A composition for modulating prr infection and uses thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a composition for regulating PRV infection and application thereof. The composition for regulating PRV infection contains Ku protein. The composition contains at least one of Ku70 protein or Ku80 protein. Compared with the prior art, the application provides the composition for inhibiting PRV infection and application thereof, which has the advantages of: (1) Ku protein is easy to obtain, non-pathogenic and high in safety; (2) Ku protein is safe and harmless in production and operation, and non-infectious; and (3) Ku protein has high specificity and high sensitivity in inhibiting PRV infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a composition for regulating PRV infection and its application. Background Technology

[0002] Pseudorabies (PR, also known as Aujeszky's disease, AD) is a viral disease caused by the pseudorabies virus (PRV). It has been circulating globally since the 1980s, and the classic pseudorabies virus was first isolated in 1902. Pseudorabies virus is highly contagious; pigs are the natural host, and infected pigs shed large amounts of the virus through their feces and other bodily secretions. Pseudorabies virus is primarily transmitted through direct contact, but can also be transmitted through air, water, and contaminated objects. Symptoms vary among pigs of different ages and health conditions. Adult pigs often exhibit respiratory symptoms, with a high morbidity but low mortality rate. Infected suckling piglets show fever and loss of appetite. As the infection progresses, infected pigs gradually exhibit central nervous system symptoms such as tremors, gyratory states, and paralysis, with a mortality rate approaching 100%. Pregnant sows infected with the virus generally experience abortion and stillbirth, easily leading to sow infertility. Immunizing pigs with the PRV live attenuated vaccine strain Bartha-K61 can effectively control the spread of the virus, but it cannot prevent viral infection.

[0003] PRV (Poxvirus) belongs to the genus Poxvirus of the subfamily Alphaherpesvirinae in the family Herpesviridae. It shares the highest evolutionary homology with human herpesvirus type 1 (HSV-1) within the same genus, belonging to the same branch. PRV particles are spherical or oval, composed, from the inside out, of viral nucleic acid, capsid, envelope, and periapsis. The nucleic acid is located within the icosahedral capsid. The envelope, located between the capsid and periapsis, has two layers, connected to capsid proteins and envelope proteins respectively. The PRV genome is 147 kb in size, presenting as linear double-stranded DNA with a G+C content as high as 74%. It has two unique regions: a long unique region (UL) and a short unique region (US), as well as internal repeat sequences (IRS) and terminal inverted repeat sequences (TRS) flanking the US. Based on expression time, PRV genes are classified into early genes, late genes, and late genes, encoding up to 70 proteins.

[0004] The early protein EP0 is a homologue of the HSV-1 protein ICP0. EP0 possesses a ring-finger domain (amino acids 1-84) at its N-terminus, highly homologous to the ring-finger region of the ICP0 protein of herpes simplex virus type 1 (HSV-1), the ORF61 protein of herpes zoster virus type ZV (VZV), and the BICP0 protein of equine herpesvirus type 1 (BHV-1). PRV EP0 and IE180 cooperate to activate viral gene transcription and enhance viral DNA infectivity. Furthermore, the ring-finger domain and acidic region (amino acids 114-242) of EP0 are essential transactivation regions. Studies of EP0-deficient recombinant pseudorabies virus have shown that the EP0 gene is non-essential for pseudorabies virus but may be important for reactivation from the latent state. EP0 can activate proteins transcribed by the TATA promoter. EP0 is a transcription factor that can activate the expression of many viral proteins such as IE180, UL23, and US4. EP0 also exhibits transcription factor activity against homologous proteins of other herpesviruses, such as the ORF29 gene of VZV virus, the UL23 (thymidine kinase) gene of HSV-1 virus, and the early gene of simian virus 40 (SV40). However, EP0 inhibits the promoter activity of the UL41 (Vhs) and gE genes.

[0005] DNA damage response (DDR) is a mechanism that cells have evolved to detect and repair DNA damage in order to combat the threat posed by DNA damage. DNA double-strand breaks are mainly repaired through two mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ simply joins the double helix with little or no processing, while HR requires specific nucleases to process the DSB ends to form a 3'-ssDNA tail, which then relies on the recognition and pairing of this 3'-ssDNA tail with a complete homologous sequence.

[0006] Non-homologous end repair (NHEJ) occurs throughout the cell cycle and is independent of homologous DNA sequences. The first step of NHEJ is the formation of a circular binding of the heterodimer proteins Ku70 and Ku80 to the damaged DNA site, thereby protecting the DNA from excessive nuclease cleavage. After binding to DNA, the Ku heterodimer recruits and binds to the catalytic subunit DNA-PKcs, forming an active DNA-PK holoenzyme. DNA-PKcs are activated upon binding to single-stranded DNA, exerting Ser / Thr kinase activity. One of its targets is XRCC4, which forms a stable complex with DNA ligase IV (LIG IV). The XRCC4-ligase IV complex binds to the terminal DNA molecule and joins the double-stranded DNA with a molecule that is complementary to the ligase end. The XRCC4-ligase IV complex cannot directly rejoin broken DNA; the DNA ends must be treated first. The processing of DSBs primarily involves the MRE11-Rad50-NBS1 complex, which utilizes exonuclease, endonuclease, nuclease, and helicase activities to remove excess DNA from the 3' end. Another protein involved in NHEJ is the Artemis protein, which possesses single-strand-specific exonuclease activity and gains endonuclease activity upon phosphorylation by DNA-PKcs. Artemis interacts with DNA-PKcs and is activated by DNA-PKcs phosphorylation, subsequently cleaving the DNA ends to form structures suitable for ligase ligation. The polymerase Polμ primarily exhibits template-independent polymerase activity, while Polλ primarily exhibits template-dependent polymerase activity, forming terminal microhomological arms. In the presence of microhomological arms, Ku protein binding to DNA is unnecessary; however, in the absence of microhomological arms, Ku protein binding to DNA ends becomes crucial, mediating the binding of the XRCC4 / ligase IV complex to DNA ends. Additionally, XLF and PAXX have also been found to assist in ligase IV ligation, and both are structurally very similar to XRCC4. Both XLF and PAXX can form homodimers that bind to Ku-DNA. Their main function is to pull the two broken ends of DNA closer together and fix them, facilitating ligase IV to perform its ligase activity and join the DNA ends. In summary, the NHEJ pathway utilizes proteins to recognize, cleave, polymerize, and join DNA ends in a flexible manner. This flexibility allows NHEJ to function across a wide range of DNA end conformations; however, the repaired DNA ligation often contains mutations. Summary of the Invention

[0007] The purpose of this invention is to provide a safe, harmless, non-infectious, highly specific and highly sensitive composition for regulating PRV infection and its application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A composition for regulating PRV infection, said composition containing Ku protein.

[0010] Furthermore, the composition contains at least one of Ku70 protein or Ku80 protein.

[0011] Furthermore, the Ku protein regulates PRV replication through the NHEJ signaling pathway.

[0012] The present invention also provides an agent for regulating PRV infection.

[0013] An agent for modulating PRV infection, comprising the Ku protein and a pharmaceutically acceptable carrier.

[0014] The present invention also provides the use of a composition for inhibiting PRV infection.

[0015] The use of a composition that modulates PRV infection, and the use of said composition in inhibiting PRV replication.

[0016] The present invention also provides the use of another composition for inhibiting PRV infection.

[0017] The use of a composition for modulating PRV infection, and the use of said composition in the preparation of anti-PRV.

[0018] The present invention also provides an application of a composition that promotes PRV replication.

[0019] The use of a composition that modulates PRV infection, and the use of said composition in promoting PRV replication.

[0020] The present invention also provides the use of a composition for inhibiting PRV infection.

[0021] The use of Ku protein in the preparation of drugs that inhibit PRV infection.

[0022] The present invention also provides the use of a composition that promotes PRV replication.

[0023] Use of Ku protein in the preparation of reagents that promote PRV replication.

[0024] Compared with the prior art, the advantages of the composition for regulating PRV infection and its application provided by the present invention are as follows:

[0025] (1) Ku protein is easy to obtain, non-pathogenic, and has high safety.

[0026] (2) Ku protein is safe, harmless and non-infectious during production and handling.

[0027] (3) Ku protein has high specificity and high sensitivity in inhibiting PRV infection. Attached Figure Description

[0028] Figure 1 This is the result of the influence of PRV on the NHEJ signaling pathway in Embodiment 6 of the present invention.

[0029] Figure 2 In Example 6 of this invention, HEp-2 cells were infected with PRV-WT or PRV-EP0 KO virus (MOI=1), and cell samples were collected at 0, 6, 12, and 24 hours, respectively. The results of Western blot detection of related proteins (top figure) and virus growth curve (bottom figure) were obtained.

[0030] Figure 3 The results of Western blot detection of related proteins (top figure) and virus growth curves (bottom figure) after PRV-WT or PRV-EP0 KO virus (MOI=1) infected CRL cells in Example 6 of this invention are shown.

[0031] Figure 4 In Example 6 of this invention, plasmids Flag-EV, Flag-EP0 (PRV), Flag-ICP0 (HSV-1), and Flag-UL18 (PRV) were transfected into HeLa cells. After 24 hours, the cells were harvested and the expression levels of endogenous Ku protein, DNA-PKcs, and exogenous viral proteins were analyzed by Western blot.

[0032] Figure 5 The diagram in Example 6 of this invention shows the alignment results of the Ring-finger amino acid sequence of PRV EP0 protein with the Ring-finger amino acid sequences of HSV-1ICP0, VZV ORF61, and BoHV1 BICP0.

[0033] Figure 6 In Example 6 of this invention, the Flag-tagged EPO, the EPO Ring-finger mutant EPO(CA), and the PRV protein UL18 expression plasmid were transfected into HeLa cells, and the cells were collected 24 hours later to detect the relevant proteins.

[0034] Figure 7 In Example 6 of this invention, the Flag-tagged EPO, the mutant EPO(CA), and the PRV protein UL18 expression plasmid were transfected into HeLa cells, and RNA was extracted 24 hours later to detect the mRNA levels of Ku70 and Ku80.

[0035] Figure 8In Example 6 of this invention, HEK293T cells were co-transfected with Flag-Ku70 / Ku80 and His-Ub, and after 6 hours, they were transfected with a plasmid expressing EP0. Then, MG132 was added for treatment, and the ubiquitination modification result of Ku protein was detected after 24 hours.

[0036] Figure 9 In Example 6 of this invention, HEK293T cells were co-transfected with HA-Ku70 / Ku80 and Flag-EP0, treated with MG132 (20 nM), and the interaction between Ku70 and EPO was detected by immunoprecipitation after 24 hours.

[0037] Figure 10 The results of U2OS cells transfected with I-SceI plasmid in Example 6 of this invention, infected with PRV-WT or PRV-EP0 KO virus (MOI=1) 48 hours later, and the amount of GFP-expressing cells was detected by flow cytometry 24 hours later.

[0038] Figure 11 This refers to the detection of Ku protein and viral protein expression in virus-infected cells in Example 6 of the present invention. The data are the results of three independent replicates, and the statistical analysis method is two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001.

[0039] Figure 12 In Example 7 of this invention, HEp-2 cells were transfected with si-Control, si-Ku70, and si-Ku80, respectively, and then infected with PRV-WT or PRV-EP0 KO (MOI=1). PRV protein expression was detected by Western blot (top and middle figures), and cell supernatant was collected to detect viral titer (bottom figure). Data represent three independent replicates. Statistical analysis was performed using a two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001.

[0040] Figure 13 In Example 7 of this invention, CRL cells were transfected with si-Control, si-Ku70, and si-Ku80, respectively, and then infected with PRV-WT or PRV-EP0 KO virus (MOI=1). PRV protein expression was detected by Western blot (top and middle figures), and cell supernatant was collected to detect viral titer (bottom figure). Data represent three independent replicates, and statistical analysis was performed using a two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001.

[0041] Figure 14 In Example 7 of this invention, HEp-2 cells were transfected with si-Control, si-Ku70 and si-Ku80 respectively, and then transfected with plasmids Flag-EV, Flag-EP0 or Flag-EP0(CA) respectively. After 24 hours of plasmid expression, Western blot was used to detect the degradation of Ku70 / Ku80.

[0042] Figure 15 In Example 7 of this invention, HEp-2 cells were transfected with si-Control, si-Ku70, or si-ku80 to knock down Ku70 and Ku80. Then, complement plasmids Flag-Ku70res and Flag-Ku80res were transfected. After the plasmids were stably expressed, PRV was infected (MOI=1). After 24 hours, the expression of viral proteins was detected by Western blot, and the viral titer was detected by collecting the cell supernatant.

[0043] Figure 16 In Example 7 of this invention, CRL cells were transfected with si-Control, si-Ku70, or si-ku80 to knock down Ku70 and Ku80, and then transfected with complement plasmids Flag-Ku70res and Flag-Ku80res. After the plasmids were stably expressed, the cells were infected with PRV (MOI=1). After 24 hours, the expression of viral proteins was detected by Western blot, and the viral titer was detected by collecting the cell supernatant.

[0044] Figure 17 In Example 8 of this invention, Ku70 and Ku80 in human laryngeal cancer epithelial cells (HEp-2) were knocked down using siRNA, followed by infection with PRV-WT virus (MOI=1). Viral protein expression was detected at 0, 6, 12, and 24 hours after viral infection.

[0045] Figure 18 In Example 8 of this invention, Ku70 and Ku80 of human laryngeal cancer epithelial cells (HEp-2) were knocked down using siRNA, and then the cells were infected with PRV-WT virus (MOI=1). The viral titer was detected 24 hours after infection.

[0046] Figure 19 In Example 8 of this invention, HEp-2 cells with knocked-down Ku70 and Ku80 were infected with PRV-EP0KO virus (MOI=1). Viral protein expression was detected at different time points after viral infection.

[0047] Figure 20 In Example 8 of this invention, HEp-2 cells with knocked-down Ku70 and Ku80 were infected with PRV-EP0KO virus (MOI=1). The viral titer was detected 24 hours after infection.

[0048] Figure 21 In Example 8 of this invention, verification was performed on pig cells. Ku70 and Ku80 in porcine alveolar macrophages (CRL) were knocked down using siRNA, and then the cells were infected with PRV-WT virus (MOI=1). Viral protein expression was detected at different time points after viral infection.

[0049] Figure 22 In Example 8 of this invention, verification was performed on pig cells. Ku70 and Ku80 in porcine alveolar macrophages (CRL) were knocked down using siRNA, and then the cells were infected with PRV-WT virus (MOI=1). The viral titer was detected 24 hours after infection.

[0050] Figure 23 In Example 8 of this invention, Ku70 and Ku80 of CRL cells were knocked down and then infected with PRV-EP0KO virus (MOI=1). Viral protein expression was detected at different time points after viral infection.

[0051] Figure 24 In Example 8 of this invention, Ku70 and Ku80 of CRL cells were knocked down and then infected with PRV-EP0KO virus (MOI=1). The viral titer was detected 24 hours after infection. Detailed Implementation

[0052] To enable those skilled in the art to better understand the technical solutions of the present invention, the following embodiments provide a more detailed description of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0053] A composition for regulating PRV infection, said composition containing Ku protein.

[0054] Furthermore, the composition contains at least one of Ku70 protein or Ku80 protein.

[0055] Furthermore, the Ku protein inhibits PRV replication through the NHEJ signaling pathway.

[0056] The present invention also provides an agent for regulating PRV infection.

[0057] An agent for modulating PRV infection, comprising the Ku protein and a pharmaceutically acceptable carrier.

[0058] The present invention also provides an application of a composition for regulating PRV infection.

[0059] The use of a composition that modulates PRV infection, and the use of said composition in inhibiting PRV replication.

[0060] The present invention also provides the use of another composition for regulating PRV infection.

[0061] The use of a composition for modulating PRV infection, and the use of said composition in the preparation of anti-PRV.

[0062] The present invention also provides an application of a composition that promotes PRV replication.

[0063] The use of a composition that modulates PRV infection, and the use of said composition in promoting PRV replication.

[0064] The present invention also provides the use of a composition for inhibiting PRV infection.

[0065] The use of Ku protein in the preparation of drugs that inhibit PRV infection.

[0066] The present invention also provides the use of a composition that promotes PRV replication.

[0067] Use of Ku protein in the preparation of reagents that promote PRV replication.

[0068] Example 1

[0069] A composition for regulating PRV infection, the composition containing a Ku protein. The composition includes a Ku70 protein. The Ku70 protein inhibits PRV replication via the NHEJ signaling pathway.

[0070] Example 2

[0071] A composition for regulating PRV infection, the composition containing a Ku protein. The composition includes a Ku80 protein. The Ku80 protein inhibits PRV replication via the NHEJ signaling pathway.

[0072] Example 3

[0073] The use of a composition that modulates PRV infection, and the use of said composition in inhibiting PRV replication.

[0074] Example 4

[0075] Use of a composition for regulating PRV infection, and use of the Ku protein in the preparation of a medicament for inhibiting PRV infection.

[0076] Example 5

[0077] Application of a composition for regulating PRV infection, specifically its use in promoting PRV replication. Reducing the amount of this composition significantly enhances viral protein expression and viral titer, and can significantly increase PRV numbers. It can be used for research and / or as a vector, etc.

[0078] Example 6

[0079] Research on the regulation of NHEJ repair pathway in PRV infection

[0080] 1. PRV infection reduces Ku protein expression

[0081] HEp-2 cells were infected with PRV and HSV-1, respectively. The results showed that HSV-1 infection significantly reduced the amount of DNA-PKcs, while the amounts of Ku70 and Ku80 remained unchanged. PRV infection did not reduce the amount of DNA-PKcs, but it did cause a significant reduction in Ku70 and Ku80. Figure 1 A). In other words, PRV infection can cause a decrease in Ku protein.

[0082] Then, infection of porcine alveolar macrophages (CRL) with PRV showed a significant reduction in both Ku70 and Ku80. Figure 1 B). PRV infection can reduce the amount of Ku protein, possibly by promoting the degradation of Ku protein at the protein level, or by inhibiting the transcription of the Ku gene at the transcriptional level.

[0083] Then, the virus-infected cells were treated with proteasome MG132, which showed the effect in virus-infected CRL cells ( Figure 1 In C), MG132 treatment for 8 hours completely inhibited the degradation of Ku70 and Ku80. This indicates that Ku proteins are degraded via the proteasome pathway during viral infection.

[0084] 2. PRV degrades Ku protein via EPO protein.

[0085] Infection of HEp-2 and CRL cells with the EP0-deficient virus showed that the deletion virus (PRV-EP0 KO) did not induce Ku protein degradation compared to the wild-type virus (PRV-WT). Figure 2 and Figure 3 The results indicate that PRV degrades Ku protein via EPO. Simultaneously, a long curve of virus generation was plotted using cell supernatant, showing the PRV-EP0 KO virus titer (PRV-WT) at 24 hours. These results suggest that PRV infection-induced Ku protein reduction is mediated by the EPO protein.

[0086] 3. The Ring-finger region of EP0 mediates the degradation of Ku proteins.

[0087] Overexpression of ICP0, EP0, and PRV protein UL18 in HeLa cells showed that EP0 could degrade Ku protein, and that ICP0 also had a degrading effect on DNA-PKcs. Figure 4 This further demonstrates that EP0 can degrade Ku proteins.

[0088] Comparison of EP0 with ICP0 and other homologous proteins of alpha-herpesviruses: ORF61 of VZV and BICP0 of BoHV1. The results show that EP0 and other homologous proteins have high homology in the ring-finger region and all contain Zn. 2+ The C3HC4 sequence was combined, but the C-terminal sequence showed significant differences. Figure 5 The glycine in the Ring-finger region of EP0 was mutated to alanine, resulting in EP0(CA), which lost its E3 ubiquitin ligase activity. Overexpression of EP0 and the mutant EP0(CA) in HeLa cells showed that the mutant could not degrade Ku protein, indicating that EP0's degradation of Ku protein is dependent on its Ring-finger region (…). Figure 6 The mRNA levels of Ku70 and Ku80 were detected, showing that neither EP0 nor EP0(CA) affected the transcription of the Ku70 and Ku80 genes. Figure 7 Therefore, EP0 mediates Ku protein degradation by exercising E3 ubiquitin ligase activity through its N-terminal Ring-finger domain.

[0089] 4. Investigation into the ubiquitination modification of Ku protein by EP0

[0090] Proteins require ubiquitination before they can be degraded via the proteasome. Ku70 and Ku80 are degraded via the proteasome under the influence of EPO, so the ubiquitination status of Ku proteins was examined.

[0091] In HEK293T cells, Ku protein and ubiquitin molecules were simultaneously overexpressed, followed by transfection with untagged EP0 or EP0(CA) plasmids. The results showed that both Ku70 and Ku80 enhanced ubiquitination in the presence of EP0, while EP0(CA) did not enhance ubiquitination in either cell. Figure 8 Therefore, the above results further indicate that Ku protein is degraded by EP0 via the ubiquitin-proteasome pathway. E3 ubiquitinase interacts with the substrate during the ubiquitin-proteasome degradation process. In HEK293T cells, EP0 and Ku proteins were simultaneously overexpressed, and the interaction between EP0 and Ku proteins was detected by immunoprecipitation (IP). The results showed that EP0 could interact with both Ku70 and Ku80, respectively. Figure 9 Therefore, EP0 directly mediates the degradation of Ku proteins and can bind to Ku70 and Ku80 respectively to mediate the degradation of both.

[0092] 5. The effect of PRV on NHEJ damage repair

[0093] PRV can degrade the key proteins Ku70 and Ku80 in the NHEJ pathway via EPO, therefore PRV infection has an important impact on the NHEJ repair pathway.

[0094] The repair efficiency of U2OS-EJ5 cells, a U2OS model cell line equipped with a GFP reporter, was directly assessed. The structure and principle of the GFP reporter are as follows: the GFP gene is constructed on a eukaryotic expression plasmid, with a Puro gene inserted in the middle. The Puro gene has I-SceI-specific restriction enzyme sites at both ends. After I-SceI cleavage, the Puro gene is removed, resulting in a double-strand break (DSB), which induces a DNA damage response in the cell. This damage is ultimately repaired using the intracellular NHEJ repair system. The repaired GFP gene can then express GFP protein normally. Therefore, the number of cells expressing GFP was quantified by flow cytometry to assess the NHEJ repair efficiency.

[0095] U2OS-EJ5 cells were seeded in 12-well plates and transfected with the I-SceI plasmid when the cell density reached 60%. Forty-eight hours later, the cells were infected with PRV-WT and PRV-EP0 KO viruses, respectively. Cells were harvested 24 hours later for flow cytometry analysis. The results showed that PRV-WT infection reduced the NHEJ repair efficiency by approximately 30%, while PRV-EP0 KO virus infection had almost no effect on NHEJ repair. Figure 10 Similarly, PRV-WT virus infection can significantly degrade the Ku protein, while PRV-EP0 KO virus cannot. Figure 11 Therefore, PRV infection inhibits the NHEJ repair pathway in cells, which is achieved by the degradation of Ku70 and Ku80 proteins by the EPO protein.

[0096] Example 7

[0097] The effect of the NHEJ pathway on PRV replication

[0098] 1. Effects of the NHEJ pathway on PRV protein expression

[0099] Ku70 and Ku80 were knocked down in HEp-2 or CRL cells using siRNA, followed by infection with PRV-WT or PRV-EP0KO virus. Viral protein expression was assessed at different time points after infection, and viral titers were measured at 24 hours. Results showed that low expression of either Ku70 or Ku80 significantly enhanced viral protein expression. Figure 12 The same phenomenon was observed in both the top and middle images (top and middle images). Figure 13(Top and middle images). Viral titer results show that when Ku protein expression is low, viral titer increases significantly, and the fold increase in PRV-EP0 KO viral titer is much greater than that of PRV-WT. Figure 12 The image below and Figure 13 (See figure below). This indicates that endogenous Ku protein plays an important role in resisting P infection, meaning that the NHEJ repair pathway does indeed inhibit PRV replication. Because EP0 can interact with both Ku70 and Ku80, the EP0-induced downregulation of Ku70 and Ku80 is related to EP0's ability to target Ku70 and Ku80 individually. Therefore, knocking down Ku70 with siRNA and overexpressing EP0 protein or its mutant EP0(CA) showed that Ku80 can be degraded by EP0. Thus, EP0 can target Ku80 alone to induce its ubiquitination and degradation. Similarly, when Ku80 is knocked down, Ku70 can still be degraded by EP0. These results show that EP0 can target either Ku70 or Ku80 individually. Figure 14 ).

[0100] 2. The effect of Ku protein replenishment on PRV protein levels

[0101] Since low expression of Ku70 or Ku80 can enhance PRV replication, Ku70 and Ku80 were reintroduced into HEp-2 cells, followed by PRV infection. Viral replication was assessed 24 hours later. The results showed that after Ku70 or Ku80 reintroduction, Ku protein significantly inhibited PRV replication at the protein expression level. Figure 15 The same phenomenon is observed in CRL cells. Figure 16 The above results demonstrate that the Ku protein does indeed inhibit viral replication, meaning that the NHEJ signaling pathway can suppress PRV replication.

[0102] The effect of Ku protein complementation on progeny virus yield was investigated. PRV virus was introduced after Ku70 and Ku80 complementation, and viral titers were measured 24 hours later. The results showed that Ku70 or Ku80 complementation significantly inhibited PRV progeny virus yield in HEp-2 cells. Figure 15 The same phenomenon is observed in CRL cells. Figure 16 The above results indicate that the Ku protein further inhibits viral replication, meaning that the NHEJ signaling pathway can suppress PRV replication.

[0103] Example 8

[0104] Reduced Ku protein expression via siRNA enhances PRV replication.

[0105] Ku70 and Ku80 in human laryngeal cancer epithelial cells (HEp-2) were knocked down using siRNA, followed by infection with PRV-WT virus (MOI=1). Viral protein expression was assessed at 0, 6, 12, and 24 hours post-infection, and viral titer was measured at 24 hours. Figures 17-18 (As shown). The results showed that reducing the amount of Ku70 or Ku80 significantly enhanced the expression of viral proteins and viral titers, with viral titers increasing by 40-fold and 45-fold, respectively.

[0106] HEp-2 cells were knocked down at Ku70 and Ku80 levels and then infected with PRV-EP0 KO virus (MOI=1). Viral protein expression was assessed at different time points after infection, and viral titers were measured at 24 hours (e.g., [missing data]). Figures 19-20 As shown in the figure, the results showed that when Ku protein was expressed at low levels, the PRV-EP0 KO virus titer was significantly increased, with an increase of much greater than that of PRV-WT, approximately 78 times and 95 times, respectively.

[0107] Subsequently, validation was performed on pig cells. Ku70 and Ku80 in porcine alveolar macrophages (CRL) were knocked down using siRNA, followed by infection with PRV-WT virus (MOI=1). Viral protein expression was assessed at different time points after infection, and viral titers were measured at 24 hours (e.g., [missing data]). Figures 21-22 (As shown). The same phenomenon was observed in CRL cells, with significantly enhanced expression of viral proteins and viral titers, increasing by approximately 65-fold and 80-fold, respectively.

[0108] Similarly, after knocking down Ku70 and Ku80 in CRL cells, they were infected with PRV-EP0 KO virus (MOI=1). Viral protein expression was assessed at different time points after infection, and viral titer was measured at 24 hours. Figures 23-24 (As shown in the image). The results showed that when Ku protein was expressed at low levels, the viral titer of PRV-EP0 KO virus was significantly increased, with increases of approximately 110-fold and 150-fold, respectively. These results indicate that endogenous Ku protein plays an important role in resisting PRV infection, meaning that the NHEJ repair pathway does indeed inhibit PRV replication.

[0109] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0110] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0111] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. Use of a Ku protein selected from the group consisting of a Ku70 protein and / or a Ku80 protein for the preparation of a medicament for inhibiting PRV infection.

2. Use according to claim 1, wherein the medicament comprises a Ku protein and a pharmaceutically acceptable carrier.

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