A method of inhibiting and promoting the multiplication of pseudorabies virus

Abstract

The application belongs to the technical field of biology, and discloses a method for inhibiting the proliferation of pseudorabies virus, which is characterized by overexpressing DDX5 gene in host cells, and using the modified host cells to inhibit the proliferation of pseudorabies virus. Meanwhile, the application also discloses a method for proliferating pseudorabies virus by knocking out or knocking down DDX5 gene in host cells. The application finds that DDX5 gene has a significant influence on the proliferation of PRV through research, and it is verified that overexpression of DDX5 gene can inhibit the proliferation of PRV, and knocking out DDX5 can promote the proliferation of PRV.

Description

A method for inhibiting and promoting the proliferation of pseudorabies virus Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for inhibiting and promoting the proliferation of pseudorabies virus. Background Technology

[0002] Herpesviruses are diverse, among which pseudorabies virus (PRV), belonging to the Alphaherpesviridae family, can cause pseudorabies (PR). PRV has a wide host range, including pigs, cattle, sheep, dogs, cats, rodents, and rabbits. PRV poses a potential risk of cross-species transmission. Pigs are the only natural host for PRV, and pigs at all stages are susceptible, leading to abortion and stillbirth in pregnant sows, infertility in boars, encephalitis and high mortality in newborn piglets, and respiratory distress and growth retardation in finishing pigs, resulting in significant economic losses. PRV can establish lifelong latent infection in the trigeminal ganglion of the pig's peripheral nervous system, and can be reactivated and intermittently shed under stress and weakened immunity, leading to recurrent outbreaks of PRV in pig farms that are difficult to control and eradicate. Currently, vaccination is the primary means of controlling porcine pseudorabies, with live attenuated vaccines being the main type of vaccine. Since the end of 2011, the emergence of PRV variants has led to relatively insufficient immunization doses and frequencies of existing vaccines, increasing the difficulty of controlling and eradicating porcine pseudorabies. In the production of live attenuated vaccines, increasing virus titer and employing efficient culture technologies to reduce production costs and increase capacity have become key issues in porcine pseudorabies vaccine development.

[0003] As parasites, viruses engage in a game-like process of infection and replication with their hosts. For the virus itself, on the one hand, it needs to utilize intracellular host factors to achieve the entire infection process, including uncoating, transcription, replication, assembly, and release. On the other hand, it also needs to resist the restrictive effects of host factors and antiviral responses to escape cellular clearance. Therefore, identifying host restrictive factors in the PRV infection process and targeting their expression has become a novel strategy for increasing viral titers and achieving efficient culture.

[0004] Therefore, the technical problem this case addresses is: how to find new strategies to modify host cells in order to achieve artificial control over viral proliferation or inhibition. Summary of the Invention

[0005] The purpose of this invention is to provide a method for inhibiting and promoting the proliferation of pseudorabies virus. Through research, this invention has found that the DDX5 gene has a significant impact on PRV proliferation. Verification has shown that overexpression of the DDX5 gene can inhibit PRV proliferation, while knocking out DDX5 can promote PRV proliferation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for inhibiting the proliferation of pseudorabies virus involves overexpressing the DDX5 gene in host cells, and using the modified host cells to inhibit the proliferation of pseudorabies virus.

[0008] Regarding the DDX5 gene, existing reports document its applications in anti-tumor and antiviral treatments. In the aquaculture industry, there are clearly documented literature reports on its use.

[0009] "A Study on the Mechanism of Host Proteins DDX5 and HOIP Affecting the Proliferation of Avian Influenza Virus in Human Cells," authored by Ren Xingxing et al., describes how DDX5 interacts with AIV polymerase, promoting AIV polymerase activity, thereby promoting AIV RNA transcription and replication, and subsequently affecting RNP nuclear export. Therefore, the DDX5 gene is one of the host proteins on which AIV's efficient replication depends. Using siRNA interference and CRISPR / Cas9 gene knockout technology to silence or knock out the expression of endogenous DDX5 protein, respectively, it was found that the replication level of influenza virus decreased significantly at 36 and 48 hours, indicating that DDX5 promotes AIV proliferation.

[0010] "The interaction between host cell protein DDX5 and Nsp9 positively regulates PRRSV replication," Zhao Shuangcheng et al., recorded that overexpression of DDX5 can significantly promote PRRSV replication.

[0011] "A Preliminary Study on the Inhibition of Host Innate Immunity by DDX5 Protein" by Ma Zhenbang et al. describes how DDX5 acts as a negative antiviral regulator in innate immunity, inhibiting the production of IFN-β and IL-6 after viral infection and promoting viral replication.

[0012] The above records all demonstrate that DDX5 can promote virus replication.

[0013] In the current technology, no research has been conducted on the association between the DDX5 gene and herpesviruses.

[0014] The study in this application found that overexpression of the DDX5 gene can inhibit PRV replication, which is contrary to the conclusions of traditional studies. This means that there are significant differences between the signaling pathway regulated by the DDX5 gene and the signaling pathway regulated by traditional methods.

[0015] We believe that the mechanism by which DDX5 inhibits PRV proliferation is as follows: DDX5 protein interacts with PRV VP16 protein and promotes its autophagy-dependent degradation. In terms of autophagy dependence, DDX5 interacts with OPTN, the autophagy receptor on which VP16 autophagy degradation depends. This further verifies that DDX5 recruits UBE3C to catalyze the ubiquitination of PRV VP16 protein to achieve autophagy degradation.

[0016] In the above method, the method for overexpressing the DDX5 gene in host cells is as follows: host cells are transfected with a plasmid containing the DDX5 gene. After successful transfection, host cells overexpressing the DDX5 gene can be obtained.

[0017] In the above method, the host cell is a 293T cell or a PK-15 cell.

[0018] In the above method, the plasmid is a plasmid into which the DDX5 gene is inserted into the HA vector.

[0019] Meanwhile, the present invention also discloses a method for promoting the proliferation of pseudorabies virus, which uses host cells with knockout or knockdown of the DDX5 gene to proliferate pseudorabies virus.

[0020] In the above methods, the method for knocking out the DDX5 gene in the host cell is as follows:

[0021] Host cells with the DDX5 gene knocked out were obtained by transfecting host cells with siDDX5 or siNC.

[0022] In the above method, the host cell is a 293T cell or a PK-15 cell.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] In the technical solution of this invention, overexpression of the DDX5 gene can inhibit PRV replication, which is contrary to the conclusions of traditional studies. This means that there are significant differences between the signaling channels regulated by the DDX5 gene and those regulated by traditional methods. Attached Figure Description

[0025] Figure 1 shows the transcriptional levels of DDX5 in PK-15 cells at 4 h, 8 h, and 12 h after PRV infection.

[0026] Figure 2 shows the transcriptional levels of DDX5 in 293T cells at 4 h, 8 h, and 12 h after PRV infection.

[0027] Figure 3 shows the relative content of PRV genomic DNA after DDX5 overexpression;

[0028] Figure 4 shows the results of PRV proliferation after DDX5 overexpression;

[0029] Figure 5 shows the silencing efficiency of siDDX5;

[0030] Figure 6 shows the relative content of PRV genomic DNA after DDX5 silencing;

[0031] Figure 7 shows the effect of knocking down DDX5 on enhancing the proliferation ability of PRV;

[0032] Figure 8a shows the results of PARP1's inability to induce VP16 protein degradation;

[0033] Figure 8b shows the results of PCBP2 failing to induce VP16 protein degradation.

[0034] Figure 8c shows the results of DDX5 inducing the degradation of VP16 protein in a dose-dependent manner;

[0035] Figure 8d shows the results of the interaction between DDX5 and VP16 proteins;

[0036] Figure 8e shows the results of DDX5 inducing the degradation of VP16 protein via autophagy;

[0037] Figure 9a shows the results of the interaction between DDX5 and OPTN;

[0038] Figure 9b shows the results of silencing OPTN blocking DDX5 degradation of VP16 protein;

[0039] Figure 10a shows the results of DDX5 promoting ubiquitination of VP16 protein;

[0040] Figure 10b shows the results of ZNF598 failing to induce VP16 protein degradation.

[0041] Figure 10c shows the results of RNF138 failing to induce VP16 protein degradation.

[0042] Figure 10d shows the results of UBE3C promoting the degradation of VP16 protein in a dose-dependent manner;

[0043] Figure 10e shows the results of the interaction between UBE3C and VP16 protein;

[0044] Figure 10f shows the results of UBE3C promoting ubiquitination of VP16 protein;

[0045] Figure 10g shows the results of the interaction between DDX5 and UBE3C;

[0046] Figure 10h shows the silencing efficiency of siUBE3C;

[0047] Figure 10i shows the results of silencing UBE3C blocking the degradation of VP16 protein by DDX5. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: Overexpression of the DDX5 gene

[0050] 1.1 Detection of the effect of PRV infection on DDX5 expression in host cells

[0051] PK-15 and 293T cells were inoculated with PRV at MOI=1, and the mRNA transcription level of DDX5 in the cells was detected by relative quantitative qPCR at different time points after virus infection. The data showed that the transcription level of DDX5 was significantly upregulated at 4 h, 8 h and 12 h after PRV infection in both PK-15 and 293T cells (Figures 1 and 2), suggesting that DDX5 may be involved in regulating PRV replication.

[0052] 1.2 Overexpression of DDX5 gene

[0053] To determine the effect of DDX5 on PRV replication, pCAGGS-HA-DDX5 and pCAGGS-HA plasmids were transfected into 293T cells, and then PRV was inoculated at an infection dose of MOI=1. Cell DNA and cell supernatant were collected 24 h later.

[0054] The specific procedure for transfecting 293T cells is as follows: Seed cells in six-well plates 18-24 hours before transfection, ensuring a cell density of approximately 70-80% by the next day. Replace the medium with fresh medium before transfection. Prepare the transfection complex by adding 125 μL of DMEM and 2.5 g of plasmid (or 100 pmol siRNA) to a 1.5 mL sterile centrifuge tube and gently mixing with a pipette. Add 4 µL of Beyotime lipo8000™ transfection reagent and gently mix with a pipette. Add the transfection mixture evenly to each well, gently mix, and continue culturing for 24 hours before proceeding with subsequent experiments.

[0055] The preparation method of HA-DDX5 is as follows:

[0056] Primers were designed based on the DDX5 gene sequence published by NCBI (GenBank: NM_001320595.2), and Sac I and Xho I restriction sites were added to the 5' ends of the upstream and downstream primers, respectively (upstream primer sequence: 5-CGACCTCtcgggttattcgagtgaccg-3 (SEQ ID NO.1), downstream primer sequence: 5-CCCTCGAGttgggaatatcctgttggcatt-3 (SEQ ID NO.2), and were sent to Shanghai Sangon Biotech Co., Ltd. for synthesis.

[0057] Using total RNA from 293T cells as a template, reverse transcription was performed followed by amplification with a high-fidelity enzyme to obtain the target fragment, which was then purified using the Gel Extraction Kit D2500 according to the instructions. The purified target fragment was double-digested with restriction endonucleases and then purified again. Then, a linearized vector and the insert fragment were ligated using T4 ligase, and the resulting plasmid was transformed into DH5α competent cells to construct a recombinant plasmid. Finally, a single colony grown on an antibiotic-resistant plate was picked and mixed in 50 μL of ultrapure water, and 1 μL was used as a template for PCR amplification. After preliminary confirmation by agarose gel electrophoresis, the plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0058] qPCR analysis showed that DDX5 overexpression significantly reduced the relative content of PRV genomic DNA (Figure 3). Similarly, TCID... 50 The assay showed that overexpression of DDX5 significantly inhibited the proliferation of PRV (Figure 4), indicating that DDX5 restricts the replication and proliferation of PRV.

[0059] Example 2: Knockdown of the DDX5 gene

[0060] Transfect siDDX5 or siNC into 293T cells;

[0061] The siRNA sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., and the sequence is shown in Table 1.

[0062] Table 1. siRNA sequences of the target genes

[0063] Gene Name Sensitive Sequence (5'-3') Antisense Sequence (5'-3') OPTNTGCACGGCAUUGUCUAAAUATT (SEQ ID NO.3) UAUUUAGACAAUGCCGUGCTT (SEQ ID NO.4) DDX5ACAUAAAGCAAGUGAGCGATT (SEQ ID NO.5) UCGCUCACUUGCUUUAUGUTT (SEQ ID NO.6) UBE3CGCAGAUAAGCAAGAAGUUCAATT (SEQ ID NO.7) UUGAACUUCUUGCUUAUCUGCTT (SEQ ID NO.8) NCUUCUCCGAACGUGUCACGUTT (SEQ ID NO.9) ACGUGACACGUUCGGAGAATT (SEQ ID NO.10) surface

[0064] The method for transfecting 293T cells with siDDX5 is as follows:

[0065] One day before transfection (18-24 hours), seed cells into six-well plates to achieve a cell density of approximately 70%-80% by the next day. Replace the medium with fresh medium before transfection. Prepare the transfection complex by adding 125 μL LMEM and 100 pmol siRNA to a 1.5 mL sterile centrifuge tube and gently mixing with a pipette. Add 4 µL of Beyotime lipo8000™ transfection reagent, gently mix with a pipette, and incubate at room temperature for 20 min. Add the transfection mixture evenly to each well, gently mix, and continue culturing for 24 h before proceeding with subsequent experiments.

[0066] After verifying the silencing efficiency of siDDX5 by qPCR (Figure 5), PRV was infected with MOI=1, and cell DNA and cell supernatant were collected after 24 h.

[0067] qPCR analysis showed that DDX5 silencing significantly increased the relative content of PRV genomic DNA (Figure 6).

[0068] TCID 50 The results showed that knocking down DDX5 significantly enhanced the proliferation ability of PRV (Figure 7).

[0069] These results indicate that silencing DDX5 enhances PRV replication and proliferation, suggesting that DDX5 is a host-limiting factor for PRV infection.

[0070] Example 3: Mechanism study of DDX5 inhibiting PRV proliferation

[0071] To further investigate the mechanism by which DDX5 inhibits PRV proliferation, this section clarifies the specific mechanisms through the relationships between DDX5-VP16 protein-autophagy-dependent degradation, OPTN-DDX5-autophagy degradation, and E3 ubiquitin ligase-DDX5-selective autophagy receptor, as detailed below:

[0072] 3.1 The interaction between the host restriction factor DDX5 and VP16 protein promotes its autophagic degradation.

[0073] Host restriction factors play a key role in recognizing pathogen proteins and activating selective autophagy.

[0074] To screen for host restriction factors that interact with the VP16 protein, this application transfected 293T cells with the Flag-VP16 plasmid or the Flag empty vector, followed by immunoprecipitation and mass spectrometry analysis. The mass spectrometry results showed that host factors such as PARP1, PCBP2, and DDX5 may interact with VP16.

[0075] In this application, different quantities (300 ng, 600 ng, 900 ng) of HA-PARP1, HA-PCBP2, HA-DDX5 and Flag-VP16 were co-transfected into 293T cells to study the effect of these host proteins on the VP16 protein content.

[0076] Western blot results showed that PARP1 and PCBP2 did not induce VP16 degradation (Figures 8a and 8b). Overexpression of DDX5 alone significantly reduced VP16 protein levels, and the protein abundance of VP16 was inversely proportional to the transfection dose of DDX5 (Figure 8c), indicating that DDX5 promotes VP16 protein degradation in a dose-dependent manner. This suggests that DDX5 may be involved in the cellular anti-PRV response.

[0077] In this study, HA-DDX5 and Flag-VP16 were co-transfected in 293T cells for CO-IP experiments. The results also confirmed the interaction between DDX5 and VP16 (Figure 8 d).

[0078] To investigate the pathway by which DDX5 degrades VP16 protein, this study added the proteasome inhibitor MG132 or the autophagy inhibitor CQ to 293T cells overexpressing HA-DDX5 and Flag-VP16, respectively, and collected cellular proteins after 6 hours of incubation. Western blotting showed that CQ blocked the degradation of VP16 by DDX5, while MG132 did not (Figure 8e). This indicates that DDX5 degrades VP16 protein through the autophagy-lysosome pathway, rather than the ubiquitin-protease system. These results demonstrate that the host DDX5 protein interacts with PRV VP16 protein and promotes its autophagy-dependent degradation.

[0079] Figure 8a shows the results of PARP1's inability to induce VP16 protein degradation;

[0080] Figure 8b shows the results of PCBP2 failing to induce VP16 protein degradation.

[0081] Figure 8c shows the results of DDX5 inducing the degradation of VP16 protein in a dose-dependent manner;

[0082] Figure 8d shows the results of the interaction between DDX5 and VP16 proteins;

[0083] Figure 8e shows the results of DDX5 inducing the degradation of VP16 protein via autophagy.

[0084] 3.2 DDX5 degrades PRV VP16 protein via the selective receptor OPTN.

[0085] To investigate the role of OPTN in DDX5-induced autophagic degradation of VP16 protein, this application transfected HA-DDX5 and Flag-OPTN in 293T cells for CO-IP assay.

[0086] The results showed that DDX5 interacts with the autophagy receptor OPTN (Figure 9a).

[0087] To further determine that DDX5-induced autophagic degradation of VP16 depends on OPTN, 293T cells were co-transfected with HA-DDX5 and Flag-VP16 after OPTN silencing, and VP16 protein levels were analyzed using Western blotting. The results showed that silencing OPTN blocked the degradation of VP16 protein by DDX5 (Figure 9b). These results indicate that DDX5 degrades PRV VP16 protein through the selective receptor OPTN.

[0088] Figure 9a shows the results of the interaction between DDX5 and OPTN;

[0089] Figure 9b shows the results of silencing OPTN blocking DDX5 degradation of VP16 protein.

[0090] 3.3 DDX5 recruits the E3 ubiquitin ligase UBE3C to ubiquitinate PRV VP16 protein

[0091] The ubiquitination of substrates by E3 ubiquitin ligase is key to the binding of selective autophagy receptors.

[0092] First, this application detected the ubiquitination level of VP16 by CO-IP and found that overexpression of DDX5 significantly increased the ubiquitination of VP16 (Figure 10a).

[0093] To screen E3 ubiquitin ligases involved in VP16 ubiquitination, mass spectrometry analysis showed that ZNF598, RNF138, and UBE3C may interact with the VP16 protein.

[0094] Therefore, this application first verifies the effect of these E3 ubiquitin ligases on VP16 protein content. Flag-VP16 and different doses (300 ng, 600 ng, 900 ng) of MYC-ZNF598, MYC-RNF138, and MYC-UBE3C were transfected into 293T cells.

[0095] Western blot results showed that overexpression of ZNF598 and RNF138 did not cause a decrease in VP16 protein content (Fig. 10b and Fig. 10c), but overexpression of UBE3C caused a dose-dependent decrease in VP16 protein content (Fig. 10d).

[0096] To investigate whether UBE3C promotes VP16 ubiquitination, this application simultaneously overexpressed MYC-UBE3C and Flag-VP16 in 293T cells.

[0097] CO-IP results showed that UBE3C and VP16 do indeed interact (Figure 10e). Notably, overexpression of UBE3C increased the ubiquitination level of VP16 (Figure 10f).

[0098] To further investigate whether UBE3C participates in the autophagic degradation of VP16 protein by DDX5, we first overexpressed MYC-UBE3C and HA-DDX5 in 293T cells and confirmed the interaction between UBE3C and DDX5 by CO-IP (Figure 10 g).

[0099] In addition, this application verified the silencing efficiency of siUBE3C at the mRNA level by transfecting siRNA or siNC (Figure 10h), and then analyzed the effect of silencing UBE3C on DDX5-induced VP16 protein degradation. Western blot results showed that knocking down UBE3C in cells restored the protein content of VP16, indicating that silencing UBE3C significantly inhibited the degradation of VP16 by DDX5 (Figure 10i).

[0100] Figure 10a shows the results of DDX5 promoting ubiquitination of VP16 protein;

[0101] Figure 10b shows the results of ZNF598 failing to induce VP16 protein degradation.

[0102] Figure 10c shows the results of RNF138 failing to induce VP16 protein degradation.

[0103] Figure 10d shows the results of UBE3C promoting the degradation of VP16 protein in a dose-dependent manner;

[0104] Figure 10e shows the results of the interaction between UBE3C and VP16 protein;

[0105] Figure 10f shows the results of UBE3C promoting ubiquitination of VP16 protein;

[0106] Figure 10g shows the results of the interaction between DDX5 and UBE3C;

[0107] Figure 10h shows the silencing efficiency of siUBE3C;

[0108] Figure 10i shows the results of silencing UBE3C blocking the degradation of VP16 protein by DDX5.

[0109] The above analysis used GraphPad Prism 8 software to perform statistical analysis on all experimental data. ** indicates P < 0.01, which is highly significant.

[0110] The above results collectively demonstrate that DDX5 recruits UBE3C to catalyze PRV VP16 protein ubiquitination.

[0111] In summary, we believe that the mechanism by which DDX5 inhibits PRV proliferation is as follows: DDX5 protein interacts with PRV VP16 protein and promotes its autophagy-dependent degradation. In terms of autophagy dependence, DDX5 interacts with OPTN, the autophagy receptor on which VP16 autophagy degradation depends. This further verifies that DDX5 recruits UBE3C to catalyze the ubiquitination of PRV VP16 protein to achieve autophagy degradation.

Claims

1. A method for inhibiting the proliferation of pseudorabies virus, characterized in that, The host cells were modified to overexpress the DDX5 gene to inhibit the proliferation of pseudorabies virus. The host cells were either 293T cells or PK-15 cells. The method for overexpressing the DDX5 gene in the host cells was as follows: the host cells were transfected with a plasmid containing the DDX5 gene. After successful transfection, host cells overexpressing the DDX5 gene were obtained. The plasmid was a plasmid into an HA vector with the DDX5 gene inserted. The gene sequence of the DDX5 gene was the nucleotide sequence with the number NM_001320595.2 in GenBank.

2. A method for promoting the proliferation of pseudorabies virus, characterized in that, Pseudorabies virus was propagated in host cells with the DDX5 gene knocked down. The method for preparing the host cells with the DDX5 gene knocked down was as follows: host cells were transfected with siDDX5 to obtain host cells with the DDX5 gene knocked out; the host cells were 293T cells or PK-15 cells; the siDDX5 consisted of a sense sequence and an antisense sequence; the nucleotide sequence of the sense sequence was shown in SEQ ID NO.5; the nucleotide sequence of the antisense sequence was shown in SEQ ID NO.6.

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