from the trans-complementary novel coronavirus infection and replication subsystem constructed by Associate Professor Ding Qiang of Tsinghua University. Reference: DOI:10.1371 / journal.ppat.1009439.

[0034] Depend on Figure 1 It can be seen that Caco-2-ACE2-N cells infected with the SARS-CoV-2-GFP strain can be observed to have green fluorescence 24 hours after infection, indicating that the cells were successfully infected with SARS-CoV-2-GFP.

[0035] 2. High-depth proteomics screening: 10X depth proteomic analysis was performed using a Thermo Orbitrap Astral mass spectrometer to compare the expression profiles of tumor suppressor proteins in the infected group versus the non-infected control group. The heatmap showed that VHL expression was significantly downregulated among tumor suppressor genes. Figure 2 This indicates that VHL was selectively downregulated due to novel coronavirus infection.

[0036] 3. VHL antiviral function verification: The experimental group was transfected with small interfering RNA (siRNA) targeting VHL and then infected with the virus, while the control group was transfected with non-targeting small interfering RNA (siNC) and then infected with the virus. Cytopathic effects and GFP expression were observed under a microscope, and total viral RNA and subgenomic RNA were detected by qPCR.

[0037] Figure 3 To determine the cytopathic effect and GFP expression status, by Figure 3 It can be seen that knocking down VHL can significantly promote cytopathic effects caused by SARS-CoV-2 infection in HEK293T-ACE2-N cells and promote the expression of SARS-CoV-2 infection GFP fluorescence, indicating that endogenous VHL protein has a role in limiting SARS-CoV-2 infection replication in this cell.

[0038] Figure 4 For the results of qPCR detection of total viral RNA, by Figure 4 It was found that the total genomic RNA of the novel coronavirus infection increased by more than 100-fold in the VHL knockdown group (p<0.001).

[0039] Figure 5 For the results of qPCR detection of viral subgenomic RNA, by Figure 5 It was found that the subgenomic RNA in the VHL knockdown group increased by more than 100-fold (p<0.001).

[0040] This indicates that the expression of endogenous VHL protein has a strong effect in limiting the RNA replication ability of novel coronavirus infection.

[0041] 4. N-terminal functional domain positioning: Discovery of the N-terminal active fragment: Cross-species specificity validation: VHL was compared with those of humans (H. sapiens, NP_000542.1), pigs (S. scrofa, XP_003483262.1), chickens (G. gallus, XP_040538194.1), and ducks (A. platyrhynchos, XP_027323646.3). The results showed that the human N-terminal homologous region has a 60-aa amino acid hypervariable region.

[0042] The process involved constructing a VHL mutant (Δ1-60aa) → transfecting cells → infecting with the virus. Results showed that, compared to wild-type VHL, the Δ1-60aa mutant lost its ability to suppress subgenomic RNA synthesis. Figure 6 This indicates that the amino acid fragment from 1 to 60 aa is an active fragment capable of inhibiting subgenomic RNA synthesis.

[0043] 5. Functional independence verification: The VHL mutant (Δ1-60aa) was constructed → transfected into 786-O cells → HIF-1α degradation was detected. Results showed that this antiviral defective mutant still mediated HIF-2α degradation. Figure 7 This indicates that the antiviral function of VHL protein differs from its classic antitumor function, which regulates the expression level of intracellular HIF protein through the action of its E3 ubiquitin ligase. The N-terminal antiviral functional region of VHL is a newly identified functional region.

[0044] 6. Verification of the mechanism by which VHL inhibits viral replication: Subgenomic RNA-specific detection: Forward primers (5'-CGATCTCTTGTAGATCTGTTCTC-3' (SEQ ID NO.2)) and reverse primers (5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO.3)) were designed to target subgenomic RNA infected with the novel coronavirus, and qPCR quantification was performed. Results showed ( Figure 6 VHL overexpression of high-dose group (wild-type WT) reduced the novel coronavirus infection subgenomic RNA by about 70% compared with the control group.

[0045] 7. Virus life cycle analysis: The expression of the SARS-CoV-2 receptor ACE2, spike protein-mediated pseudoviral invasion, and negative-strand RNA synthesis in SARS-CoV-2-infected cells were detected in VHL-knockdown HEK293T-ACE2-N cells. Results showed (e.g.) Figure 8As shown), VHL does not affect the early steps of the virus life cycle ( p >0.05), which only inhibits the negative strand RNA replication phase.

[0046] Figure 8 In diagram A, knocking down VHL in HEK293T-ACE2-N cells does not affect the expression level of total intracellular ACE2 protein. In diagram B, knocking down VHL in HEK293T-ACE2-N cells does not affect the expression level of cell surface ACE2 protein. In diagrams C and D, knocking down VHL in HEK293T-ACE2-N cells does not affect VSV-G or Spike-mediated pseudovirus invasion (indicating that VHL does not restrict the invasion steps of SARS-CoV-2 infection). In diagram E, it is a schematic diagram of the experimental steps for detecting negative-strand RNA of SARS-CoV-2 infection. In diagram F, knocking down VHL in HEK293T-ACE2-N cells promotes the synthesis of negative-strand RNA at an early time point of SARS-CoV-2 infection, indicating that VHL does not affect the early steps of the viral life cycle, but only inhibits the negative-strand RNA replication stage.

[0047] Therefore, the present invention utilizes the above-mentioned novel coronavirus infection restriction factor and its application, which can inhibit coronavirus replication by upregulating the expression level of VHL protein or the N-terminal active fragment of VHL protein in host cells, thereby enabling the preparation of antiviral drug formulations.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for inhibiting coronavirus replication, characterized in that: Coronavirus replication is inhibited by upregulating the expression levels of VHL protein or the N-terminal active fragment of VHL protein in host cells.

2. The method for inhibiting coronavirus replication according to claim 1, characterized in that: The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

3. The method for inhibiting coronavirus replication according to claim 1, characterized in that: The coronavirus in question is SARS-CoV-2.

4. A coronavirus inhibitory factor, characterized in that: It is VHL protein or the N-terminal active fragment of VHL protein or a formulation containing the N-terminal active fragment of VHL protein; The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

5. The coronavirus inhibitory factor according to claim 4, characterized in that: The coronavirus in question is SARS-CoV-2.

6. The use of a coronavirus inhibitory factor as described in claim 4 or 5 in the preparation of an agent for inhibiting the replication of SARS-CoV-2 virus.

7. The application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in the preparation of antiviral drugs, characterized in that: The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

8. The application of VHL protein and / or the N-terminal active fragment of VHL protein as an antiviral target in screening antiviral drugs, characterized in that: The NCBI accession number for the VHL protein is NP_000542.1, and the amino acid sequence of the N-terminal active fragment of the VHL protein is MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR.

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