Cecropin D-modified antiviral peptides and their application in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Abstract

The application discloses an anti-virus polypeptide based on Cecropin D (CD) modification and application thereof in inhibition of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection. The amino acid sequence of the anti-virus polypeptide is shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. Based on the balance between hydrophilicity and hydrophobicity, the application designs and synthesizes three derivatives CD-2, CD-3 and CD-4 by modifying the parent peptide CD. Compared with the research of other known anti-virus polypeptides, the polypeptide of the application exhibits significant anti-PRRSV activity at a lower concentration (100 μg / mL), which indicates that the polypeptide designed and synthesized by the application has higher safety and application potential in anti-PRRSV drug development. The application provides a new technical means for inhibiting PRRSV infection.

Description

Technical Field

[0001] This invention relates to a novel antiviral polypeptide based on Cecropin D modification, and also to the application of this antiviral polypeptide in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Viral infections in pigs are one of the major challenges facing the global swine industry. Widely prevalent swine pathogens include pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV), and porcine reproductive and respiratory syndrome virus (PRRSV), causing significant economic losses to the industry. PRRSV, in particular, poses a substantial threat to the global swine industry, making vaccination a primary means of disease control. However, despite the approval and widespread clinical use of various traditional PRRSV vaccines, many challenges remain. Traditional inactivated vaccines, while posing no biosafety risks, exhibit low levels of neutralizing antibodies, fail to induce cellular immunity, and require multiple vaccinations. In contrast, while live vaccines provide immune protection, they offer insufficient protection against heterologous strains and suffer from issues such as virulence reversion, recombination between vaccine and wild-type strains, and a lack of markers. Furthermore, while subunit vaccines and virus-like particle vaccines are non-pathogenic, their protective ability in susceptible animals is limited. Nucleic acid vaccines, although simple to produce, pose potential biosafety risks, such as the possibility of exogenous DNA integrating into the host genome. At the same time, the high mutation and recombination rates of PRRSV make existing vaccines prone to ineffectiveness, further increasing the difficulty of vaccine development.

[0003] Due to the significant challenges posed by PRRSV globally, an increasing number of peptides with anti-PRRSV activity have been discovered and designed. Antimicrobial peptides are small protein molecules widely found in animals, plants, and microorganisms. They possess potential antibacterial, antiviral, antifungal, antitumor, and antiparasitic activities. They can inhibit viral replication by interfering with multiple stages of the viral life cycle, and their mechanisms of action are diverse, mainly including disrupting the viral envelope, preventing the virus from binding to host cells, and interacting with specific receptors on host cells.

[0004] Cecropin D (CD) belongs to the Cecropin family and was originally isolated from the silkworm moth [HULTMARK D, ENGSTROMA, BENNICH H, et al. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae[J]. Eur J Biochem, 1982, 127(1): 207-17]. It is a multifunctional antimicrobial peptide with multiple effects such as antiviral, antifungal, antitumor and immunomodulatory properties.

[0005] This invention first studied the antiviral effect of Cecropin D, and then further enhanced the anti-PRRSV activity of Cecropin D by introducing different cationic amino acids, providing a new technical means to inhibit PRRSV infection. Summary of the Invention

[0006] The purpose of this invention is to provide an antiviral polypeptide based on Cecropin D and its application in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] This invention, based on a balance between hydrophilicity and hydrophobicity, designed and synthesized Cecropin D and its three derivatives CD-2 to CD-4. Specifically, CD-2 incorporates more positively charged lysine residues to enhance the peptide's interaction with the membrane, while CD-3 incorporates highly hydrophobic and positively charged tryptophan. In CD-4, valine (V) is replaced with phenylalanine (F) to promote secondary structure stability. HPLC and CD spectroscopy results show that the designed peptides have high purity and all form significant α-helical structures. Especially under simulated cell membrane conditions, the peptides exhibit significant structural changes, verifying their ability to form stable secondary structures. Studies have shown that α-helices can insert into the viral envelope, causing membrane disruption and interfering with its structural integrity, thereby inhibiting viral infection. [HUANGY, HE L, LI G, et al. Role of helicity of alpha-helical antimicrobial peptides to improve specificity[J]. Protein Cell, 2014, 5(8): 631-42. HUAN Y, KONG Q, MOU H, et al. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields[J]. Front Microbiol, 2020, 11(582-779.] G W. Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles[J]. The Journal of Biological Based on these structural characteristics, it was inferred that they should possess good antiviral activity. Therefore, we tested their antiviral activity using various experimental methods. The results showed that the peptides of this invention exhibited significant anti-PRRSV activity at a low concentration (100 μg / mL). These peptides inhibit PRRSV replication by blocking key steps in PRRSV invasion of cells.

[0009] Therefore, based on the above research, this invention proposes an antiviral polypeptide modified with Cecropin D, the amino acid sequence of which is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] Furthermore, the present invention also proposes the application of the aforementioned antiviral peptide in the preparation of drugs that inhibit porcine reproductive and respiratory syndrome virus (PRRSV).

[0011] Preferably, the porcine reproductive and respiratory syndrome virus (PRRSV) is a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).

[0012] Preferably, the antiviral peptide inhibits PRRSV replication by blocking key steps in PRRSV invasion of cells.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] This invention, based on a balance between hydrophilicity and hydrophobicity, designed and synthesized three derivatives by modifying the parent peptide Cecropin D. Compared with studies of other known antiviral peptides, the peptides of this invention exhibit significant antiviral activity at lower concentrations. For example, Liu et al. demonstrated that the peptide Cecropin D exhibited effective antiviral activity against PRRSV infection and replication at an in vitro concentration of 300 μg / mL. [ XL,CG,YH,et al.Inhibition of porcinereproductive andrespiratory syndrome virus by Cecropin D in vitro[J].Infection,genetics and evolution:journal of molecular epidemiology andevolutionary genetics in infectious diseases,2015, ] The peptide designed in this invention exhibits excellent anti-PRRSV activity at a concentration of 100 μg / mL. This indicates that the peptide designed and synthesized in this invention has high safety and application potential in the development of anti-PRRSV drugs, and the invention provides a new technical means for inhibiting PRRSV infection. Attached Figure Description

[0015] Figure 1A -D represents the high-performance liquid chromatogram and mass spectrum of polypeptide CD and CD-2 to CD-4;

[0016] Figure 1E The CD values ​​are for the polypeptide CD and CD-2 to CD-4 in aqueous solution;

[0017] Figure 1F The CD values ​​of peptides and CD-2 to CD-4 in a simulated cell membrane environment;

[0018] Figure 2 The results show the toxicity of peptide CD and CD-2 to CD-4 to MARC-145 cells.

[0019] Among them, ad represents the toxicity results of peptide CD and CD-2 to CD-4 on MARC-145 cells, respectively.

[0020] Figure 3 The results show the detection of antiviral activity of peptide CD and CD-2 to CD-4 direct action;

[0021] Where A represents the relative expression level of PRRSV mRNA; B represents the Western blot results of PRRSV-N protein expression; and C represents the bar chart of the Western blot results of PRRSV-N protein expression.

[0022] Figure 4 The results show the antiviral activity of peptide CD and CD-2 to CD-4 during the pretreatment stage;

[0023] In this diagram, A represents the relative expression level of PRRSV mRNA; B represents the Western blot results of PRRSV-N protein expression; C represents the bar chart of the Western blot results of PRRSV-N protein expression; and D represents the results of indirect immunofluorescence detection.

[0024] Figure 5 The results show the antiviral activity of peptide CD and CD-2 to CD4 during the adsorption phase.

[0025] Where A represents the relative expression level of PRRSV mRNA; B represents the Western blot results of PRRSV-N protein expression; and C represents the bar chart of the Western blot results of PRRSV-N protein expression.

[0026] Figure 6 The results show the antiviral activity of peptide CD and CD-2 to CD-4 during the invasion phase;

[0027] In this diagram, A represents the relative expression level of PRRSV mRNA; B represents the Western blot results of PRRSV-N protein expression; C represents the bar chart of the Western blot results of PRRSV-N protein expression; and D represents the results of indirect immunofluorescence detection. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0029] Example 1: Synthesis of antiviral peptides and their application in combating PRRSV infection

[0030] 1. Materials and Methods

[0031] 1.1 Cells and Viruses

[0032] African green monkey kidney epithelial cells (MeatAnimal Research Center-145, MARC-145) are preserved in our laboratory.

[0033] The strain: High pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV) was preserved in this laboratory.

[0034] 1.2 Synthesis and purification of antiviral peptides

[0035] Based on the structural characteristics of Cecropin D (CD) and the membrane structure of PRRSV, CD was cationicized, and three derivatives, CD-2 to CD-4, were designed and synthesized. Subsequently, these antiviral peptides were synthesized using solid-phase synthesis technology, and purified and analyzed for purity by high-performance liquid chromatography, yielding antiviral peptides with a purity of up to 95%.

[0036] 1.3 Structural Analysis

[0037] The secondary structures of these antiviral peptides were characterized by circular dichroism spectroscopy. We used a circular polarization spectroscopy system (JASCO) to dissolve these antiviral peptides in water and 50% trifluoroethanol, respectively, and measured their CD spectra in the range of 190 nm to 260 nm.

[0038] 1.4 Cytotoxicity assay

[0039] To assess the toxicity of CD4 and CD2 through CD4 to MARC-145 cells, a cell viability assay (MTS) was used. MARC-145 cells in the logarithmic growth phase (density 1 × 10⁶) were used. 6Cells (cells / mL) were seeded into 96-well plates and cultured for 24 hours until cell confluence reached 80%–90%. Peptide CD and CD-2 to CD-4 were diluted proportionally starting at 100 μg / mL, the supernatant was discarded, and 100 μL of peptide solution was added to each well. Cells were then cultured for another 24 hours. 10 μL of MTS solution was added to each well, and the cells were cultured for another 1–2 hours. The absorbance at 490 nm was then measured using a microplate reader, and the percentage of relative cell viability was calculated.

[0040] 1.5 Anti-PRRSV virus experiment

[0041] 1.5.1 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0042] MARC-145 cells were divided at a ratio of 1×10 6 Cells were seeded at a density of 1 cell / mL in 6-well plates and treated with peptide CD and CD-2 to CD-4, respectively, and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). After 18 hours, RNA was extracted from the cells, reverse transcribed to obtain cDNA, and detected by real-time quantitative PCR. The reaction program and primers are shown in Table 1. Each sample was tested in triplicate, and the data were analyzed.

[0043] Table 1 RT-qPCR primers

[0044]

[0045] 1.5.2 Western blotting (WB)

[0046] MARC-145 cells were seeded in 6-well plates. When the cell confluence reached 70%, the cells were treated. After 18 hours, the culture medium was discarded, and the cells were treated with 5× loading buffer and denatured at 100°C for 10 minutes. The sample was added to the wells of the protein gel and electrophoresed at 80V. The protein was then transferred to an NC membrane by electroporation. After transfer, the membrane was blocked with 5% skim milk for 2 hours. Next, PRRSV-N protein antibody (primary antibody) was prepared at a dilution of 1:1000 and incubated overnight at 4°C. The membrane was washed 5 times for 3 minutes each time, and HRP-labeled anti-rabbit IgG secondary antibody at a dilution of 1:2000 was added. After incubation at room temperature for 1 hour, the membrane was washed 5 more times. Finally, chemiluminescence buffer was prepared, and the membrane was placed in a chemiluminescence imaging system for detection.

[0047] 1.5.3 Indirect immunofluorescence assay (IFA)

[0048] MARC-145 cells were loaded into 6-well plates at a rate of 1 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL. When cells reached 70% confluence, they were treated. After 18 hours, the culture medium was discarded, and the cells were washed three times with PBS for 3 minutes each time. Cells were then fixed with 4% paraformaldehyde for 15 minutes and washed again with PBS. Next, cells were treated with 0.1% Triton X-100 for 15 minutes and washed three times with PBS. Cells were blocked with 5% BSA and incubated at 37°C for 1 hour, after which the blocking solution was discarded. Primary antibody (1:100 dilution) was added at 500 μL per well, and the cells were incubated overnight at 4°C, followed by washing with PBS. Secondary antibody (1:500 dilution) was added at 500 μL per well, and the cells were incubated at 37°C in the dark for 1 hour, after which the solution was discarded, and the cells were washed five times with PBS. DAPI was added to each well, and the cells were incubated at room temperature for 10 minutes, then discarded. The cells were then washed three times with PBS, and finally observed under a fluorescence microscope.

[0049] 2 Results Analysis

[0050] 2.1 Design and synthesis of antiviral peptides

[0051] Based on the structural features of CD and the membrane structure of PRRSV, CD was cationicized to design and synthesize three derivatives: CD-2, CD-3, and CD-4. The antiviral peptide sequences are shown in Table 2.

[0052] Table 2. Peptide sequences and their physicochemical properties

[0053]

[0054] a: Molecular weight (MW) of peptides were measured by ESI-MS.

[0055] b:Retention time of peptides were determined by RP-HPLC.

[0056] 2.2 Peptide Purity and Structure Analysis

[0057] Antiviral peptides CD and CD-2 to CD-4 were synthesized using solid-phase synthesis technology, and purified and purified using high-performance liquid chromatography and mass spectrometry. The results are as follows: Figure 1A As shown in Figure -D, the results indicate that the purity of all peptides exceeded 95%. Subsequently, circular dichroism spectroscopy was used to analyze the structure of the synthesized peptides, and the results are as follows... Figure 1EAs shown, this result indicates that in aqueous solution, the four polypeptides exhibit a random coiled structure. However, as... Figure 1F As shown, the four peptides underwent significant structural changes in a simulated cell membrane environment, exhibiting two typical negative peaks at wavelengths of 208 nm and 222 nm, indicating significant α-helix structure formation. To simulate protein behavior in the cell membrane, we selected hydrophobic 50% trifluoroethanol (TFE) as the simulation environment and found significant changes in the circular dichroism chromatograms of the four peptides. This indicates that the TFE environment can induce significant changes in peptide structure, promoting the formation of more stable α-helix structures in the cell membrane environment.

[0058] 2.3 Cytotoxicity

[0059] The cytotoxicity of peptide CD and CD-2 to CD-4 to MARC-145 cells was evaluated using the MTS assay. Results are as follows: Figure 2 As shown in the diagram, the results indicate that peptides CD and CD-2 to CD-4 exhibited low toxicity to MARC-145 cells within the effective concentration range. Even at a final peptide concentration of 100 μg / mL, the toxicity of all four peptides remained low, with cell viability maintained above 90%. This result demonstrates that even at higher concentrations, peptides CD and CD-2 to CD-4 have minimal impact on cells and do not significantly inhibit cell proliferation and growth. Based on these results, 100 μg / mL was determined as the highest test concentration in the antiviral activity evaluation experiment to ensure the antiviral efficacy of peptides CD and CD-2 to CD-4 was assessed at the maximum effective concentration while maintaining good cell viability.

[0060] 2.4 Evaluation of anti-PRRSV activity

[0061] 2.4.1 The peptide has a direct inactivating effect on PRRSV.

[0062] To investigate whether the peptides CD and CD-2 to CD-4 have a direct viral inactivation effect on PRRSV, HP-PRRSV was incubated with 100 μg / mL of CD, CD-2, CD-3, and CD-4 at 37°C for 2 h. The incubation was then applied to cells, and the antiviral activity of the peptides was assessed by viral titer. RT-qPCR and Western blot were used to quantify viral RNA levels and PRRSV-N protein expression. Results are as follows: Figure 3 As shown, the results indicate that none of the four peptides exhibited a significant viral inactivation effect. RT-qPCR data showed no significant decrease in viral RNA levels between the treated and control groups. Figure 3 A). Furthermore, the results of the Western blot analysis ( Figure 3 As shown in B and C), there was no significant difference in the expression level of PRRSV-N protein between the peptide-treated group and the control group. This indicates that under these conditions, the peptide did not produce a significant direct antiviral effect against PRRSV.

[0063] 2.4.2 Peptide inhibits PRRSV infection of cells

[0064] To evaluate whether the designed peptides CD and CD-2 to CD-4 could protect MARC-145 cells from PRRSV infection, the preventive effects of peptides CD and CD-2 to CD-4 on MARC-145 were investigated. Cells were treated with 100 μg / mL of CD, CD-2, CD-3, and CD-4 for 2 h, respectively. The supernatant was then removed, and the cells were washed twice with serum-free DMEM. Subsequently, cells were infected with HP-PRRSV (MOI=1) for 2 h, washed, and cultured for another 18 h before sample preparation and detection of viral load. RT-qPCR results are shown below. Figure 4 As shown in Figure A, the results indicate that viral RNA levels were significantly lower in the CD and CD-2 treatment groups compared to the control group, showing a clear difference. Western blot analysis results are as follows: Figure 4 As shown in B and C, the results indicate that the expression levels of PRRSV-N protein were significantly different between the CD and CD-2 treatment groups and the control group, while there were no significant differences in the other treatment groups. Indirect immunofluorescence detection results are shown below. Figure 4 As shown in Figure D. The above results indicate that the parent peptide CD and the structurally optimized peptide CD-2 have a certain protective effect on cells, and PRRSV is effectively inhibited.

[0065] 2.4.3 Peptide inhibits PRRSV adsorption to cells

[0066] MARC-145 cells were pre-cooled at 4°C for 30 min, then infected with HP-PRRSV (MOI=1) for 2 h at 4°C with CD4+ (100 μg / mL), CD2+ (100 μg / mL), CD3+ (25 μg / mL), and CD4+ (25 μg / mL) in DMEM. The supernatant was discarded, and the cells were washed twice with serum-free DMEM to prepare samples. The viral load in the cells was detected using Western blot and RT-qPCR. The results are shown below. Figure 5 As shown, the results indicate that the parent peptide CD exhibits good antiviral activity during the virus adsorption phase. However, the results of the other treatment groups showed no significant difference in viral RNA levels compared to the control group, and Western blot results showed that the expression of PRRSV-N protein was not significantly inhibited.

[0067] 2.4.4 Peptide Inhibits PRRSV Invasion of Cells

[0068] MARC-145 cells were pre-cooled at 4°C and then infected with HP-PRRSV (MOI=1) at 4°C for 2 hours. After infection, the supernatant was discarded, and the cells were washed twice with serum-free DMEM to remove unbound viral particles. Subsequently, the cells were incubated at 37°C for 2 hours with 100 μg / mL of CD3, CD2, CD3, and CD4 peptides, respectively. Samples were collected for antiviral activity assays. Results are as follows: Figure 6 As shown, the experimental results indicate that, through Western blot and RT-qPCR analysis, all four peptides significantly inhibited HP-PRRSV replication. RT-qPCR results showed that ( Figure 6 A) The viral RNA levels in all peptide-treated groups were significantly lower than those in the control group, demonstrating a significant inhibitory effect of the peptides on viral invasion. The parent peptide CD showed an inhibition rate of 50.42% against HP-PRRSV, which was effectively improved after structural optimization. The inhibition rates of CD-2, CD-3, and CD-4 against HP-PRRSV were 72.06%, 72.12%, and 71.92%, respectively. Furthermore, Western blot results further support this conclusion. Figure 6 B, C), and all four peptides significantly reduced the expression of PRRSV-N protein, indicating that the virus was also inhibited at the protein level. Indirect immunofluorescence assay results are as follows: Figure 6 As shown in Figure D. These results indicate that these peptides inhibit PRRSV replication by blocking key steps in PRRSV invasion of cells.

Claims

1. An antiviral peptide based on Cecropin D (CD) modification, characterized in that, The amino acid sequence of the antiviral polypeptide is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. The use of the antiviral polypeptide of claim 1 in the preparation of a drug for inhibiting highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).

3. The application as described in claim 2, characterized in that, The antiviral peptide described herein inhibits HP-PRRSV replication by blocking key steps in HP-PRRSV's invasion of cells.

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