Application of MG132 in preparation of product for cyprinid herpesvirus type II

Abstract

The invention belongs to the fields of agriculture, fishery and breeding industry, and particularly relates to application of MG132 in preparation of a product for cyprinid herpesvirus II. The invention finds that the MG132 can effectively inhibit the infection replication of CyHV-2 virus in cells, can be used for inhibiting the CyHV-2 virus, can also be used for preventing and treating the infection of the cyprinid herpesvirus II and hematopoietic necrosis induced by the infection of the cyprinid herpesvirus II, and provides a new method for preventing and treating the cyprinid herpesvirus II.

Description

Technical Field

[0001] The present invention belongs to the technical fields of agriculture, fishery, aquaculture, and prevention and control of aquaculture diseases. Specifically, it relates to a new use of MG132 - its application in the preparation of an inhibitor of Cyprinid herpesvirus 2. Background Art

[0002] Cyprinid herpesvirus 2 (CyHV-2) mainly infects crucian carp and goldfish, inducing herpesviral haematopoietic necrosis (HVHN) in diseased fish, which is an important problem for the healthy development of the Chinese crucian carp aquaculture industry. CyHV-2 belongs to the order Herpesvirales, family Alloherpesviridae, genus Cyvirus, and is a large linear double-stranded DNA enveloped virus. Studies have confirmed that CyHV-2 infection of hosts can cause acute infection and establish latency and persistent infection in surviving fish with asymptomatic infection. Surviving fish with asymptomatic infection may become important sources of infection (Chai W, Qi L, Zhang Y, et al. Evaluation of Cyprinid Herpesvirus 2 Latency and Reactivation in Carassius gibel [J]. Microorganisms, 2020, 8(3): 445.). This complex and variable infection characteristic makes the virus easy to spread, and virus prevention and control are more difficult. At present, there is no effective commercial vaccine and effective drug to control the spread of the virus. One of the main reasons is that its pathogenic mechanism is still unclear. Therefore, it is urgent to take measures to reduce the economic losses of the crucian carp aquaculture industry caused by CyHV-2.

[0003] The ubiquitin-proteasome system (UPS) is the main pathway for protein degradation in cells and plays a crucial role in a variety of basic cellular processes, including gene transcription regulation, cell cycle, autophagy, development and differentiation, and immune response regulation (Ben-Nissan G, Sharon M. Regulating the 20S proteasome ubiquitin-independent degradation pathway [J]. Biomolecules, 2014, 4(3): 862-884). Increasing evidence indicates that the UPS is essential for virus infection, and it can affect virus entry, gene transcription, assembly, release, and immune evasion. A variety of viruses, especially large DNA viruses such as herpesviruses, utilize the UPS system at different stages of their life cycles (Gu H, Jan Fada B. Specificity in ubiquitination triggered by virus infection [J]. International journal of molecular sciences, 2020, 21(11): 4088). Studies have shown that the proteasome degradation mechanism plays an important role in the process of virus infection. Based on the specific inhibitors of this pathway, the most common one is MG132, whose chemical name is carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (Z-Leu-Leu-Leu-CHO), which is a peptide aldehyde with a CAS number of 1211877-36-9, namely (R)-MG-132 (an enantiomer of MG-132 with relatively weak cytotoxicity), and its chemical formula is C 26 H 41 N3O5, and its structural formula is shown in Formula 1.

[0004]

[0005] MG132 is a reversible and cell-permeable proteasome inhibitor. Its mechanism of action is to covalently bind to the active site of the β subunit of the 20S proteasome, inhibit its proteolytic activity, and then effectively block the function of the 26S proteasome complex. MG132 can induce apoptosis in a variety of cells. Its mechanism involves upregulating pro-apoptotic proteins (such as Bax, caspase-3) and downregulating anti-apoptotic proteins (such as Bcl-2, XIAP), and is mostly used to study the mechanism of apoptosis induced by the proteasome pathway; it can activate autophagy, but at the same time block the autophagic flux, resulting in the accumulation of autophagosomes; it can also inhibit the activation of the NF-κB signaling pathway, thereby affecting cell survival and inflammatory responses (Guo N, Peng Z. MG132, a proteasome inhibitor, induces apoptosis in tumor cells[J]. Asia-Pacific Journal of Clinical Oncology, 2013, 9(1): 6-11). Research shows that MG-132 inhibits the degradation activity of 20S proteasome ZLLL-MCA, with an IC50 of 100 nM; it inhibits calpain, with an IC50 of 1.2 μM; MG-132 induces neurite outgrowth in PC12 cells at an optimal concentration of 20 nM (Tsubuki S, Saito Y, Tomioka M, et al. Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine[J]. The journal of biochemistry, 1996, 119(3): 572-576). MG-132 effectively inhibits TNF-α-induced NF-κB activation, IL-8 gene transcription, and protein release in A549 cells by inhibiting proteasome-mediated IκBα degradation at a concentration of 10 μM (Fiedler MA, Wernke-Dollries K, Stark JM. Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132[J]. Am J Respir Cell Mol Biol. 1998, 19(2): 259-268).MG-132 treatment effectively induces p53-dependent apoptosis in KIM-2 cells by inhibiting the 26S proteasome (MacLaren A P, Chapman R S, Wyllie A H, et al. p53-dependent apoptosis induced by proteasome inhibition in mammary epithelial cells[J]. Cell Death & Differentiation, 2001, 8(3): 210-218). In view of the above research, targeting the host UPS may be a potential antiviral strategy, and there is currently no report on the inhibition of CyHV-2 replication by MG132. Summary of the Invention

[0006] The object of the present invention is to provide the application of MG132, especially its application in preventing and treating Cyprinid herpesvirus type II (CyHV-2) infection, so as to provide an alternative solution for the prevention and treatment of hematopoietic organ necrosis disease.

[0007] MG132 can effectively inhibit the infection and replication of CyHV-2 virus in GiCF cells, can be used for the inhibition of CyHV-2 virus, and can also be used for preventing and treating Cyprinid herpesvirus type II infection and hematopoietic organ necrosis disease induced by Cyprinid herpesvirus type II infection.

[0008] The technical solution of the present invention is the application of MG132 in preparing at least one of the following products for Cyprinid herpesvirus type II:

[0009] (1) A product for inhibiting Cyprinid herpesvirus type II;

[0010] (2) A product for preventing or treating Cyprinid herpesvirus type II infection;

[0011] (3) A product for preventing or treating hematopoietic organ necrosis disease induced by Cyprinid herpesvirus type II infection.

[0012] The products include but are not limited to reagents, drugs, feeds, additives, water quality improvers, bottom quality improvers, etc.

[0013] The products are applicable to agriculture, fishery, aquaculture, especially applicable to aquaculture, and more preferably applicable to fish farming in aquaculture. The fish are crucian carp or goldfish.

[0014] The present invention also provides at least one of the following products for Cyprinid herpesvirus type II containing MG132:

[0015] (1) A product for inhibiting Cyprinid herpesvirus type II;

[0016] (2) A product for preventing or treating Cyprinid herpesvirus type II infection;

[0017] (3) Products for the prevention or treatment of hematopoietic organ necrosis disease induced by Cyprinid herpesvirus type II.

[0018] The products include, but are not limited to, reagents, drugs, feeds, additives, water quality improvers, bottom quality improvers, etc.

[0019] The products are applicable to agriculture, fishery, aquaculture, especially applicable to aquaculture, and more preferably applicable to fish farming in aquaculture. The fish are crucian carp or goldfish.

[0020] The present invention also provides an operation method for preventing and treating Cyprinid herpesvirus type II infection. The steps include: adding MG132 or the above-mentioned product for Cyprinid herpesvirus type II containing MG132 to the target environment.

[0021] The present invention first applies MG132 in the preparation of an inhibitor of Cyprinid herpesvirus type II. When MG132 acts on the GiCF cell model infected with CyHV-2, it can effectively reduce the copy number of CyHV-2, achieving the effect of hindering virus replication. The present invention provides a new method for preventing and treating Cyprinid herpesvirus type II.

[0022] The technical solution of the present application has the following beneficial effects:

[0023] 1. Applying MG132 to the prevention and treatment of Cyprinid herpesvirus type II, thereby providing a new treatment plan for CyHV-2.

[0024] 2. After using MG132 in vitro, it effectively reduces the copy number of CyHV-2 in cells and is expected to become a candidate drug for preventing and treating CyHV-2. Description of the Drawings

[0025] Figure 1 It is the detection result of the virus copy number in cells after MG132 treatment.

[0026] Figure 2 It is the detection result of the virus gene transcription and expression in cells after MG132 treatment.

[0027] Figure 3 It is to determine the effect of MG132 on the replication and localization of CyHV-2 in cells by immunofluorescence staining combined with AIE probe labeling. Detailed Embodiments

[0028] 1 Experimental Materials

[0029] 1.1 Experimental Viruses and Cells

[0030] The caudal fin cell line of Carassius auratus gibelio (GiCF) was constructed by our laboratory and stored in liquid nitrogen. The medium used for cell resuscitation was M199 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the cells were cultured in a constant temperature incubator at 27°C. The CyHV-2 YC-01 isolate (GenBank accession number: MN593216; hereinafter referred to as CyHV-2) was used to infect the cells at 25°C. After all the cells died, the cell supernatant was collected and filtered through a 0.22 μm needle filter to obtain the pure virus solution, which was stored at -80°C for later use.

[0031] 1.2 Experimental reagents

[0032] M199 medium, fetal bovine serum, and 100× penicillin-streptomycin were purchased from Gibco; MG132 was purchased from Selleck; DMSO was purchased from Sigma; the cell genomic DNA extraction kit (DP304) was purchased from Tiangen; DEPC-treated water, RNase-free double-distilled water, PBS, and 4% paraformaldehyde fixation solution were purchased from Sangon Biotech; Trizol was purchased from Invitrogen; chloroform was purchased from Shanghai Keling's Reagent Co., Ltd.; absolute ethanol and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; TB Premix ExTaq TM II (RR820A), PrimeScript TM RT Master Mix (RR036A) was purchased from TaKaRa; AIE green nuclear probe and AIE red cell membrane probe were purchased from the Institute of Advanced AIE Aggregation-Induced Emission; the polyclonal rabbit antibody against ORF150 polypeptide was prepared by Gil Biochemical; 405-conjugated Goat anti-Rabbit IgG(H+L) was purchased from ABclonal; QuickBlock TM Blocking solution was purchased from Beyotime; polylysine-coated 12-well cell slides were purchased from WHBSCIENTIFIC.

[0033] 2 Experimental methods

[0034] 2.1 MG132 inhibition experiment

[0035] The experimental concentrations of MG132 were 10 μM, 20 μM, and 50 μM, and the DMSO treatment was used as the control group. First, GiCF cells were pretreated with the inhibitor for 2 h, and then infected with CyHV-2 (MOI = 0.5) for 72 h. After that, the cells were collected to extract DNA and total RNA to detect the virus copy number and virus gene expression.

[0036] 2.2 DNA Extraction

[0037] Collect the cell lysate after drug treatment into a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant and transfer it to a new centrifuge tube, and extract the genomic DNA of the cells with reference to the Cell Genomic DNA Extraction Kit (DP304). Add 30 μL of RNase-free double-distilled water to dissolve the DNA.

[0038] 2.3 Total RNA Extraction from Cells

[0039] Collect the cells after drug treatment, add 1 mL of Trizol and pipette to mix well; add 200 μL of chloroform and vortex for 15 s, let stand for 3 min, and centrifuge at 12000×g for 15 min at 4°C. Take the supernatant and add an equal volume of isopropanol, let stand for 10 min, and centrifuge at 12000×g for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC-treated water), and centrifuge at 7500×g for 5 min at 4°C. Add 20 μL of DEPC-treated water to dissolve the RNA.

[0040] 2.4 Reverse Transcription to cDNA by RT-PCR

[0041] Refer to the PrimeScript TM RT Master Mix (RR036A) instruction manual to prepare the reverse transcription reaction system

[0042] (Table 1):

[0043]

[0044] Gently mix and centrifuge briefly.

[0045] Reaction program: 37°C, 15 min; 85°C, 5 s; 4°C, ∞.

[0046] 2.5 Fluorescent Quantitative PCR Detection (qPCR)

[0047] The qPCR reaction system is prepared with reference to the TB Green Premix Ex Taq II Kit (RR820A), as shown in Table 2.

[0048]

[0049]

[0050] The qPCR reaction conditions are shown in Table 3.

[0051]

[0052] The primer sequences used in qPCR are shown in Table 4.

[0053]

[0054] 2.6 Detection of viral copy number

[0055] Based on ORF79 (catalytic subunit of DNA polymerase) encoded by CyHV-2, the laboratory established a standard curve between the copy number of the target gene and the Ct value as follows: y = -3.1394x + 38.9 (y is the Ct; x is the lg value of the viral copy number; R 2 = 0.9974). Using cellular DNA as a template, qPCR was performed with the same primers as the standard curve, and the Ct value was substituted into the standard curve to calculate the viral copy number.

[0056] 2.7 Determination of the effect of MG132 on CyHV-2 replication and localization in cells by immunofluorescence staining combined with AIE probe labeling

[0057] GiCF cells were seeded in 12-well plates and pretreated with 20 μM MG132, and DMSO treatment served as the control group. Then, after infecting with CyHV-2 (MOI = 1) for 48 h, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min, and then blocked with QuickBlock TM Blocking Buffer for 10 min. The above cell samples were incubated overnight at 4 °C with ORF150 rabbit primary antibody (1:1000), washed with PBS, and then incubated with 405-conjugated Goat anti-Rabbit IgG(H+L) at room temperature for 2 h. After washing with PBS, the cells were sequentially counterstained with AIE nuclear green probe C 33 H 30 N4O2 2+ and AIE cell membrane red probe C 35 H 26 N3S2 for 10 min. Images were recorded using a Leica SP8 confocal microscope system and analyzed using LASX Office software.

[0058] 3 Results

[0059] As Figure 1 shown, compared with the DMSO treatment group, MG132 treatment could significantly inhibit the CyHV-2 copy number in cells in a dose-dependent manner. At the same time, the transcription of viral genes in cells was also significantly downregulated in a dose-dependent manner, including immediate-early genes (ORF121), early genes (ORF62, ORF92), and late genes (ORF33, ORF46), as Figure 2As shown. In addition, the replication of the virus in cells was observed by immunofluorescence staining, indicating that viral proteins were significantly inhibited in the presence of MG132, while in the DMSO control, viral proteins filled the entire cell. The above results indicate that MG132 can significantly inhibit the copy number of CyHV-2 and the transcriptional level of viral genes in cells, indicating that the host proteasome is crucial for CyHV-2 infection.

Claims

1. Use of MG132 in the preparation of at least one of the following products for use with carp herpesvirus type II: (1) Products that inhibit carp herpesvirus type II; (2) Products for the prevention or treatment of carp herpesvirus type 2 infection; (3) Products for the prevention or treatment of hematopoietic necrosis induced by herpesvirus type 2 infection.

2. The use according to claim 1, characterized in that: The product includes at least one of reagents, medicines, feeds, additives, water quality improvers, and bottom quality improvers.

3. The use according to claim 1, characterized in that: The product is suitable for agriculture, fishery and breeding.

4. At least one of the following products for use with carp herpesvirus type II, characterized in that: Contains MG132: (1) Products that inhibit carp herpesvirus type II; (2) Products for the prevention or treatment of carp herpesvirus type 2 infection; (3) Products for the prevention or treatment of hematopoietic necrosis induced by herpesvirus type 2 infection.

5. The product according to claim 4, characterized in that The product includes at least one of reagents, medicines, feeds, additives, water quality improvers, and bottom quality improvers.

6. The product according to claim 4, characterized in that The product is suitable for agriculture, fishery and breeding.

7. A method for preventing and treating carp herpesvirus type II infection, characterized in that: The steps include: Add MG132 or the product for carp herpes virus type II described in any one of claims 4-6 to the target environment.

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