Establishment and application of flavivirus replication-dependent RNA (Ribonucleic Acid) recombination experiment system

Abstract

The invention discloses establishment and application of a flavivirus replication-dependent RNA (Ribonucleic Acid) recombination experiment system, and belongs to the technical field of biology. The RNA recombination system comprises two components: a tembusu virus E gene deleted subgenome replicon CQW1-delta E, and a defective virus genome CQW1-delta NS1 or MM1775-delta NS1 with a part of NS1 gene sequence deleted. The two components cannot generate progeny viruses independently, but after the two components are co-transfected, a large number of recombinant progeny viruses can be generated within several days after transfection due to RNA recombination, the recombination efficiency is extremely high, continuous passage is not needed, the construction period is greatly shortened, and the production cost is saved; meanwhile, the generated progeny virus can form plaques on cells, and by means of plaque counting, the method can be applied to quantitative measurement of the RNA recombination efficiency and analysis of key factors influencing the tembusu virus RNA recombination.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an RNA recombination experimental system on which flavivirus replication depends, and an establishment method and application thereof. Background Art

[0002] TMUV belongs to the Flaviviridae family ( Flaviviridae ) Orthoflavivirus ( OrthoFlavivirus Flavivirus (FLV) is an enveloped, positive-sense RNA virus. Its genomic RNA (vRNA) is approximately 11,000 nt long and contains a single open reading frame, encoding a 3145-aa polyprotein. Posttranslationally, it is cleaved by host signal peptidases and viral proteases NS2B / 3 to produce three structural proteins: C, prM, and E, and seven nonstructural proteins: NS1, NS2A / 2B, NS3, NS4A / 4B, and NS5. The 5′ end of the genome contains a type I cap structure, a 5′ UTR of approximately 95 nt, and a 3′ UTR of approximately 600 nt, but lacks a poly(A) tail. The 3′ UTR contains multiple conserved RNA secondary structures, which play a key role in viral genome circularization and RNA replication, and influence viral host specificity. During flavivirus RNA replication, the genomic RNA must undergo circularization to facilitate the synthesis of the viral NS5 initiator RNA.

[0003] Recombination is very common in RNA viruses, but its occurrence presupposes that different viruses simultaneously infect a single cell in order to produce recombinant hybrid progeny viruses. To date, the virology community has proposed two distinct models of genomic RNA recombination mechanisms, namely replication-dependent RNA recombination and replication-independent RNA recombination. RNA recombination models can be widely used in the study of viruses. The trans-complementing recombination trap method reported in flaviviruses (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.) requires continuous passage and serial dilution to screen for a very small number of recombinants. This method has a long construction cycle and low recombination efficiency. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned problems existing in the prior art and to provide the establishment and application of an RNA recombination experimental system that is dependent on the replication of flaviviruses. The RNA recombination experimental system has an extremely high efficiency in producing or screening recombinants, and can produce a large number of recombinants within only a few days after transfection without the need for continuous passage.

[0005] The technical solution adopted in the present invention is as follows: A flavivirus replication-dependent RNA recombination experimental system comprises components A and B; component A is a replicon plasmid of the Tembusu virus lacking amino acid residues 11-472 of the E protein; and component B is a defective Tembusu virus genome plasmid lacking nt 437-878 of the NS1 gene sequence or a defective Tembusu virus genome plasmid lacking nt 434-869 of the NS1 gene sequence.

[0006] Preferably, the component A is pACYC-CQW1-ΔE plasmid, and the gene sequence is shown in SEQ ID NO.2.

[0007] Preferably, the preparation method of the pACYC-CQW1-ΔE plasmid is: using the infectious clone plasmid pACYC-FL-CQW1 plasmid of the Tembusu virus CQW1 strain as a template, amplifying the DNA fragment of CQW1-P1-ΔE, the gene sequence of which is shown in SEQ ID NO.1; double-digesting pACYC-CQW1-P2-6 with SpeI and XhoI to obtain a linearized vector, and connecting the CQW1-P1-ΔE fragment with the linearized pACYC-CQW1-P2-6 vector by homologous recombination to construct the pACYC-CQW1-ΔE plasmid.

[0008] Preferably, the defective Tembusu virus genome plasmid lacking nt 437-878 of the NS1 gene sequence is a pACYC-CQW1-ΔNS1 plasmid; Preparation method: Using the infectious clone plasmid of Tembusu virus CQW1 strain as a template, the CQW1-P2-ΔNS1 fragment was amplified, the sequence of which is shown in SEQ ID NO. 3; the pACYC-FL-CQW1 plasmid was double-digested with XhoI and PmlI to obtain a linearized vector, and the CQW1-P2-ΔNS1 fragment was ligated with the linearized pACYC-FL-CQW1 by homologous recombination. After single clone screening, the pACYC-CQW1-ΔNS1 plasmid was obtained.

[0009] Further preferably, the defective Tembusu virus genome plasmid lacking nt 434-869 of the NS1 gene sequence is pACYC-MM1775-ΔNS1 plasmid; Preparation method: Using the Tembusu virus MM1775 strain as a template, the MM1775-P1-ΔNS1 fragment was amplified, and the sequence was shown in SEQ ID NO.4; the pACYC-MM1775-P2-3 plasmid was linearized by double enzyme digestion with NruI and SacI, and the pACYC-MM1775-P2-3 plasmid sequence was shown in SEQ ID NO.5; the MM1775-P1-ΔNS1 fragment was ligated with the linearized pACYC-MM1775-P2-3 by homologous recombination, and the pACYC-MM1775-ΔNS1 plasmid was obtained by single clone screening.

[0010] The present invention also provides the application of the above-mentioned flavivirus replication-dependent RNA recombination experimental system in a flavivirus RNA recombination model, wherein the RNA transcripts of component A and component B are co-transfected into cells at a ratio of 1:2, and recombinant progeny viruses are obtained 3-6 days after transfection.

[0011] The beneficial effects of the present invention are: (1) The flavivirus replication-dependent RNA recombination experimental system established by the present invention includes two key components: the replicable CQW1-ΔE replicon and the defective genome CQW1-ΔNS1 or MM1775-ΔNS1 with a missing NS1 gene sequence. CQW1-ΔE is only capable of RNA replication due to the lack of structural genes and can serve as a donor when template jumping occurs during RNA recombination. The defective viral genome CQW1-ΔNS1 or MM1775-ΔNS1 with a missing ΔNS1 cannot replicate RNA due to the missing NS1 sequence and resulting frameshift, but it retains the conserved RNA cyclization sequence of flavivirus and can serve as a receptor when template jumping occurs during RNA recombination. Neither of them can produce progeny viruses alone, but after co-transfection, progeny viruses can be produced and plaques can be formed due to RNA recombination. By counting plaques, the efficiency of RNA recombination can be quantitatively measured and the key factors affecting Tembusu virus RNA recombination can be analyzed. Due to the similarity of the genomes of flaviviruses, this system can also be applied to other mosquito-borne flaviviruses.

[0012] (2) The flavivirus replication-dependent RNA recombination experimental system established in the present invention can produce a large number of recombinants within only a few days after transfection, with extremely high recombination efficiency and no need for continuous passage, which greatly shortens the construction cycle and saves production costs. At the same time, this experimental system can accurately evaluate or screen and confirm the key factors affecting RNA recombination through plaque counting. Therefore, it can be widely used in the establishment and research of flavivirus RNA recombination models. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the recombination system of the present invention.

[0014] Figure 2 Identification results of the RNA recombination system; (A) plaque identification; (B) recombinant type.

[0015] Figure 3 These are the application results of the recombination experimental system; among them, (A) the effect of sequence homology on recombination efficiency; (B) the effect of RNA replication on recombination efficiency; (C) the effect of NS5 mutation on recombination efficiency.

[0016] Figure 4 This is the plasmid map of pACYC-CQW1-ΔE.

[0017] Figure 5 This is the plasmid map of pACYC-CQW1-ΔNS1 plasmid.

[0018] Figure 6 It is the pACYC-MM1775-ΔNS1 plasmid.

[0019] Plasmids pACYC-FL-CQW1, pACYC-CQW1-P2-6, and pACNR-FL-MM1775 were all deposited in Sichuan Agricultural University.

[0020] Plasmid pACYC-FL-CQW1 and plasmid pACYC-CQW1-P2-6 are both disclosed in the following documents: He Yu. Research on attenuation strategy of duck Tembu virus based on abnormal C protein function[D]. Sichuan Agricultural University, 2022.DOI:10.27345 / d.cnki.gsnyu.2022.001185. Plasmid pACNR-FL-MM1775 is disclosed in the following document: 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

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1

[0023] 1. Construction of CQW1-ΔE replicon plasmid The schematic diagram of the construction of CQW1-ΔE replicon plasmid is shown in Figure 1Using the infectious clone plasmid pACYC-FL-CQW1 of the Tembusu virus CQW1 strain as a template, a fusion PCR method was used to generate the DNA fragment CQW1-P1-ΔE, shown in SEQ ID NO. 1. This fragment lacks the gene sequence corresponding to amino acid residues 11-472 of the E protein. Simultaneously, pACYC-CQW1-P2-6 was double-digested with SpeI and XhoI to obtain a linearized vector. The pACYC CQW1-P2-6 plasmid (which lacks only the P1 fragment compared to the full-length infectious clone plasmid) was derived from the infectious clone plasmid pACYC FL-CQW1, which contains the full-length cDNA of the CQW1 strain. The SpeI and XhoI restriction sites were retained to facilitate the insertion of the P1 fragment. Subsequently, the CQW1-P1-ΔE fragment was ligated with the linearized pACYC-CQW1-P2-6 vector by homologous recombination. The pACYC-CQW1-ΔE plasmid was screened by single clone, as shown in SEQ ID NO. 2, and sequenced to confirm that there was no mutation. The plasmid map is shown in the attached figure. Figure 4 shown.

[0024] 2. Construction of the CQW1-ΔNS1 replication-defective viral genome The schematic diagram of the construction of the defective genome plasmid of CQW1-ΔNS1 is shown in Figure 1 As shown. By fusion PCR method, the infectious clone plasmid pACYC-FL-CQW1 of Tembusu virus CQW1 strain was used as a template to obtain the CQW1-P2-ΔNS1 fragment (with 437-878 nt of NS1 deleted), the sequence of which is shown in SEQ ID NO.3; the pACYC-FL-CQW1 plasmid was double-digested with XhoI and PmlI to obtain a linearized vector. Subsequently, the CQW1-P2-ΔNS1 fragment was connected to the linearized pACYC-FL-CQW1 by homologous recombination method, and the pACYC-CQW1-ΔNS1 plasmid was screened by single clone and confirmed to have no mutations by sequencing. The plasmid map is shown in the attached figure. Figure 5 shown.

[0025] Example 2

[0026] The construction of CQW1-ΔE replicon plasmid was the same as in Example 1.

[0027] Construction of the MM1775-ΔNS1 replication-defective viral genome The schematic diagram of the construction of MM1775-ΔNS1 defective genome plasmid is shown in Figure 1As shown. Using the infectious clone plasmid pACNR-FL-MM1775 of the Tembusu virus MM1775 strain as a template, the MM1775-P1-ΔNS1 fragment (with the 434-869 nt of NS1 deleted) was obtained by PCR amplification, and the sequence is shown in SEQ ID NO.4; the pACYC-MM1775-P2-3 plasmid (the sequence is shown in SEQID NO.5, containing the 3328-11002 nt of the MM1775 genome) was linearized by double enzyme digestion with NruI and SacI. Subsequently, the MM1775-P1-ΔNS1 fragment was connected to the linearized pACYC-MM1775-P2-3 by homologous recombination. The pACYC-MM1775-ΔNS1 plasmid was screened by single clone and confirmed to have no mutations by sequencing. The plasmid map is shown in the attached. Figure 6 shown.

[0028] Example 3

[0029] The recombinant experimental system was verified based on Examples 1 and 2.

[0030] 1. Components and Working Principles of the Recombinant Experimental System The experimental system consists of two components: CQW1-ΔE-RNA and ΔNS1-RNA. The former, lacking the E protein, is only capable of RNA replication and cannot assemble into virions. However, it can replicate RNA normally and serve as a donor for the key enzyme NS5 during RNA recombination. ΔNS1, however, lacks a portion of the NS1 gene sequence and undergoes a subsequent frameshift, resulting in only partial translation of the final protein, making it incapable of RNA replication and viral assembly. However, its genome can circularize normally, allowing it to act as a receptor for NS5 detached from CQW1-ΔE-RNA and complete the recombination process. However, theoretically, neither of these two components produces virions or plaques upon transfection into cells. However, when co-transfected, viral plaques form only after RNA recombination has occurred and progeny viruses have been produced.

[0031] 2. Obtaining RNA transcripts Purify the pACYC-CQW1-ΔE, pACYC-CQW1-ΔNS1, and pACYC-MM1775-ΔNS1 plasmids using an endotoxin-free plasmid extraction kit or a plasmid miniprep kit for future use. Prior to in vitro transcription, linearize the plasmids using SmaI or NotI-HF restriction enzymes. Purify and recover the linearized plasmids using the TaKaRa MiniBEST DNA Fragment Purification Kit and concentrate to a concentration above 200 ng / µL. Subsequently, perform in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit. Set up the reaction mixture in a PCR tube according to the kit instructions:

[0032] After gently pipetting to mix, incubate at 37°C for 3 h. Then, add 1 μL of TURBO DNase to digest the DNA template. RNA transcripts are then purified using lithium chloride precipitation: (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; (2) Freeze at -20°C for 2 h or overnight; (3) Centrifuge at 12,000 rpm and 4°C for 15 min to allow the RNA to precipitate to the bottom. (4) Remove the supernatant, add 70% ethanol to wash the RNA precipitate, and then centrifuge at 12,000 rpm and 4°C for 15 min; (5) Carefully remove the 70% ethanol and carefully air-dry the RNA at room temperature until it becomes transparent. Then add an appropriate amount of Nuclease-free Water to dissolve the precipitate.

[0033] Finally, the concentration was measured by NanoDrop2000 and the samples were aliquoted and stored at -80°C.

[0034] 3. Validation of the Recombinant Experimental System BHK-21 cells were pre-plated in 6-well plates. When cells reached approximately 90% confluence, CQW1-ΔE and MM1775-ΔNS1 RNA transcripts were co-transfected into the cells at a 1:2 ratio using Lipofectamine MessengerMAX transfection reagent according to the reagent's instructions. Single RNA transcript transfection and blank control groups were also established. Six hours after transfection, the cell culture medium was carefully removed and a 2 mL overlay of 1% methylcellulose was added to each well. The plate was returned to the CO2 incubator and cultured for six consecutive days. After removal, the methylcellulose overlay was carefully aspirated and the cells were carefully rinsed twice with PBS. 500 µL / well of 4% formaldehyde in PBS was then added and fixed for 20 minutes at room temperature. The fixative was removed and an appropriate amount of 1% crystal violet stain was added to each well for 1 minute. The stain was then carefully rinsed off with running water. Plaques were then visualized.

[0035] The experimental results showed that no plaques were produced after single transfection of CQW1-ΔE or MM1775-ΔNS1 RNA, indicating that no infectious progeny viruses were produced. However, when CQW1-ΔE and MM1775-ΔNS1 RNA were co-transfected, plaques were clearly visible, indicating that progeny virus particles were produced (see attached). Figure 2 A).

[0036] To further verify whether the co-transfection group did produce recombinant progeny viruses, the co-transfection experiment was repeated without adding methylcellulose covering solution. On the 3rd to 4th day after transfection, the cell supernatant was harvested and passaged once in BHK-21 cells to eliminate the influence of transfected RNA transcripts. The cell supernatant after passage was harvested, and viral RNA was extracted using a virus whole genome extraction kit and reverse transcribed into cDNA as a template. The recombinant crossover region sequence ( Figure 2 B) primers for RT-PCR amplification, the amplified DNA fragments were recovered and purified and then directly connected to the T vector. Subsequently, the ligation product was transformed into E. coli competent cells and spread on agar plates with ampicillin resistance. After the single clones grew out, a large number of single clones were selected and sent directly for sequencing confirmation. The results showed that, as expected, the genomic sequences of all recombinants were derived from MM1775 at the 5 end and CQW1 at the 3 end; and the recombination intersection sites were all located in the putative recombination intersection region. According to the sequence characteristics of the recombination intersection region, recombinants can be divided into three types: precise (without any sequence redundancy or deletion), recombinants with sequence redundancy, and recombinants with sequence deletion ( Figure 2 B).

[0037] The above results demonstrate that RNA recombination successfully and efficiently occurred in this experimental system, and progeny viral particles were produced. The efficiency of obtaining recombinants was extremely high, and a large number of recombinants could be produced within just a few days after transfection, without the need for continuous passaging. In addition, this experimental system can accurately evaluate or screen and confirm key factors affecting RNA recombination through plaque counting.

[0038] Example 4

[0039] Application of Recombinant Experimental System On the basis of Examples 1-3, the recombination experimental system of the present invention was used to analyze the key factors affecting the recombination efficiency of Tembusu virus RNA.

[0040] (1) Sequence homology To compare whether there are differences in the efficiency of recombination within the homologous strain CQW1 virus and between different strains (MM1775), CQW1-ΔE was combined with CQW1-ΔNS1 and MM1775-ΔNS1, respectively, and co-transfected into cells. The results showed that the plaques produced by the CQW1-ΔE + CQW1-ΔNS1 group were significantly more than those of the CQW1-ΔE + MM1775-ΔNS1 group, indicating that sequence homology is a key factor affecting RNA recombination ( Figure 3 A).

[0041] (2) RNA replication level To verify the effect of viral RNA replication on RNA recombination, we co-transfected CQW1-ΔE and CQW1-ΔNS1 into cells and added different concentrations of the antiviral inhibitor ribavirin (which inhibits RNA replication) to analyze its effect on RNA recombination efficiency. The results showed that the addition of ribavirin significantly reduced the number of plaques, indicating that RNA replication is crucial for RNA recombination. Figure 3 B).

[0042] (3) NS5 mutation NS5 is the viral RNA polymerase and plays a key role in the synthesis of viral RNA. G643R and L612F amino acid substitutions were introduced into the NS5 gene sequence of CQW1-ΔNS1, followed by RNA recombination experiments. The results showed that both substitutions significantly reduced plaque formation. This suggests that amino acid substitutions in NS5 affect the efficiency of RNA recombination. Figure 3 C).

[0043] In summary, the present invention discloses a method for establishing and applying a Tembusu virus replication-dependent RNA recombination experimental system. Using reverse genetics techniques, the present invention constructs the Tembusu virus E gene-deficient subgenomic replicon CQW1-ΔE, as well as defective viral genomes CQW1-ΔNS1 or MM1775-ΔNS1, each lacking a portion of the NS1 gene sequence. CQW1-ΔE, lacking structural genes, is only capable of RNA replication and can serve as a donor for template jumping during RNA recombination. The defective viral genome lacking ΔNS1, however, is incapable of RNA replication due to the frameshift caused by the NS1 sequence loss. However, it retains the conserved RNA cyclization sequence of flaviviruses and can serve as a receptor for template jumping during RNA recombination. Neither of these two systems can produce progeny viruses individually, but when co-transfected, progeny viruses can be produced due to RNA recombination, forming plaques. Plaque counting can quantitatively measure the efficiency of RNA recombination and analyze key factors influencing Tembusu virus RNA recombination. This system can also be applied to mosquito-borne flaviviruses.

[0044] The description and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative work, and all of them are within the scope of protection of the present invention.

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 the Tembusu virus lacking the 11-472 amino acid residues of the E protein; component B is a defective Tembusu virus genome plasmid lacking the 437-878 nt of the NS1 gene sequence or a defective Tembusu virus genome plasmid lacking the 434-869 nt of the NS1 gene sequence.

2. The flavivirus replication-dependent RNA recombination experimental system according to claim 1, characterized in that: The component A is the pACYC-CQW1-ΔE plasmid, and the gene sequence is shown in SEQ ID NO.

2.

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

4. The flavivirus replication-dependent RNA recombination experimental system according to claim 1, characterized in that: The defective Tembusu virus genome plasmid lacking nt 437-878 of the NS1 gene sequence is the pACYC-CQW1-ΔNS1 plasmid; The preparation method of the pACYC-CQW1-ΔNS1 plasmid comprises the following steps: using the infectious clone plasmid of the Tembusu virus CQW1 strain as a template, amplifying a CQW1-P2-ΔNS1 fragment, the sequence of which is shown in SEQ ID NO. 3; double-digesting the pACYC-FL-CQW1 plasmid with XhoI and PmlI to obtain a linearized vector; ligating the CQW1-P2-ΔNS1 fragment with the linearized pACYC-FL-CQW1 by homologous recombination, and obtaining the pACYC-CQW1-ΔNS1 plasmid through single clone screening.

5. The flavivirus replication-dependent RNA recombination experimental system according to claim 1, characterized in that: The defective Tembusu virus genome plasmid lacking nt 434-869 of the NS1 gene sequence is the pACYC-MM1775-ΔNS1 plasmid; The preparation method of the pACYC-MM1775-ΔNS1 plasmid comprises the following steps: using the Tembusu virus MM1775 strain as a template, amplifying and obtaining the MM1775-P1-ΔNS1 fragment, the sequence of which is shown in SEQ ID NO.4; linearizing the pACYC-MM1775-P2-3 plasmid by double enzyme digestion with NruI and SacI, the sequence of which is shown in SEQ ID NO.5; and connecting the MM1775-P1-ΔNS1 fragment with the linearized pACYC-MM1775-P2-3 by homologous recombination, and obtaining the pACYC-MM1775-ΔNS1 plasmid through single clone screening.

6. Use of the flavivirus replication-dependent RNA recombination experimental system according to any one of claims 1 to 5 in a flavivirus RNA recombination model, characterized in that: The RNA transcripts of component A and component B are co-transfected into cells at a ratio of 1:

2. Recombinant progeny viruses are obtained 3-6 days after transfection.

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