Targeted G protein palmitoylated polypeptide and application thereof in resisting aquatic rhabdovirus

Abstract

The invention provides a targeted G protein palmitoylated polypeptide and an application of the targeted G protein palmitoylated polypeptide in resisting aquatic rhabdovirus, and belongs to the technical field of aquatic virus prevention and treatment. The polypeptide provided by the invention performs competitive inhibition on palmitoylation modification of G protein during virus infection and induces the G protein to be degraded by lysosome, so that the purpose of inhibiting virus proliferation is achieved. Through verification, the targeted polypeptide designed for the SVCV G protein and the IHNV G protein can respectively generate competitive inhibition on the G proteins of the two viruses, SVCV and IHNV infection can be safely and efficiently inhibited, and the survival rate of fishes infected with the SVCV and IHNV viruses is effectively improved. The invention provides a theoretical and application basis for developing aquatic animal virus disease treatment medicines taking palmitoylation modification as a target spot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquatic virus prevention and treatment, and particularly relates to a polypeptide targeting viral G protein palmitoylation and application thereof in resisting aquatic rhabdovirus. BACKGROUND

[0002] Spring viremia of carp (SVC) is an acute and highly lethal infectious disease caused by spring viremia of carp virus (SVCV), which has been listed as one of the diseases that must be reported by the World Organization for Animal Health. The main pathological symptoms of the diseased fish are hemorrhage of liver, spleen and kidney tissues, severe peritonitis and hemorrhagic enteritis, etc. According to the report of “2022 Analysis of Important Epidemic Diseases of Aquatic Animals in China”, the monitoring results from 2005 to 2021 showed that SVC was widely distributed, mainly in Liaoning Province and Heilongjiang Province in the northeast; Tianjin, Hebei Province and Inner Mongolia Autonomous Region in the north; Shaanxi Province, Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region in the northwest; Henan Province, Hubei Province and Hunan Province in central China; among them, SVCV positive samples were found in Hubei Province for 5 consecutive years. There are about 10 seedling farms per year in China that are SVCV positive, and among the positive samples monitored, carp accounts for 70.7%, koi carp 12.0%, goldfish 8.0%, crucian carp 5.2%, grass carp 1.8%, silver carp 1.4%, bighead carp 0.2%, and other varieties 0.7%.

[0003] Infectious hematopoietic necrosis (IHN) is a viral disease caused by infectious hematopoietic necrosis virus (IHNV) that leads to high mortality in young salmonids, with a mortality rate of up to 100%. After the fish is infected with the virus, symptoms such as spiral swimming, fecal tailing, blackening of the skin, protruding eyeballs, abdominal distension and external bleeding may occur. In the case of fish survival, spinal deformity may become apparent. The World Health Organization has listed this disease as an animal epidemic that must be reported, and the disease has been widely distributed in many countries around the world, causing huge economic losses and threats to salmon and trout farming.

[0004] Existing research has found that viruses cannot encode palmitoyl transferase, but during the process of infecting host cells, they can widely use the palmitoylation of host cells to modify the proteins necessary for their own replication and infection of host cells. In 1979, researchers first discovered that Sindbis virus (SbV) envelope glycoprotein and vesicular stomatitis virus (VSV) envelope glycoprotein were palmitoylated by using 3H-palmitic acid as a metabolic marker of host cells through autoradiography. Sindbis virus Vesicular stomatitis virus ​, VSV) glycoprotein G can be S-palmitoylated. During viral infection, SARS-CoV-2 S protein undergoes extensive palmitoylation modification, especially the rapid palmitoylation of SARS-CoV-2 S protein on 10 cytosolic cysteines in the endoplasmic reticulum and Golgi. Further studies have shown that SARS-CoV-2 S-palmitoylation generates a cholesterol-rich lipid domain in the viral envelope, and palmitoylation modification and lipid biosynthesis pathways promote the infectivity of the new coronavirus. Thorp et al. found that low-dose 2-BP affected alphavirus and flavivirus infection, greatly reduced the specific infectivity of mouse coronavirus, and found that palmitoylation on the spike protein of mouse coronavirus was essential for virus assembly and infectivity. These studies suggest that viral protein palmitoylation modification and lipid biosynthesis enzymes are novel antiviral therapeutic targets. However, there is no report on therapeutic drugs targeting palmitoylation modification in aquatic viruses. SUMMARY

[0005] The present application first found that polypeptides targeting viral G protein palmitoylation can significantly inhibit the replication of certain aquatic rhabdoviruses in fish in vivo and in vitro, and significantly improve the survival rate of fish after infection with aquatic viruses, providing a research basis for the development of anti-aquatic virus drugs targeting palmitoylation modification.

[0006] The technical solutions of the present application are as follows: In a first aspect, the present application provides two polypeptides targeting palmitoylation modification of aquatic rhabdovirus G protein, specifically: Polypeptide A targeting SVCV G protein, containing an amino acid sequence as shown in SEQ ID NO. 1; Polypeptide B targeting IHNV G protein, containing an amino acid sequence as shown in SEQ ID NO. 2.

[0007] Further, in specific applications, other polypeptide sequences can be connected to the above-mentioned polypeptide sequences to form a fusion polypeptide according to actual needs. For example, in some embodiments of the present application, a fusion polypeptide containing the above-mentioned polypeptide sequence and a cell-penetrating peptide is constructed. Further, the cell-penetrating peptide TAT protein transduction peptide has an amino acid sequence as shown in SEQ ID NO. 3.

[0008] In a second aspect, the present application provides the use of the above-mentioned polypeptide targeting viral G protein palmitoylation in the preparation of drugs for preventing and treating aquatic rhabdovirus, wherein the aquatic rhabdovirus includes SVCV and IHNV.

[0009] Experimental data show that the peptides targeting palmitoylation of SVCV G protein and IHNV G protein provided in this invention, through competition, can inhibit palmitoylation modification of G protein during viral infection and reduce the expression level of G protein during viral infection. Injecting these peptides into fish can inhibit viral replication, significantly improve the survival rate of fish after viral infection, and reduce the viral load in organs (including spleen, intestine, and brain) of fish infected with aquatic viruses.

[0010] Furthermore, the aforementioned drugs may also contain pharmaceutically acceptable excipients.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the palmitoylation modification of viral G proteins, this invention designs two novel anti-palmitoylation peptides targeting SVCV and IHNV, respectively, using the principle of competitive inhibition. The peptides provided by this invention can inhibit palmitoylation modification of viral G proteins and promote the degradation and clearance of viral G proteins, thereby inhibiting viral proliferation and significantly improving the survival rate of fish infected with the virus. Therefore, designing peptides targeting G protein palmitoylation can be developed as drugs for virus control in aquaculture, which is of great significance for the prevention and control of aquatic rhabdoviruses (especially SVCV). Attached Figure Description

[0012] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0013] Figure 1 The graph shows the detection results of the cytotoxicity of different concentrations of peptides CPP-GS1 and CPP-GI1 on ZF4 cells in Example 2. Figure 2 The figure shows the detection results of the inhibition of palmitoylation modification of SVCV G protein by peptide CPP-GS1 and the degradation of SVCV G protein in Example 3. Figure 3 This is a graph showing the detection results of different concentrations of peptide CPP-GS1 inhibiting SVCV proliferation in Example 4; Figure 4 The image shows the protective effect of peptide CPP-GS1 against SVCV infection in zebrafish in Example 5. Figure 5 This is a graph showing the detection results of different concentrations of peptide CPP-GI1 inhibiting IHNV proliferation in Example 6; Figure 6This is a diagram showing the protective effect of peptide CPP-GI1 against IHNV infection in rainbow trout in Example 7. Detailed Implementation

[0014] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the following embodiments are exemplary and are only used to explain this invention, and should not be construed as limiting the invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0016] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0017] Example 1 This example provides palmitoylated peptides that target the SVCV G protein and the infectious hematopoietic necrosis virus G protein, respectively, as shown below: Peptide A, which targets the SVCV G protein, contains the following amino acid sequence: LILIFLLIRCCVACTYLMKR(SEQ ID NO.1); Peptide B, which targets the IHNV G protein, contains the following amino acid sequence: LVLLLAVAACCCWSGRPPTPS (SEQ ID NO. 2).

[0018] Furthermore, cell-penetrating peptides can be linked to the aforementioned polypeptide sequences to construct fusion polypeptides. In this example, the cell-penetrating peptide is a TAT protein transduction peptide (YGRKKRRQRRR, SEQ ID NO.3), resulting in the following two fusion polypeptides: CPP-GS1:YGRKKRRQRRRLILIFLLIRCCVACTYLMKR; CPP-GI1:YGRKKRRQRRRLVLLLAVAACCCWSGRPPTPS.

[0019] Example 2 In this example, the MTT assay was used to detect the toxicity of peptides CPP-GS1 and CPP-GI1. The specific experiments are as follows: Zebrafish embryo fibroblast-like cells (ZF4) in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted. 200 μL of culture medium was added to each well of a 96-well plate, maintaining a cell density of 1000-5000 cells / well. The edge wells were filled with sterile PBS. After incubation at 28°C with 5% CO2 until cell adhesion (approximately 6-8 hours), a concentration gradient of peptides was added, with 6 replicates per gradient. Incubation continued for 48 hours, and cell status was observed under an inverted microscope. 20 μL of MTT solution (5 mg / ml, i.e., 0.5% MTT) was added to each well, and culture continued for 4 hours. Culture was then terminated, and the culture medium was carefully aspirated from the wells. 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was placed on a decolorizing shaker and shaken slowly for 10 minutes to fully dissolve any crystals. The absorbance of each well was measured at OD490 nm using an ELISA reader, and cell viability was calculated.

[0020] This experiment also includes zeroing wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, solvent of the corresponding concentration, culture medium, MTT, dimethyl sulfoxide).

[0021] The results are as follows Figure 1 As shown, the cell viability of ZF4 cells treated with various concentrations of peptides CPP-GS1 and CPP-GI1 for 48 hours did not change significantly compared with the control group, indicating that the peptides used had no toxic effect on ZF4 cells.

[0022] Example 3 This example demonstrates the inhibition of palmitoylation modification and expression of SVCV G protein by the peptide CPP-GS1 through the following experiments.

[0023] (1) Inhibition of palmitoylation modification of SVCV G protein by peptide CPP-GS1.

[0024] ZF4 cells were seeded in 12-well plates. After reaching 70-80% confluence, the SVCV G eukaryotic expression plasmid was transfected into the cells. After 24 h of overexpression, complete culture medium containing specified concentrations of peptides CPP-GS1, CPP-GS2 (peptides with palmitoylation site mutations) or DMSO was added. The cells were then cultured at 16°C in a 5% CO2 incubator for another 24 h (three independent replicates were set up for each treatment). Cell protein samples were collected after 12 h, and the effect of the peptides on the palmitoylation modification of SVCV G protein was detected by IP-ABE method.

[0025] The results are as follows Figure 2As shown in Figure A, compared with the control group, treatment with peptide CPP-GS1 significantly reduced palmitoylation modification of SVCV G protein.

[0026] (2) Degradation of SVCV G protein by peptide CPP-GS1.

[0027] ZF4 cells were seeded in 12-well plates. After reaching 70-80% confluence, the SVCV G eukaryotic expression plasmid was transfected into the cells. After 24 hours of overexpression, complete culture medium containing specified concentrations of peptides CPP-GS1, CPP-GS2 (peptides with palmitoylation site mutations), or DMSO was added. The cells were then cultured at 16°C in a 5% CO2 incubator for another 24 hours (three independent replicates were set up for each treatment). Cell protein samples were collected after 12 hours, and the effect of peptides on SVCV G protein expression was detected by Western blotting.

[0028] The results are as follows Figure 2 As shown in Figure B, compared with the control group, treatment with peptide CPP-GS1 significantly reduced the expression level of SVCV G protein.

[0029] Example 4 The peptide CPP-GS1 can effectively inhibit the proliferation of SVCV, which was specifically verified in this case through the following experiments: ZF4 cells were seeded in 12-well plates. After reaching 70-80% confluence, the culture medium was aspirated, and complete culture medium containing different concentrations of peptide CPP-GS1 or DMSO was added for pretreatment for 2 hours. The culture medium was then aspirated, and cells were adsorbed with 1 mL of 0.1 MOI SVCV. The cells were incubated at 16°C for 1 hour, and free viruses were washed away. Complete culture medium containing the corresponding concentration of peptide or DMSO was added again, and the cells were incubated at 16°C in a 5% CO2 incubator for another 24 hours (each treatment had three independent replicates). After 24 hours, cells and culture supernatant were collected, and SVCV proliferation was detected using qRT-PCR and plaque assays. The sequences of the primers used for quantitative detection of the SVCV-G gene are shown in SEQ ID NO. 4-5 of the sequence listing.

[0030] The results are as follows Figure 3 As shown, compared with the control group, treatment with peptide CPP-GS1 significantly reduced the viral load of SVCV ( P The result was <0.001, indicating that the peptide CPP-GS1 can significantly inhibit the proliferation of SVCV at the cellular level.

[0031] Example 5 This example uses zebrafish to investigate the effect of intraperitoneal injection of the peptide CPP-GS1 on SVCV infection in fish. The specific experiment is as follows: Adult zebrafish aged three months were purchased from the National Zebrafish Resource Center and acclimatized for two weeks at 28℃, fed with brine shrimp twice daily (morning and evening). After the acclimatization period, healthy, disease-free zebrafish of similar size and uniformity were selected. The selected zebrafish were then cooled by 1℃ daily, gradually decreasing the water temperature from 28℃ to 16℃ and maintaining it at 16℃ for subsequent experiments. Dissolved oxygen levels in the culture water were maintained at 6-14 mg / L using an air pump, and the pH was controlled between 6.8 and 7.8. The zebrafish were divided into a control group and a polypeptide injection group (50 μg / g), with the control group receiving the same volume of DMSO as a control. SVCV virus suspension was injected into the zebrafish along the base of the pelvic fins, and survival was observed and recorded over 14 days.

[0032] See results Figure 4 A. The survival rate of zebrafish infected with SVCV in the control group was 20.00%, while the survival rate of zebrafish infected with SVCV significantly increased to 56.67% after injection of peptide CPP-GS1, indicating that peptide CPP-GS1 can improve the survival rate of zebrafish infected with SVCV.

[0033] Spleen, intestine, and brain of zebrafish infected with SVCV for 5 days in the above experiment were collected, and total RNA was extracted. After reverse transcription, absolute quantification of SVCV-G was performed using fluorescence quantitative PCR. The primers used for quantification of the SVCV-G gene were the same as in Example 4. Results are shown below. Figure 4 B indicates that the peptide CPP-GS1 can significantly reduce the viral load in the spleen, intestine, and brain of zebrafish infected with SVCV.

[0034] Example 6 The peptide CPP-GI1 can effectively inhibit the proliferation of IHNV, which was specifically verified in this case through the following experiments: ZF4 cells were seeded in 12-well plates. After reaching 70-80% confluence, the culture medium was aspirated, and complete culture medium containing different concentrations of peptide CPP-GI1 or DMSO was added for pretreatment for 2 hours. The culture medium was then aspirated, and cells were adsorbed with 1 mL of 0.1 MOI IHNV. The cells were incubated at 16°C for 1 hour to wash away free virus. Complete culture medium containing the corresponding concentrations of peptide and DMSO was added, and the cells were incubated at 16°C in a 5% CO2 incubator for another 48 hours (each treatment had three independent replicates). After 48 hours, cells and culture supernatant were collected, and IHNV proliferation was detected using qRT-PCR and plaque assays. The sequences of the primers used for quantitative detection of the IHNV-G gene are shown in SEQ ID NO. 6-7 of the sequence listing.

[0035] The results are as follows Figure 5 As shown, compared with the control group, treatment with peptide CPP-GI1 significantly reduced the viral load of IHNV (P The result was <0.001, indicating that the peptide CPP-GI1 can significantly inhibit the proliferation of IHNV at the cellular level.

[0036] Example 7 This example uses rainbow trout to investigate the effect of peptide CPP-GI1 on IHNV infection in fish. The specific experiment is as follows: Rainbow trout measuring 6-7 cm were purchased from a fish farm in Meishan, Sichuan Province. They were acclimatized for two weeks at 16℃, fed twice daily (morning and evening). After the acclimatization period, healthy, disease-free rainbow trout of similar size and specifications were selected and maintained at 16℃ for subsequent experiments. Dissolved oxygen levels in the culture water were maintained at 6-14 mg / L using an air pump, and the pH was controlled at 6.8-7.8. The rainbow trout were divided into a control group and a polypeptide injection group (50 μg / g). The control group received the same volume of DMSO as a control. IHNV virus suspension was injected into infected zebrafish along the base of the pelvic fin, and survival was observed and recorded over 14 days.

[0037] See results Figure 6 A. The survival rate of rainbow trout infected with IHNV was 16.67%, while the survival rate of rainbow trout infected with IHNV significantly increased to 46.67% after injection of peptide CPP-GI1, indicating that peptide CPP-GI1 can improve the survival rate of rainbow trout infected with IHNV.

[0038] Spleen, intestine, and brain of rainbow trout infected with IHNV for 5 days in the above experiment were collected, and total RNA was extracted. IHNV-G was then quantitatively detected using reverse transcription followed by quantitative real-time fluorescence assay (primers used were the same as in Example 6). Results are shown below. Figure 6 B shows that the peptide CPP-GI1 significantly reduces the viral load in the spleen, intestine, and brain of rainbow trout infected with IHNV.

[0039] In summary, the peptides CPP-GS1 and CPP-GI1 provided by this invention can competitively inhibit the palmitoylation modification of SVCV G protein and IHNV G protein during viral infection, respectively, and induce the degradation of G protein by lysosomes, thereby safely and efficiently inhibiting the proliferation of SVCV and IHNV, reducing the infection effect, and can be developed as therapeutic drugs for viral diseases in aquatic animals.

[0040] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Use of a polypeptide targeting palmitoylation of viral G protein in the preparation of a medicament for preventing and treating aquatic rhabdovirus.

2. Use according to claim 1, characterized in that, The polypeptide is at least one of the following: a polypeptide A comprising an amino acid sequence as shown in SEQ ID NO. 1; a polypeptide B comprising an amino acid sequence as shown in SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, The polypeptide A and / or polypeptide B is a fusion polypeptide further comprising a cell-penetrating peptide.

4. Use according to claim 3, characterized in that, The cell-penetrating peptide is a TAT protein transduction peptide having an amino acid sequence as shown in SEQ ID NO.

3.

5. The use according to claim 1, characterized in that, The aquatic rhabdovirus includes spring viremia of carp virus and infectious hematopoietic necrosis virus.

6. Use according to claim 1, characterized in that, The polypeptide inhibits palmitoylation modification of G protein during viral infection by competition and reduces the expression level of G protein during viral infection.

7. An agent against aquatic viruses, characterized by, The polypeptide A and / or polypeptide B of claim 2.

8. The medicament according to claim 7, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

9. The medicament according to claim 7, characterized in that, The aquatic virus includes spring viremia of carp virus and infectious hematopoietic necrosis virus.

10. The medicament according to claim 9, characterized in that, The medicament inhibits the proliferation of the aquatic virus and reduces the viral load in the organs of fish infected with the aquatic virus, the organs including spleen, intestinal tract and brain.

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