Establishment and application of an RNA recombination experiment system dependent on replication of flavivirus

Abstract

The application discloses a kind of establishment and application of flavivirus replication-dependent RNA recombination experimental system, belong to the field of biotechnology.The RNA recombination system includes two components: tanbusu virus E gene deletion subgenomic replicon CQW1-ΔE, and the defective virus genome CQW1-ΔNS1 or MM1775-ΔNS1 missing partial NS1 gene sequence.The two components cannot produce progeny virus alone, but after co-transfection, due to RNA recombination, a large number of recombinant progeny viruses can be produced within a few days after transfection, the recombination efficiency is extremely high, and continuous passage is not required, greatly shortening the construction cycle and saving production costs;At the same time, the progeny virus can form plaques on cells, and the efficiency of RNA recombination can be quantitatively measured by plaque counting, and the key factors affecting tanbusu virus RNA recombination can be analyzed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a replication-dependent RNA recombination experimental system of flavivirus and a method for establishing the same and application thereof. BACKGROUND

[0002] TMUV belongs to the family Flaviviridae Flaviviridae ), genus Flavivirus OrthoFlavivirus ), is an enveloped positive-sense RNA virus. Its genomic RNA (vRNA) is about 11000 nt long, containing only one open reading frame, encoding a 3145 aa long polyprotein, which is cleaved by host signal peptidase and viral protease NS2B / 3 after translation, to produce 3 structural proteins C, prM, E, and 7 non-structural proteins NS1, NS2A / 2B, NS3, NS4A / 4B, NS5. The 5' end of the genome has a type I cap structure, the 5' UTR is about 95 nt long, and the 3' UTR is about 600 nt long, but there is no poly(A) tail at the 3' end; the 3' UTR contains multiple conserved RNA secondary structures, which play a key role in the circularization of viral genomes and RNA replication, and affect viral host specificity. During the replication of flavivirus RNA, the genomic RNA must be circularized to initiate RNA synthesis in combination with viral NS5.

[0003] Recombination is very common in RNA viruses, but the prerequisite for its occurrence is that different viruses simultaneously infect a single cell, which can produce hybrid offspring viruses. So far, the virology community has proposed two distinct models of genomic RNA recombination mechanisms, namely replication-dependent RNA recombination and non-replication-dependent RNA recombination. The RNA recombination model can be widely applied to the study of viruses. Currently, the trans-complementing recombination trap method (TAUCHER C, BERGERA, MANDL C W. A trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination [J]. J Virol, 2010, 84(1): 599-611.) has been reported in flavivirus, which requires continuous passage and continuous dilution method to screen a very small number of recombinants, and the construction cycle is long, and the recombination efficiency is low. SUMMARY

[0004] The application aims to solve the above problems in the prior art, and provides a method for establishing and applying a flavivirus replication-dependent RNA recombination experimental system.

[0005] The technical scheme adopted by the application is as follows:

[0006] The RNA recombination experimental system comprises components A and B.

[0007] Preferably, the component A is a pACYC-CQW1-ΔE plasmid, and the gene sequence is shown in SEQ ID NO. 2.

[0008] Preferably, the preparation method of the pACYC-CQW1-ΔE plasmid comprises the following steps: taking a Tanbussu virus CQW1 strain infectious clone plasmid pACYC-FL-CQW1 as a template to amplify a CQW1-P1-ΔE DNA fragment, and the gene sequence is shown in SEQ ID NO. 1; performing double enzyme cutting on pACYC-CQW1-P2-6 by using SpeI and XhoI to obtain a linearized vector, and connecting the CQW1-P1-ΔE fragment and the linearized pACYC-CQW1-P2-6 vector by using a homologous recombination method to construct the pACYC-CQW1-ΔE plasmid.

[0009] Preferably, the defective Tanbussu virus genome plasmid in which the NS1 gene sequence is deleted from 437nt to 878nt is a pACYC-CQW1-ΔNS1 plasmid.

[0010] Preferably, the preparation method comprises the following steps: taking a Tanbussu virus CQW1 strain infectious clone plasmid as a template to amplify a CQW1-P2-ΔNS1 fragment, and the sequence is shown in SEQ ID NO. 3; performing double enzyme cutting on pACYC-FL-CQW1 by using XhoI and PmlI to obtain a linearized vector, and connecting the CQW1-P2-ΔNS1 fragment and the linearized pACYC-FL-CQW1 by using a homologous recombination method to obtain the pACYC-CQW1-ΔNS1 plasmid through single clone screening.

[0011] Further preferably, the defective Tanbussu virus genome plasmid in which the NS1 gene sequence is deleted from 434nt to 869nt is a pACYC-MM1775-ΔNS1 plasmid.

[0012] Preparation method: the MM1775-P1-ΔNS1 fragment is amplified by taking the Tangu virus MM1775 strain as a template, and the sequence is shown as SEQ ID NO. 4; the pACYC-MM1775-P2-3 plasmid is linearized by double enzyme cutting of NruI and SacI, and the sequence of the pACYC-MM1775-P2-3 plasmid is shown as SEQ ID NO. 5; the MM1775-P1-ΔNS1 fragment is connected with the linearized pACYC-MM1775-P2-3 by using the homologous recombination method, and the pACYC-MM1775-ΔNS1 plasmid is obtained by monoclonal screening.

[0013] The application further provides application of the replication-dependent RNA recombination experimental system of the above-mentioned flavivirus in a flavivirus RNA recombination model.

[0014] The application has the following beneficial effects:

[0015] (1) The replication-dependent RNA recombination experimental system of the flavivirus established in the application includes two key components: the replicable CQW1-ΔE replicon and the defective genome CQW1-ΔNS1 or MM1775-ΔNS1 with a sequence of NS1 gene missing. The CQW1-ΔE can only replicate RNA due to the missing structural gene, and can be used as a donor when template jumping occurs in the RNA recombination process; and the defective virus genome CQW1-ΔNS1 or MM1775-ΔNS1 with the sequence of NS1 missing cannot replicate RNA due to the missing NS1 sequence and the frame shift, but retains the conserved RNA circularization sequence of the flavivirus, and can be used as a receptor when template jumping occurs in the RNA recombination process. The two components cannot produce progeny viruses alone, but can produce progeny viruses and form plaques after being co-transfected. The RNA recombination efficiency can be quantitatively measured by plaque counting, and the key factors affecting the RNA recombination of the Tangu virus can be analyzed. Due to the similarity of the virus genomes of the flavivirus, the system can also be applied to other mosquito-borne flaviviruses.

[0016] (2) The replication-dependent RNA recombination experimental system of the flavivirus established in the application can produce a large number of recombinants within a few days after transfection, the recombination efficiency is extremely high, and continuous passage is not required, which greatly shortens the construction period and saves the production cost; meanwhile, the experimental system can accurately evaluate or screen and confirm the key factors affecting the RNA recombination by plaque counting, and therefore, can be widely applied to the establishment and research of the flavivirus RNA recombination model. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Schematic diagram of the recombinant system of the application.

[0018] Figure 2 Identification results of the RNA recombination system; wherein, (A) plaque identification; (B) recombinant type.

[0019] Figure 3 Application results of the recombination experimental system; wherein, (A) influence of sequence homology on recombination efficiency; (B) influence of RNA replication on recombination efficiency; (C) influence of NS5 mutation on recombination efficiency.

[0020] Figure 4 Plasmid map of pACYC-CQW1-ΔE.

[0021] Figure 5 Plasmid map of pACYC-CQW1-ΔNS1 plasmid.

[0022] Figure 6 pACYC-MM1775-ΔNS1 plasmid.

[0023] The plasmid pACYC-FL-CQW1, the plasmid pACYC-CQW1-P2-6 and the plasmid pACNR-FL-MM1775 are all preserved in Sichuan Agricultural University.

[0024] The plasmid pACYC-FL-CQW1 and the plasmid pACYC-CQW1-P2-6 are disclosed in the following literature:

[0025] He Y. Study on attenuated strategy of duck Tembusu virus based on abnormal function of C protein [D]. Sichuan Agricultural University, 2022. DOI:10.27345 / d.cnki.gsnyu.2022.001185.

[0026] The plasmid pACNR-FL-MM1775 is disclosed in the following literature:

[0027] WANG X, HE Y, GUO J, et al. Construction of an Infectious Clone for Mosquito-Derived Tembusu Virus Prototypical Strain [J]. Virol Sin, 2021, 36(6): 1678-1681. DETAILED DESCRIPTION

[0028] The application will be further described below in combination with the drawings and specific embodiments.

[0029] Example 1

[0030] 1. Construction of CQW1-ΔE replicon plasmid

[0031] The schematic diagram of construction of CQW1-ΔE replicon plasmid is shown in Figure 1 SEQ ID NO. 1. The fragment lacks the gene sequence corresponding to amino acid residues 11-472 of E protein. Meanwhile, pACYC-CQW1-P2-6 plasmid (which is only missing P1 fragment from full-length infectious clone plasmid) was obtained by double digestion of pACYC-CQW1-P2-6 with SpeI and XhoI. The pACYC-CQW1-P2-6 plasmid was modified from the full-length cDNA infectious clone plasmid pACYC-FL-CQW1 of CQW1 strain, and the SpeI and XhoI enzyme digestion sites were retained to facilitate the insertion of the P1 fragment. Subsequently, the CQW1-P1-ΔE fragment was ligated to the linearized pACYC-CQW1-P2-6 vector by homologous recombination, and the pACYC-CQW1-ΔE plasmid was obtained by single colony screening, as shown in SEQ ID NO. 2, and no mutation was confirmed by sequencing. The plasmid map is shown in Figure 4 .

[0032] 2. Construction of CQW1-ΔNS1 replication-defective virus genome

[0033] The schematic diagram of construction of CQW1-ΔNS1 replication-defective virus genome is shown in Figure 1 SEQ ID NO. 3; and the pACYC-FL-CQW1 plasmid was double-digested with XhoI and PmlI to obtain a linearized vector. Subsequently, the CQW1-P2-ΔNS1 fragment was ligated to the linearized pACYC-FL-CQW1 by homologous recombination, and the pACYC-CQW1-ΔNS1 plasmid was obtained by single colony screening, and no mutation was confirmed by sequencing. The plasmid map is shown in Figure 5 .

[0034] Example 2

[0035] The construction of CQW1-ΔE replicon plasmid was the same as in Example 1.

[0036] Construction of MM1775-ΔNS1 replication-defective virus genome

[0037] The schematic diagram of construction of MM1775-ΔNS1 defective genome plasmid is shown in Figure 1 The MM1775-P1-ΔNS1 fragment (deletion of 434-869 nt of NS1) was obtained by PCR amplification using the infectious clone plasmid pACNR-FL-MM1775 of the Tanapoxvirus MM1775 strain as a template, and the sequence is shown in SEQ ID NO. 4; the pACYC-MM1775-P2-3 plasmid (the sequence is shown in SEQ ID NO. 5, containing 3328-11002 nt of the MM1775 genome) was linearized by double digestion with NruI and SacI. Subsequently, the MM1775-P1-ΔNS1 fragment was ligated to the linearized pACYC-MM1775-P2-3 by homologous recombination, and the pACYC-MM1775-ΔNS1 plasmid was screened by monoclonal selection, and no mutation was confirmed by sequencing, and the plasmid map is shown in FIG. 2. Figure 6

[0038] Example 3

[0039] Verification of the recombinant experimental system was performed on the basis of Example 1 and Example 2.

[0040] 1. Components and working principle of the recombinant experimental system

[0041] The experimental system consists of two components, CQW1-ΔE-RNA and ΔNS1-RNA. The former can only replicate RNA and cannot assemble into virions due to the deletion of the E protein, but can normally replicate RNA and serve as a donor to provide key enzyme protein NS5 during RNA recombination; and ΔNS1 can only translate part of the resulting protein due to the deletion of part of the NS1 gene sequence and subsequent frameshift, and cannot perform subsequent RNA replication and virus assembly process, but its genome can be normally circularized, so it can receive NS5 dropped from CQW1-ΔE-RNA as a receptor to complete the recombination process. However, neither of the two can produce virions when transfected into cells, and cannot form plaques. When co-transfected, only when RNA recombination occurs and offspring viruses are produced, viral plaques will be formed.

[0042] 2. Obtaining of RNA transcripts

[0043] ​The pACYC-CQW1-ΔE plasmid, pACYC-CQW1-ΔNS1 plasmid and pACYC-MM1775-ΔNS1 plasmid were purified using an endotoxin-free plasmid extraction kit or a plasmid extraction kit for standby use. Before performing the in vitro transcription reaction, the plasmid was linearized using SmaI or NotI-HF restriction endonuclease, and the linearized plasmid was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit, and the concentration was concentrated to more than 200 ng / µL. Subsequently, the in vitro transcription reaction was performed using the mMESSAGE mMACHINE T7 Transcription kit, and the reaction system was configured in a PCR tube according to the reagent instructions:

[0044]

[0045] After gently blowing and mixing, incubate at 37 ℃ for 3 h. Then add 1 μL TURBO DNase to digest the DNA template. Subsequently, the RNA transcript was purified using lithium chloride precipitation:

[0046] (1) Transfer the reaction product to a 1.5 mL RNase-free EP tube, then add 30 μL of Nuclease-free Water and 30 μL of LiCl precipitation reagent;

[0047] (2) Freeze at -20 ℃ for 2 h, or overnight;

[0048] (3) Centrifuge at 12000 r / min, 4 ℃ for 15 min to precipitate the RNA to the bottom;

[0049] (4) Remove the supernatant, wash the RNA precipitate with 70% ethanol, then centrifuge at 12000 r / min, 4 ℃ for 15 min;

[0050] (5) Carefully remove the 70% ethanol, and carefully air dry the RNA at room temperature until transparent, then add an appropriate amount of Nuclease-free Water to dissolve the precipitate.

[0051] After the final concentration determination by NanoDrop2000, aliquot and store at -80 ℃.

[0052] 3. Verification of the recombinant experimental system

[0053] BHK-21 cells were pre-plated in 6-well plates, and when the cells were about 90% confluent, CQW1-ΔE and MM1775-ΔNS1 RNA transcripts were co-transfected into the cells at a ratio of 1:2 using Lipofectamine Messenger MAX transfection reagent, according to the manufacturer's instructions; at the same time, single RNA transcript transfection and blank control groups were set up. Six hours after transfection, the cell culture medium was carefully removed and 1% methylcellulose was added to each cell well at 2 mL. The cell plate was placed back in the carbon dioxide incubator, and after 6 days of continuous culture, it was removed, the methylcellulose cover liquid was carefully aspirated, and the cells were carefully rinsed with PBS twice, and then 500 μL / well of 4% formaldehyde (prepared with PBS) was added to fix the cells at room temperature for 20 min. The fixing solution was removed, and each well was added with an appropriate amount of 1% crystal violet for 1 min, and then the staining solution was carefully washed off with running water, and then the plaque situation was observed.

[0054] The experimental results show that: after single transfection of CQW1-ΔE and MM1775-ΔNS1 RNA, no plaque was produced, indicating that no infectious progeny virus was produced. However, when CQW1-ΔE and MM1775-ΔNS1 RNA were co-transfected, plaques were clearly visible, indicating that progeny virus particles were produced (see Figure 2 A).

[0055] To further verify whether the co-transfection group indeed produced recombinant progeny virus, the co-transfection experiment was repeated, but no methylcellulose cover liquid was added, and on the 3rd to 4th day after transfection, the cell supernatant was harvested, and the BHK-21 cells were passaged once to exclude the influence of the transfected RNA transcript. The cell supernatant after passage was harvested, and the viral RNA was extracted using a viral whole genome extraction kit and reverse transcribed into cDNA as a template, and RT-PCR amplification was performed using primers covering the recombinant crossover region sequence (see Figure 2 B). The amplified DNA fragments were recovered and purified and directly ligated to a T vector. Then, the ligation product was transformed into E. coli recipient cells and spread on agar plates with ampicillin resistance, and when single colonies grew, a large number of single colonies were directly sent for sequencing to confirm. The results showed that, as expected, the genomic sequences of all recombinants were 5' from MM1775, and the 3' sequence was from CQW1; and the recombinant crossover sites were all located in the putative recombinant crossover region. According to the sequence characteristics of the recombinant crossover region, the recombinants can be divided into three types: precise (without any sequence redundancy or deletion), sequence redundancy, and sequence deletion recombinants (see Figure 2 B).

[0056] The above results show that the RNA recombination is successfully and efficiently occurred in the experimental system, and the progeny virions are produced, and the efficiency of obtaining the recombinants is very high, and a large number of recombinants can be produced within a few days after transfection, and continuous passage is not required; in addition, the experimental system can accurately evaluate or screen and confirm the key factors affecting RNA recombination through plaque counting.

[0057] Example 4

[0058] Application of the recombinant experimental system

[0059] On the basis of Examples 1-3, the key factors affecting the efficiency of Tangu virus RNA recombination are analyzed by using the recombinant experimental system of the application.

[0060] (1) Sequence homology

[0061] In order to compare whether there is a difference in the efficiency of recombination between the internal CQW1 virus and different strains (MM1775 strain), CQW1-ΔE is combined with CQW1-ΔNS1 and MM1775-ΔNS1 respectively, and co-transfected into cells. The results show that the number of plaques produced by CQW1-ΔE+CQW1-ΔNS1 is significantly more than that of CQW1-ΔE+MM1775-ΔNS1 group, indicating that sequence homology is a key factor affecting RNA recombination. Figure 3 A).

[0062] (2) RNA replication level

[0063] In order to verify the influence of the replication ability of viral RNA on RNA recombination, after co-transfecting CQW1-ΔE and CQW1-ΔNS1 into cells, different concentrations of antiviral inhibitor ribavirin (inhibiting RNA replication) are added, and the influence of ribavirin on the efficiency of RNA recombination is analyzed. The results show that the addition of ribavirin significantly reduces the number of plaques, indicating that the replication of RNA is crucial for RNA recombination. Figure 3 B).

[0064] (3) NS5 mutation

[0065] NS5 is the RNA polymerase of the virus, which plays a key role in the synthesis of viral RNA. G643R amino acid substitution and L612F amino acid substitution are introduced into the NS5 gene sequence of CQW1-ΔNS1, and then RNA recombination experiment is carried out. The results show that both of them significantly reduce the formation of plaques. It is shown that the amino acid substitution of NS5 will affect the efficiency of RNA recombination. Figure 3 C).

[0066] In summary, the application discloses a method for establishing a replication-dependent RNA recombination experimental system of a Tangu virus and application. Based on reverse genetics technology, the application respectively constructs a subgenomic replicon CQW1-ΔE of the Tangu virus with an E gene deleted and a defective virus genome CQW1-ΔNS1 or MM1775-ΔNS1 with a partial NS1 gene sequence deleted. The CQW1-ΔE can only carry out RNA replication due to the deletion of a structural gene, and can be used as a donor in a template jump in the RNA recombination process. The defective virus genome with the ΔNS1 deleted cannot carry out RNA replication due to the deletion of the NS1 sequence and a frame shift, but retains a conserved RNA circularization sequence of the flavivirus, and can be used as a receptor in the template jump in the RNA recombination process. Neither of the two can produce progeny viruses, but after being co-transfected, the RNA recombination can produce progeny viruses to form plaques. Through plaque counting, the efficiency of the RNA recombination can be quantitatively measured, and key factors influencing the RNA recombination of the Tangu virus can be analyzed. The system can also be applied to mosquito-borne flaviviruses.

[0067] The description and drawings of the present application are considered illustrative and not restrictive in character, and the changes and modifications that can be suggested by the technological content disclosed can be carried out by a person skilled in the art without creative labor, and are within the scope of the present application.

Claims

1. A flavivirus replication-dependent RNA recombination experimental system, characterized in that, It includes component A and component B; component A is a replicon plasmid of Tembusu virus with amino acid residues 11-472 of the E protein missing; component B is a defective Tembusu virus genome plasmid with NS1 gene sequence deletion 437-878nt or a defective Tembusu virus genome plasmid with NS1 gene sequence deletion 434-869nt. Component A is the pACYC-CQW1-ΔE plasmid, and its gene sequence is shown in SEQ ID NO.2; The defective Tembusu virus genome plasmid that lacks the NS1 gene sequence from 437 to 878 nt is pACYC-CQW1-ΔNS1 plasmid. The preparation method of the pACYC-CQW1-ΔNS1 plasmid is as follows: using the infectious clone plasmid of Tembusu virus CQW1 strain as a template, the CQW1-P2-ΔNS1 fragment is amplified, and the sequence is shown in SEQ ID NO.3; the pACYC-FL-CQW1 plasmid is double-digested with XhoI and PmlI to obtain a linearized vector; the CQW1-P2-ΔNS1 fragment is ligated to the linearized pACYC-FL-CQW1 using homologous recombination; and the pACYC-CQW1-ΔNS1 plasmid is obtained by single-clone screening. The defective Tembusu virus genome plasmid that lacks the NS1 gene sequence from 434 to 869 nt is pACYC-MM1775-ΔNS1 plasmid. The preparation method of the pACYC-MM1775-ΔNS1 plasmid is as follows: using Tembusu virus strain MM1775 as a template, the MM1775-P1-ΔNS1 fragment is amplified, and its sequence is shown in SEQ ID NO.4; the pACYC-MM1775-P2-3 plasmid is linearized by double digestion with NruI and SacI, and the sequence of the pACYC-MM1775-P2-3 plasmid is shown in SEQ ID NO.5; the MM1775-P1-ΔNS1 fragment is ligated to the linearized pACYC-MM1775-P2-3 using homologous recombination, and the pACYC-MM1775-ΔNS1 plasmid is obtained by single cloning screening.

2. The experimental system for replication-dependent RNA recombination of flaviviruses according to claim 1, characterized in that, The preparation method of the pACYC-CQW1-ΔE plasmid is as follows: using the infectious clone plasmid of Tembusu virus CQW1 strain as a template, the DNA fragment of CQW1-P1-ΔE is amplified, and the gene sequence is shown in SEQ ID NO.1; pACYC-CQW1-P2-6 is double-digested with SpeI and XhoI to obtain a linearized vector, and the CQW1-P1-ΔE fragment is ligated with the linearized pACYC-CQW1-P2-6 vector by homologous recombination to construct the pACYC-CQW1-ΔE plasmid.

3. The application of the flavivirus replication-dependent RNA recombination experimental system as described in claim 1 or 2 in flavivirus RNA recombination models, characterized in that, RNA transcripts from component A and component B were co-transfected into cells at a 1:2 ratio. Recombinant progeny viruses were obtained 3-6 days after transfection.

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