HCoV-NL63 pseudovirus as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an HCoV-NL63 pseudovirus as well as a preparation method and application thereof, and several HCoV-NL63 pseudoviruses (NL63pp) containing truncated S protein without partial C-terminal amino acid are compared and analyzed. It is found that the NL63pp infection titer based on VSV can be remarkably improved by about 10 times by deleting 14 amino acid sequences (D14) at the C terminal of the S protein, and the NL63pp infection titer is higher than the titer of NL63pp packaged by the S protein with 18 or 24 amino acids deleted at the C terminal. In addition, a VSV-G label (D14V) is added to the C end of D14, so that the infection titer can be improved by about 30%. Subsequently, an HCoV-NL63C3 subtype double report pseudovirus system C3-D14V is constructed, and it is found that the C3-D14V has higher infection efficiency. By means of the system, the susceptibility of several cell lines is compared, and it is found that cells from the liver (Huh7.5. 1), the small intestine (Caco-2) and the lung (Calu-3) show higher susceptibility. In addition, several bisbenzylisoquinoline alkaloids are evaluated by applying the system. It is worthy of noting that the inhibition efficiency of cepharanthine on NL63pp infection is the highest, and IC50 is 0.61 [mu] M. In a word, it is determined that the S protein with 14 amino acids deleted at the C terminal remarkably improves the infection titer of the HCoV-NL63 pseudovirus, and an effective HCoV-NL63 double-report pseudovirus system based on the VSV vector is provided for various applications such as drug screening and antibody vaccine development in the future.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and virology, and in particular to an HCoV-NL63 pseudovirus and a preparation method and application thereof. Background Art

[0002] The coronavirus HCoV-NL63 was first isolated and identified in the Netherlands in 2004. Its genome contains multiple open reading frames, encoding S / E / M / N structural proteins and several non-structural proteins such as 3a / 4a; it belongs to the coronavirus family like the new coronavirus. HCoV-NL63 generally only causes self-limiting common colds, which can be cured without special treatment, but in infants and people with low immunity, HCoV-NL63 infection may cause severe pneumonia, so the monitoring and research of HCoV-NL63 is very important.

[0003] Pseudovirus is a particle that simulates the structure of a real virus but does not contain an active viral genome. For enveloped viruses, human immunodeficiency virus (HIV) lentivirus or vesicular stomatitis virus (VSV) is usually selected as a pseudovirus vector. The outer membrane uses the envelope protein of the target virus. The spike protein (S) is an important protein on the surface of the coronavirus, which binds to the host cell and mediates the entry of the virus into the cell. The preparation of pseudovirus avoids the risk of using real viruses. The S protein of coronaviruses (such as HCoV-NL63, SARS-CoV-2, etc.) is selected as the outer membrane protein for packaging pseudoviruses. Researchers can simulate viral infection in the laboratory, thereby testing the inhibitory effects of different drugs / vaccines on viral infection and transmission.

[0004] There are few studies on the preparation of pseudoviruses for HCoV-NL63, and even fewer studies on HCoV-NL63 drugs or vaccines using pseudovirus systems. Although this virus can be operated in a Level 2 laboratory, there is still a risk of infection. In our early exploration, we also found that the packaging titer of the existing HCoV-NL63 pseudovirus system is not high, so there is an urgent need to develop a high-titer HCoV-NL63 pseudovirus system. Summary of the invention

[0005] The study of HCoV-NL63 virus is crucial to understanding respiratory infections caused by coronavirus and its potential impact. The present invention is aimed at the urgent need to establish a safe, widely applicable, high-titer HCoV-NL63 pseudovirus production system. Through comparative analysis of seven HCoV-NL63S protein optimized sequences, it is found that deleting the 14 amino acid sequences at the C-terminus of the S protein can significantly increase the infection titer of the HCoV-NL63 pseudovirus packaged by VSV by nearly 10 times, and adding VSV-G tag protein at the C-terminus of the S protein further increases the infection titer by about 30%. In addition, by comparing the HCoV-NL63 virus C1 type and C3 type pseudoviruses, it is found that the infection titer of the C3 type-D14V pseudovirus is much higher than that of the C1 type. At the same time, it is found that the VSV pseudovirus packaging system has a higher infection titer of pseudoviruses than the HIV lentivirus packaging system. The present invention utilizes the pseudovirus system to compare the susceptibility of several cell lines, and observes that cells from the liver (Huh7.5.1), small intestine (Caco-2) and lung (Calu-3) show higher sensitivity to HCoV-NL63 pseudovirus. In addition, the present invention uses the system to evaluate the inhibition efficiency of several bis-benzylisoquinoline alkaloids on HCoV-NL63 pseudovirus, indicating that cephalothin and neferine have good inhibition efficiency on HCoV-NL63 pseudovirus infection. In summary, the present invention can significantly enhance the infection titer of HCoV-NL63 pseudovirus by truncating 14 amino acids at the C-terminus of the S protein and expressing it by fusion with the VSV-G tag polypeptide, providing an efficient HCoV-NL63 pseudovirus model for various applications such as drug screening and antibody development.

[0006] Preferably, in the HCoV-NL63 pseudovirus packaging system, the HCoV-NL63 envelope protein is selected from the C1 type NL63 spike protein S, named WT; the HCoV-NL63 envelope protein is selected from the sequence corresponding to the 3' end of the C1 type spike protein S after 14 amino acids are deleted, named D14; the HCoV-NL63 envelope protein is selected from the 3' end of the C1 type spike protein S after 14 amino acids are deleted and the coding sequence corresponding to the VSV-G tag protein (amino acid sequence: YTDIEMNRLGK) is inserted, named D14V; the HCoV-NL63 envelope protein is selected from the sequence corresponding to the 3' end of the C1 type spike protein S after 18 amino acids are deleted, named D18; the HCoV-NL63 envelope protein is selected from the 3' end of the C1 type spike protein S The 'end of the HCoV-NL63 protein is deleted after 18 amino acids are deleted, and the sequence corresponding to the VSV-G tag protein (amino acid sequence: YTDIEMNRLGK) is inserted, and it is named D18V; the HCoV-NL63 envelope protein is selected from the sequence corresponding to the 3' end of the C1 type spike protein S after 24 amino acids are deleted, and it is named D24; the HCoV-NL63 envelope protein is selected from the sequence corresponding to the 3' end of the C1 type spike protein S after 24 amino acids are deleted, and the VSV-G tag protein (amino acid sequence: YTDIEMNRLGK) is inserted, and it is named D24V; the HCoV-NL63 envelope protein is selected from the sequence corresponding to the 3' end of the C3 type spike protein S after 14 amino acids are deleted, and the VSV-G tag protein (amino acid sequence: YTDIEMNRLGK) is inserted, and it is named C3-D14V, that is, the sequence of SEQ ID NO: 1, and the amino acid encoded by it is the sequence of SEQ ID NO: 2;

[0007] Preferably, the HCoV-NL63 pseudovirus packaging system adopts a VSV dual reporter system, which comprises an mCherry fluorescent protein reporter gene and a Luc2 luciferase reporter gene, which are connected and co-expressed via T2A, and the amino acid sequence of the dual reporter gene is SEQ ID NO: 3;

[0008] According to the present invention, a HCoV-NL63 pseudovirus packaging system NL63pp involved in the invention is characterized in that the optimized S protein optimal sequence, i.e., the S protein end is deleted after 14 amino acid sequences are deleted and the VSV-G tag protein sequence is introduced at the same time.

[0009] Beneficial effects of the present invention:

[0010] (1) The dual reporter system containing a fluorescent reporter gene and luciferase constructed by the present invention can intuitively display the pseudovirus infection effect while using luciferase to quantitatively detect the difference values ​​of each group, comprehensively reflecting the intra-group and inter-group differences when the reporter system is used; (2) The S protein of the recently popular C3 subtype is used to construct the HCoV-NL63 pseudovirus, and the terminal 14 amino acids of the S protein are deleted and replaced with the VSV-G tag protein. The packaging infection efficiency of the VSV pseudovirus system of HCoV-NL63 is greatly improved; (3) The HCoV-NL63 pseudovirus system of the present invention can be used to perform preliminary screening of HCoV-NL63 susceptible cells, and the invention can also be used to screen some antiviral drugs, providing technical support for the future development of drugs or vaccines that meet the requirements of HCoV-NL63, and has important potential social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific embodiments or implementations of the present invention, the following is a brief introduction to the drawings used in the description of the embodiments or implementations. The drawings of the present invention are only used to describe the embodiments or implementations for the purpose of demonstration. Without departing from the principles of the present invention, those skilled in the art can obtain other drawings based on the following drawings without creative work.

[0012] Figure 1 Schematic diagram of seven different S protein truncation design methods and comparison of pseudovirus efficiency.

[0013] Figure 2 Comparison of packaging efficiency of two pseudovirus systems.

[0014] Figure 3 To compare the differences in the infection efficiency of HCoV-NL63 pseudovirus in different cells.

[0015] Figure 4 This is an analysis of the inhibitory effects of four small molecule drugs on HCoV-NL63 pseudovirus infection. DETAILED DESCRIPTION

[0016] Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. On the contrary, the purpose of providing these examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Based on the examples in the application, all other embodiments obtained by those of ordinary skill in the art without making creative work premises, all belong to the scope of protection of this application.

[0017] 1. Comparison of the efficiency of seven S protein pseudoviruses

[0018] a. Construction of seven S protein plasmids and cell transfection

[0019] Primers were designed to clone the full length of seven codon-optimized S protein sequences into the pCMV vector. The design of each group of S proteins is shown below: Figure 1 Seven S protein plasmids were transfected into HEK293T cells. After 48 hours, the cells were harvested and lysed for protein immunoblotting analysis. The results of S protein expression are shown in Figure 1 As shown, the S protein was expressed in all groups, with less expression in the WT group. The expression of D14V after adding the VSV-G protein tag was higher than that of D14S protein.

[0020] b. Comparison of infection titers of seven S protein pseudoviruses

[0021] We used a VSV pseudovirus packaging system containing the fluorescent protein mCherry and luciferase 2 (Luc2) dual reporter genes to generate these 7 S protein pseudoviruses. After infecting cells with each group of pseudoviruses, we detected the activity of luciferase in the cells and the expression of fluorescent protein. Comparing the luciferase activity results of each group, it was found that the VSV pseudovirus in which 14 amino acids were deleted from the S protein had a higher efficiency in infecting cells, among which the luciferase activity of the D14 group was nearly 10 times that of the WT group. The VSV-G tag protein (D14V) also increased the infection efficiency of the VSV pseudovirus by about 30% compared with D14, but the other two VSV-G tag groups did not show a significant improvement ( Figure 1 C). Similarly, the fluorescence results are shown in Figure ID, and the number of fluorescent cells in the D14 and D14V groups is larger. Therefore, the D14V S protein is more conducive to the packaging and infection of the NL63 pseudovirus.

[0022] 2. Comparison of pseudovirus packaging systems of VSV and HIV

[0023] The three S protein (WT, D14V, C3-D14V) plasmids were packaged into pseudoviruses using the VSV pseudovirus system or the HIV lentivirus system, and then the packaging and infection efficiencies of different pseudoviruses were compared. The specific operation method is as follows:

[0024] a.VSV pseudovirus packaging:

[0025] First, HEK293T cells were transfected with 5 μg of S protein plasmid. After 24 hours, the cells were inoculated with VSVpp (VSV-G-VSV*ΔG-mCherry-Luc2) at an MOI of 2 and infected for 1.5 hours. Subsequently, the cells were washed 5 times with PBS to eliminate residual VSVpp. Pseudoviruses were collected 24 hours, 36 hours, and 48 hours after VSVpp infection, then filtered through a 0.45 μm filter, and finally aliquoted and stored at -80°C for further use.

[0026] b. HIV pseudovirus packaging:

[0027] First, HEK293T cells were co-transfected with three plasmids, psPAX2 (4 μg), pLDR (4 μg) and different S protein plasmids (2 μg), totaling 10 μg; then, the pseudovirus in the supernatant was collected 24 hours, 36 hours, 48 ​​hours and 60 hours after transfection, and the supernatant was further removed of cell debris by centrifugation and passed through a 0.45 μm filter. After aliquoting, it was frozen at -80°C for subsequent use.

[0028] c. Comparison of infection efficiency of different pseudoviruses

[0029] 15,000 HEK293T-ACE2 cells were added to each well of a 96-well plate and cultured for 16 hours. 20 μL of pseudovirus was added to each well to infect the cells. Three replicates of each pseudovirus group were added, and cell fluorescence and luciferase activity were evaluated at specific time points (60 hours after HIV lentivirus infection or 24 hours after VSV pseudovirus infection). Intracellular fluorescence was observed using an Olympus IX71 fluorescence microscope, and Steady-Lumi TM The firefly luciferin reporter gene assay kit was used to quantify the luciferin expression level in each group.

[0030] The three S proteins were packaged using VSV pseudovirus and HIV lentivirus system respectively. The prepared pseudoviruses were added to HEK293T-ACE2 cells to detect packaging and infection efficiency. The fluorescence and luciferase detection results are shown in Figure 2 After comparison, the group with the highest infection efficiency was the C3-D14V group, and the pseudovirus of this group was named NL63pp.

[0031] 3. Comparison of the infection efficiency of different cells to HCoV-NL63 pseudovirus

[0032] a. Cell culture

[0033] 1 Vero E6 African green monkey kidney cells 2 HEK293T-hACE2 Human embryonic kidney cells (ACE2 overexpression) 3 Caco2 Human colorectal adenocarcinoma cells 4 Calu-3 Human lung adenocarcinoma cells (pleural effusion) 5 Huh7.5.1 Human liver cancer cells 6 L132 Human cervical cancer cells (Hela derivative line)

[0034] All the above cell lines were preserved in the laboratory. The cells were cultured in an incubator at 37°C and 5% CO2, and cultured using DMEM complete medium, which consisted of DMEM basal medium (#SH30022.01, purchased from Cytiva), 10% fetal bovine serum (#F0926, purchased from Millipore Sigma), and 100U / mL penicillin-streptomycin double antibody.

[0035] b. Pseudovirus infection

[0036] The above 7 types of cells were plated in 96-well plates, with 3 replicate wells for each type of cell. When the cell density reached 80%, 40ul of NL63pp was added to each well. After incubation for 5 hours, the medium was changed to 5% FBS-DMEM. After culturing the cells for another 19 hours, the expression of fluorescent protein and luciferase was observed. The results are shown in Figure 3 As shown, HCoV-NL63 pseudovirus is susceptible to lung-derived Calu-3 cells, liver-derived Huh7.5.1 cells, and intestinal-derived Caco2 cells.

[0037] 4. Analysis of the inhibitory effects of four small molecule drugs on HCoV-NL63 pseudovirus infection

[0038] The present invention selects four bisphenylisoquinoline compounds (hanfangji amine, heshi thalidomide, neferine and methyl neferine) to evaluate their inhibitory effects on the entry of HCoV-NL63 pseudovirus into cells. 15,000 HEK293T-hACE2 cells were inoculated in each well of a 96-well plate. After 16 hours of culture, four natural small molecules were added at concentrations ranging from 100 to 0.1 μM and incubated for 7 hours. Subsequently, after an additional 17 hours of culture, the CCK-8 test evaluated cell viability, with 6 replicate wells for each small molecule concentration, and expressed cell viability as a percentage. Cells were prepared using the same method as above, and the concentrations of neferine, methyl neferine and heshi thalidomide were diluted from 30 μM to 0.03 μM, covering seven concentrations. The dosing concentration of hanfangji amine ranged from 10 μM to 0.01 μM, also diluted to seven concentrations. Four natural small molecules at different concentrations were added and incubated with the cells for 1 hour. Then NL63pp was added and incubated for 6 hours. After 17 hours, the luciferase activity was measured. Three replicate wells were set for each natural small molecule concentration in each sample. EC was performed using [Agonist] vs. response-Variable slope (four parameters) 50 The result is as follows Figure 4 As shown, the CC of cepharanthine, neferine, thalidomide and tetrandrine 50 The values ​​were 64.72μM, 114.3μM, 69.72μM and 20.0μM, respectively. The EC 50 The values ​​were 0.61μM, 2.40μM, 2.48μM and 2.77μM, respectively. All four small molecules effectively inhibited the infection of HCoV-NL63 pseudovirus in cells, as calculated by CC 50 / EC 50The selectivity index (SI) of each natural small molecule was obtained, and the SIs of the four small molecules were 105.41, 47.64, 28.09 and 7.22 respectively. Among them, cepharanthine performed particularly well, with a selectivity index as high as 105.41.

[0039] The above are only preferred implementations of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A HCoV-NL63 pseudovirus based on a VSV vector, characterized in that: The HCoV-NL63 pseudovirus uses an optimized C3 subtype S protein as a pseudovirus outer membrane protein, and the S protein optimization includes gene coding sequence optimization, while deleting the coding sequence of 14 amino acids at the C-terminus of the S protein, and the C-terminus may also introduce a VSV-G tag protein coding sequence to further enhance its packaging and infection efficiency. The optimized S protein final coding sequence has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the nucleotide sequence shown in SEQ ID NO.

1.

2. The antigen encoded by SEQ ID NO. 1 of claim 1 has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO.

2.

3. The HCoV-NL63 pseudovirus described in claim 1 adopts a VSV dual reporter system, which comprises an mCherry fluorescent protein reporter gene and a Luc2 luciferase reporter gene, both of which are connected and co-expressed by T2A, and the dual reporter gene amino acid sequence has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO.

3.

4. Application of the HCoV-NL63 pseudovirus packaging system described in claim 1 and claim 2 in screening of drugs, vaccines, etc.

5. The VSV dual reporter system described in claim 3 can be replaced with other commonly used reporter genes, such as eGFP, Nluc, etc.

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