Method for inhibiting and promoting proliferation of pseudorabies virus

Abstract

The invention belongs to the technical field of biology, and discloses a method for inhibiting proliferation of pseudorabies virus, DDX5 gene overexpression transformation is carried out on host cells, and the transformed host cells are used for inhibiting proliferation of pseudorabies virus. Meanwhile, the invention further discloses a method for proliferating the pseudorabies virus through the host cell with the DDX5 gene knocked out or knocked down. Research finds that the DDX5 gene has obvious influence on PRV proliferation, and verification finds that over-expression of the DDX5 gene can inhibit PRV proliferation, and knockout of the DDX5 gene can promote PRV proliferation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for inhibiting and promoting the proliferation of pseudorabies virus. Background Art

[0002] There are many types of herpes viruses, among which pseudorabies virus (PRV) belongs to the alphaherpesviridae family and can infect and cause pseudorabies (PR). PRV has a wide range of hosts, including pigs, cattle, sheep, dogs, cats, mice, rabbits, etc. PRV has a potential risk of cross-species transmission. Pigs are the only natural host of PRV, and pigs at all stages are susceptible, causing abortion and stillbirth in pregnant sows, infertility in boars, encephalitis and high mortality in newborn piglets, and breathing difficulties and growth stagnation in fattening pigs. PRV can establish a lifelong latent infection in the trigeminal ganglia of the pig's peripheral nervous system, and reactivate and intermittently excrete toxins under stress, reduced immunity, etc., which leads to repeated epidemics of PRV in pig farms, which is difficult to control and eradicate. At present, vaccination is an important means of preventing and controlling pseudorabies in pigs, and the types of vaccines are mainly attenuated live vaccines. Since the end of 2011, the emergence of PRV variants has resulted in relatively insufficient immunization doses and immunization frequencies of the original vaccines, increasing the difficulty of preventing and purifying pseudorabies in pigs. In the production process of live attenuated vaccines, virus titer enhancement and efficient culture technology help reduce production costs and increase production capacity, becoming key issues in the development of pseudorabies vaccines in pigs.

[0003] As a parasite, the infection and replication process of the virus is actually a game reaction between the virus and the host. For the virus itself, on the one hand, it needs to use the host factors in the cell to realize the entire infection process of the virus itself, including uncoating, transcription, replication, assembly and release. On the other hand, it also needs to resist the restriction and antiviral response of host factors and escape the clearance of cells. Based on this, discovering the host restriction factors in the PRV infection process and targeting and inhibiting their expression has become a new strategy for increasing virus titer and efficient culture.

[0004] Therefore, the technical problem solved in this case is: how to find new strategies to transform host cells to achieve artificial control of viral proliferation or inhibition. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inhibiting and promoting the proliferation of pseudorabies virus. The present invention has found through research that the DDX5 gene has a significant effect on the proliferation of PRV. It has been verified that overexpression of the DDX5 gene can inhibit the proliferation of PRV, and knocking out DDX5 can promote the proliferation of PRV.

[0006] To achieve the above object, the present invention provides the following technical solutions:

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

[0008] Regarding the DDX5 gene, existing reports have documented its application in anti-tumor and anti-viral aspects. In the aquaculture industry, there are obvious reports in the literature:

[0009] "Research on the mechanism by which host proteins DDX5 and HOIP affect the proliferation of avian influenza virus in human cells", written by Ren Xingxing et al., records that DDX5 interacts with AIV polymerase, promotes the activity of AIV polymerase, thereby promoting the transcription and replication of AIV RNA, and further affecting the nuclear export of RNP. Therefore, the DDX5 gene is one of the host proteins that AIV relies on for efficient replication. The expression of endogenous DDX5 protein was silenced or knocked out using siRNA interference and CRISPR / Cas9 gene knockout technology, respectively. It was found that the replication level of influenza virus decreased significantly at 36 and 48 hours, indicating that DDX5 promotes the proliferation of AIV.

[0010] "Interaction between host cell protein DDX5 and Nsp9 positively regulates the replication of PRRSV", written by Zhao Shuangcheng et al., records that overexpression of DDX5 can significantly promote the replication of PRRSV.

[0011] "Preliminary Study on DDX5 Protein Inhibiting Host Innate Immunity", written by Ma Zhenbang et al., records that DDX5 acts as a negative antiviral regulatory factor in innate immunity to inhibit the production of IFN-β and IL-6 after viral infection and promote viral replication.

[0012] The above records all prove that DDX5 can promote viral replication.

[0013] In the prior art, no one has studied the relationship between the DDX5 gene and herpes virus.

[0014] In the research of the present application, it was found that overexpression of the DDX5 gene can inhibit the replication of PRV, which is contrary to the conclusion of traditional research, which means that there are obvious differences between the signal pathway regulated by the DDX5 gene and the signal pathway of traditional methods.

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

[0016] In the above method, the method for overexpressing the DDX5 gene in the host cell is: using a plasmid containing the DDX5 gene to transfect the host cell, and after successful transfection, the host cell overexpressing the DDX5 gene can be obtained.

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

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

[0019] At the same time, the present invention also discloses a method for promoting the proliferation of pseudorabies virus, which uses host cells with knocked-out or knocked-down DDX5 genes to propagate pseudorabies virus.

[0020] In the above method, the method for knocking out the DDX5 gene of the host cell is:

[0021] The host cells were transfected with siDDX5 or siNC to obtain host cells with DDX5 gene knocked out.

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

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] In the technical solution of the present invention, overexpression of the DDX5 gene can inhibit the replication of PRV, which is contrary to the conclusion obtained from traditional studies, which means that there are obvious differences between the signal pathway regulated by the DDX5 gene and the signal pathway of traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The result graph is the transcription level of DDX5 in PK-15 cells at 4h, 8h and 12h after PRV infection;

[0026] Figure 2 The result graph is the transcription level of DDX5 in 293T cells at 4h, 8h and 12h after PRV infection;

[0027] Figure 3 The result graph is the relative content of PRV genomic DNA after DDX5 overexpression;

[0028] Figure 4 The result diagram is the proliferation ability of PRV after overexpression of DDX5;

[0029] Figure 5 This is a result diagram of the silencing efficiency of siDDX5;

[0030] Figure 6The result graph is the relative content of PRV genomic DNA after DDX5 silencing;

[0031] Figure 7 This is a graph showing the results of knocking down DDX5 to enhance the proliferation ability of PRV;

[0032] Figure 8a This is the result diagram showing that PARP1 cannot induce VP16 protein degradation;

[0033] Figure 8b This is the result diagram showing that PCBP2 cannot induce VP16 protein degradation;

[0034] Figure 8c The figure shows the result that DDX5 induces the degradation of VP16 protein in a dose-dependent manner;

[0035] Figure 8d This is the result diagram showing the interaction between DDX5 and VP16 protein;

[0036] Figure 8e The figure shows the result of DDX5 inducing the degradation of VP16 protein through the autophagy pathway;

[0037] Figure 9a This is the result diagram showing the interaction between DDX5 and OPTN;

[0038] Figure 9b This is the result diagram showing that silencing OPTN can block DDX5 from degrading VP16 protein;

[0039] Fig.10a This is a result diagram showing that DDX5 promotes the ubiquitination of VP16 protein;

[0040] Fig.10b This is the result diagram showing that ZNF598 cannot induce VP16 protein degradation;

[0041] Fig.10c The result figure shows that RNF138 cannot induce VP16 protein degradation;

[0042] Fig.10d The figure shows that UBE3C promotes the degradation of VP16 protein in a dose-dependent manner;

[0043] Fig.10e This is the result diagram showing the interaction between UBE3C and VP16 protein;

[0044] Fig.10f This is a result diagram showing that UBE3C promotes the ubiquitination of VP16 protein;

[0045] Figure 10g This is the result diagram showing the interaction between DDX5 and UBE3C;

[0046] Fig.10h The result diagram is the silencing efficiency of siUBE3C;

[0047] Fig.10i This figure shows that silencing UBE3C can block the degradation of VP16 protein by DDX5. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1 Overexpression of 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 cells at different time points after virus infection was detected by relative quantitative qPCR. The data showed that in PK-15 and 293T cells, the transcription level of DDX5 could be significantly upregulated at 4h, 8h and 12h after PRV infection ( Figure 1 and Figure 2 ), which suggests that DDX5 may be involved in regulating the replication of PRV.

[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, respectively, and then PRV was inoculated at an infection dose of MOI = 1. Cell DNA and cell supernatant were collected after 24 h.

[0054] The specific operation of transfection into 293T cells is as follows: the cells are inoculated into a six-well plate one day before transfection (18h-24h), so that the cell density can reach about 70%-80% on the next day, and fresh culture medium is replaced before transfection. Prepare the transfection complex, add 125L DMEM and 2.5g plasmid (or 100pmol siRNA) into a 1.5mL sterile centrifuge tube, and mix it gently with a gun. Then add 4μL of Bio-Tech Lipo8000 TM Add the transfection reagent and mix it with a gun. Add the transfection mixture evenly to the entire well, then mix it gently and continue to culture for 24 hours before conducting subsequent experiments.

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

[0056] Primers were designed according to 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 primer and downstream primer, respectively (upstream primer sequence: 5-CGACCTCtcgggttattcgagtgaccg-3, downstream primer sequence: 5-CCCTCGAGttgggaatatcctgttggcatt-3), and sent to Shanghai Shenggong Biological Company for synthesis.

[0057] Using the total RNA of 293T cells as a template, after reverse transcription, a high-fidelity enzyme was used for amplification reaction to obtain the target fragment, and gel recovery and purification were performed according to the instructions of the Gel Extraction Kit D2500 gel recovery kit. The purified target fragment was double-digested with nucleic acid restriction endonucleases and recovered and purified. Then, the linearized vector and the fragment to be inserted were connected using T4 ligase, and transferred into DH5α competent cells to construct a recombinant plasmid. Finally, a single colony grown on the resistant plate was picked in 50 μL ultrapure water, blown and mixed, and 1 μL was taken from it as a PCR amplification template. After preliminary identification by agarose gel electrophoresis, the plasmid was extracted and sent to Shanghai Shenggong Biological Company 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 DDX5 gene

[0060] 293T cells were transfected with siDDX5 or siNC;

[0061] The siRNA sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and are shown in Table 2.

[0062] Table 2 siRNA sequences of target genes

[0063]

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

[0065] The day before transfection (18h-24h), cells were seeded in a six-well plate so that the cell density could reach about 70%-80% on the next day. Fresh culture medium was replaced before transfection. Prepare transfection complexes by adding 125μL DMEM and 100pmol siRNA to a 1.5mL sterile centrifuge tube and gently pipetting to mix. Then add 4μL Biotech Lipo8000 TM The transfection reagent was gently blown with a gun to mix, and incubated at room temperature for 20 minutes. The above transfection mixture was evenly added to the entire well, and then gently mixed. After continuing to culture for 24 hours, subsequent experiments can be carried out.

[0066] After verifying the silencing efficiency of siDDX5 by qPCR ( Figure 5 ) were infected with PRV at an MOI of 1, and cell DNA and cell supernatant were collected 24 hours later.

[0067] qPCR analysis showed that silencing of DDX5 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 the replication and proliferation of PRV, suggesting that DDX5 is a host restriction factor for PRV infection.

[0070] Example 3 Study on the mechanism of DDX5 inhibiting PRV proliferation

[0071] In order to further study the mechanism by which DDX5 inhibits PRV proliferation, this section clarifies the specific mechanism through the relationship between DDX5-VP16 protein-autophagy-dependent degradation, the relationship between OPTN-DDX5-autophagy degradation, and the relationship between E3 ubiquitin ligase-DDX5-selective autophagy receptors, as follows:

[0072] 3.1 Host restriction factor DDX5 interacts with VP16 protein to promote its autophagic degradation

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

[0074] In order to screen host restriction factors that interact with VP16 protein, the present application transfected Flag-VP16 plasmid or Flag empty vector into 293T cells, and performed mass spectrometry analysis after immunoprecipitation. The mass spectrometry results showed that host factors such as PARP1, PCBP2, and DDX5 may interact with VP16.

[0075] In the present application, different masses (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 effects of these host proteins on the VP16 protein content.

[0076] WB results showed that PARP1 and PCBP2 could not induce the degradation of VP16 ( Figure 8a and 8b ). Only when DDX5 was overexpressed, the protein level of VP16 was significantly reduced, and the protein abundance of VP16 was inversely proportional to the transfection dose of DDX5 ( Figure 8c ), indicating that DDX5 promoted the degradation of VP16 protein in a dose-dependent manner. This suggests that DDX5 may be involved in the anti-PRV response of cells.

[0077] In this study, HA-DDX5 and Flag-VP16 were co-transfected into 293T cells for CO-IP experiments. The results also confirmed that DDX5 and VP16 interact with each other ( Figure 8d ).

[0078] To explore the pathway of DDX5 degradation of VP16 protein, the present application added proteasome inhibitor MG132 or autophagy inhibitor CQ to 293T cells overexpressing HA-DDX5 and Flag-VP16, respectively, and collected cell proteins after incubation for 6 hours. WB showed that CQ could block the degradation of VP16 by DDX5, while MG132 could not ( Figure 8e ). This indicates that DDX5 achieves VP16 protein degradation through the autophagy-lysosome pathway rather than the ubiquitin-protease system. The above results indicate that the host DDX5 protein interacts with the PRV VP16 protein and promotes its autophagy-dependent degradation.

[0079] Figure 8a This is the result diagram showing that PARP1 cannot induce VP16 protein degradation;

[0080] Figure 8b This is the result diagram showing that PCBP2 cannot induce VP16 protein degradation;

[0081] Figure 8c The figure shows the result that DDX5 induces the degradation of VP16 protein in a dose-dependent manner;

[0082] Figure 8d This is the result diagram showing the interaction between DDX5 and VP16 protein;

[0083] Figure 8e This figure shows the result that DDX5 induces the degradation of VP16 protein through the autophagy pathway.

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

[0085] In order to explore the role of OPTN in DDX5-induced autophagic degradation of VP16 protein, the present invention transfected HA-DDX5 and Flag-OPTN in 293T cells for CO-IP detection.

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

[0087] In order to further confirm that DDX5-induced autophagic degradation of VP16 depends on OPTN, the present application silenced OPTN, co-transfected HA-DDX5 and Flag-VP16 into 293T cells, and analyzed the protein level of VP16 by WB. The results showed that silencing OPTN could block the degradation of VP16 protein by DDX5 ( Figure 9b ). The above results indicate that DDX5 degrades PRV VP16 protein through the selective receptor OPTN.

[0088] Figure 9a This is the result diagram showing the interaction between DDX5 and OPTN;

[0089] Figure 9b This figure shows the result that silencing OPTN can block DDX5 from degrading VP16 protein.

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

[0091] Ubiquitination modification of substrates by E3 ubiquitin ligase is the key to the binding of selective autophagy receptors to them.

[0092] First, the present invention detected the ubiquitination degree of VP16 by CO-IP and found that overexpression of DDX5 significantly increased the ubiquitination of VP16 ( Fig.10a ).

[0093] In order to screen the E3 ubiquitin ligase involved in VP16 ubiquitination, mass spectrometry analysis results showed that ZNF598, RNF138, and UBE3C may interact with VP16 protein.

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

[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 can cause a dose-dependent decrease in VP16 protein content ( Fig.10d ).

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

[0097] The CO-IP results showed that UBE3C and VP16 do interact with each other ( Fig.10e ). Notably, overexpression of UBE3C increased the ubiquitination of VP16 ( Fig.10f ).

[0098] In order to further explore whether UBE3C is involved in the autophagic degradation of VP16 protein by DDX5, the present application first overexpressed MYC-UBE3C and HA-DDX5 in 293T cells, and confirmed by CO-IP that UBE3C interacted with DDX5 ( Figure 10g ).

[0099] In addition, the present application verifies the silencing efficiency of siUBE3C at the mRNA level by transfecting siRNA or siNC ( Fig.10h ) to analyze the effect of silencing UBE3C on DDX5-induced VP16 protein degradation. WB results showed that knocking down cell UBE3C could restore the protein content of VP16, indicating that silencing UBE3C significantly inhibited the degradation of VP16 by DDX5 ( Fig.10i ).

[0100] Fig.10a This is a result diagram showing that DDX5 promotes the ubiquitination of VP16 protein;

[0101] Fig.10b This is the result diagram showing that ZNF598 cannot induce VP16 protein degradation;

[0102] Fig.10c The result figure shows that RNF138 cannot induce VP16 protein degradation;

[0103] Fig.10d The figure shows that UBE3C promotes the degradation of VP16 protein in a dose-dependent manner;

[0104] Fig.10e This is the result diagram showing the interaction between UBE3C and VP16 protein;

[0105] Fig.10fThis is a result diagram showing that UBE3C promotes the ubiquitination of VP16 protein;

[0106] Figure 10g This is the result diagram showing the interaction between DDX5 and UBE3C;

[0107] Fig.10h The result diagram is the silencing efficiency of siUBE3C;

[0108] Fig.10i This figure shows that silencing UBE3C can block 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, and the difference is extremely significant.

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

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

Claims

1. A method for inhibiting the proliferation of pseudorabies virus, characterized in that: The DDX5 gene is overexpressed in host cells, and the modified host cells are used to inhibit the proliferation of pseudorabies virus.

2. The method according to claim 1, characterized in that The method for overexpressing the DDX5 gene in the host cell is: using a plasmid containing the DDX5 gene to transfect the host cell, and after successful transfection, the host cell overexpressing the DDX5 gene can be obtained.

3. The method according to claim 1, characterized in that The host cell is 293T cell or PK-15 cell.

4. The method according to claim 2, characterized in that: The plasmid is a plasmid obtained by inserting the DDX5 gene into the HA vector.

5. A method for promoting the proliferation of pseudorabies virus, characterized in that: The pseudorabies virus is propagated in host cells in which the DDX5 gene is knocked out or knocked down.

6. The method according to claim 5, characterized in that The method for knocking out the DDX5 gene in the host cell is: The host cells were transfected with siDDX5 or siNC to obtain host cells with DDX5 gene knocked out.

7. The method according to claim 5, characterized in that The host cell is 293T cell or PK-15 cell.

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