Antiviral polypeptides and uses thereof

Abstract

The invention relates to the technical field of biology, and discloses an antiviral polypeptide and application thereof. According to the antiviral polypeptide provided by the invention, the host protein ARF4 is innovatively taken as a target spot, the purpose of resisting virus infection or relieving pathological lesions caused by virus infection is achieved by adjusting the function of the host protein, and the antiviral polypeptide has an excellent antiviral effect. The host protein is selected as a target spot, so that the antiviral polypeptide provided by the invention can well avoid the problem of vaccine or antiviral drug failure caused by strong RNA virus variability, and has higher stability and wider application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antiviral polypeptide and use thereof. Background Art

[0002] Zika virus (ZIKV) and influenza virus (Influenza viruses) are both RNA viruses. With the impact of climate change and increased population mobility, they have the risk of large-scale epidemics, posing a major threat to human health. At present, the prevention, control and treatment measures for viral epidemics mainly include vaccination and the use of antiviral drugs.

[0003] However, RNA viruses have a high mutation rate, which makes them prone to antigenic shift and antigenic drift, thus reducing the effectiveness of existing vaccines and antiviral drugs against RNA viruses in large-scale, long-term RNA virus epidemics. Moreover, the high mutation rate is also prone to the emergence and prevalence of drug-resistant strains, which also limits the use of existing antiviral drugs.

[0004] In order to cope with the large-scale epidemic caused by RNA virus epidemics, it is imperative to explore new and more stable antiviral targets and develop new vaccines and antiviral drugs targeting these targets. Summary of the invention

[0005] The purpose of the present invention is to overcome the above problems existing in the prior art and provide an antiviral polypeptide and its use. The antiviral polypeptide provided by the present invention innovatively targets the host protein ARF4, which can effectively inhibit the in vivo and in vitro infection of Zika virus and influenza virus, and alleviate the pathological lesions caused by viral infection.

[0006] In order to achieve the above object, the first aspect of the present invention provides an antiviral polypeptide, wherein the antiviral polypeptide has an amino acid sequence represented by the general formula (1):

[0007] Z-VX 1 PX 2 Formula (1);

[0008] Among them, V is valine, P is proline, and X is 1 and X 2 Each represents an arbitrary amino acid, and Z represents an arbitrary amino acid or a polypeptide composed of multiple arbitrary amino acids.

[0009] The second aspect of the present invention provides an antiviral infection composition, which comprises the antiviral polypeptide described in the first aspect.

[0010] The third aspect of the present invention provides the use of the antiviral polypeptide described in the first aspect, or the composition described in the second aspect, in the preparation of a medicament for preventing and / or treating diseases caused by viral infection.

[0011] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0012] (1) The antiviral polypeptide provided by the present invention has a short amino acid sequence, is convenient for artificial synthesis, and has great potential for large-scale production and application.

[0013] (2) The antiviral polypeptide provided by the present invention has been verified through in vivo and in vitro experiments to be able to effectively inhibit the infection of Zika virus and influenza virus, and can effectively alleviate the physiological lesions caused by viral infection, and has good preventive and therapeutic effects.

[0014] (3) Unlike existing vaccines and antiviral drugs that mainly target target proteins in viruses to inhibit viral infection or provide antiviral treatment, the antiviral polypeptide provided by the present invention innovatively uses the ARF4 protein in host cells as a target, avoiding the problem of antigenic shift and antigenic drift caused by viral mutations that lead to the failure of vaccines and antiviral drugs, and the antiviral effect is more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the experimental process of the Zika virus in vivo inhibition experiment in Example 2.

[0016] Figure 2 This is a comparison chart of the viremia detection results of mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS in Example 2.

[0017] Figure 3 This is a comparison chart of the testicular viral load detection results of mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS in Example 2.

[0018] Figure 4 It is a comparison chart of the detection results of pathological lesions in the brain tissue and testicular tissue of mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS in Example 2.

[0019] Figure 5 Schematic diagram of the experimental flow of the influenza virus in vivo inhibition experiment in Example 3.

[0020] Figure 6 This is a comparison chart of the survival rate test results of the mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS after being inoculated with influenza virus in Example 3.

[0021] Figure 7This is a comparison chart of the lung virus load test results of mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS in Example 3.

[0022] Figure 8 and 9 This is a comparison chart of the detection results of pathological lesions in the lung tissues of mice in the experimental group injected with ARF4TP-4 and the control group injected with PBS in Example 4. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] Most current vaccines use antigenic genes or proteins and their fragments in the virus to stimulate the immune system to produce immune protection, thereby preventing viral infection; antiviral drugs usually target certain key proteins in the virus, inhibiting their physiological functions to inhibit viral infection and relieve the pathological changes caused by the virus. However, due to the high mutation rate of RNA viruses, drugs and vaccines that use viral fragments as targets are easily ineffective when responding to mutant strain infections, and are also likely to cause the virus to produce highly resistant mutant strains, further limiting the effectiveness of antiviral drugs.

[0025] The inventors of the present invention have conducted long-term and in-depth research on the interaction between viruses and host cell factors, and accidentally discovered that ARF4, a member of the ADP-ribosylation factor (ARFs) family, plays an important role in the infection process of Zika virus and influenza virus. After further research, the inventors found that although ARFs are considered to be key initiating factors in physiological processes including secretion, endocytosis, and phagocytosis, ARF4 is basically silent in normal cells and does not play its biological function. Zika virus and influenza virus use this feature to successfully complete their own infection and replication without affecting the normal physiological processes of cells. This feature also makes ARF4 a potential and excellent target for antiviral drugs.

[0026] Based on the above research results, the inventors conducted further in-depth research and found that polypeptides with certain characteristics can target ARF4, thereby inhibiting the infection of Zika virus and influenza virus, and alleviating the physiological pathological changes caused by their infection, and have the potential to prepare new vaccines and / or antiviral drugs.

[0027] Based on the above findings, the first aspect of the present invention provides an antiviral polypeptide having an amino acid sequence represented by the general formula (1):

[0028] Z-VX 1 PX 2 Formula (1);

[0029] Among them, V is valine, P is proline, and X is 1 and X 2 Each represents an arbitrary amino acid, and Z represents an arbitrary amino acid or a polypeptide composed of multiple arbitrary amino acids.

[0030] In the antiviral polypeptide provided by the present invention, X 1 and X 2 It can be the same or different.

[0031] According to a preferred embodiment of the present invention, wherein X 1 Any one selected from serine (S), threonine (T) and tyrosine (Y).

[0032] According to a preferred embodiment of the present invention, wherein X 2 Any one selected from alanine (A), valine (V), leucine (L) and isoleucine (I).

[0033] According to a preferred embodiment of the present invention, Z at least includes asparagine (N) and / or glutamine (Q). Preferably, Z may also include at least one of S, T and V. It should be noted that only the types of amino acids that can be contained in the Z portion of the antiviral polypeptide of the present invention are listed here, but there is no particular limitation on the specific number of each amino acid that can be contained in this portion and the specific combination of the above-mentioned amino acids.

[0034] Preferably, Z is selected from any one of N, Q and a polypeptide having an amino acid sequence as shown in SEQ ID NO:6.

[0035] SVSTSQ (SEQ ID NO:6)

[0036] According to some preferred embodiments of the present invention, the antiviral polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-5.

[0037] Peptide name serial number sequence ARF4TP-1 SEQ ID NO:1 QVSPA ARF4TP-2 SEQ ID NO:2 SVSTSQVSPA ARF4TP-3 SEQ ID NO:3 NVTPV ARF4TP-4 SEQ ID NO:4 QVY ARF4TP-5 SEQ ID NO:5 NVTPI

[0038] The above polypeptides all have the effect of inhibiting Zika virus and influenza virus infection and can be used directly. However, it should be understood that on the basis of the above polypeptides, adding several amino acids to their N-terminus or C-terminus, or combining two or several of them, or repeatedly connecting a certain polypeptide, the polypeptides that can still inhibit Zika virus and influenza virus infection also belong to the scope of the present invention.

[0039] Preferably, the amino acid sequence of the antiviral polypeptide is as shown in any one of SEQ ID NOs: 1-5.

[0040] The second aspect of the present invention provides an antiviral infection composition, which comprises the antiviral polypeptide described in the first aspect.

[0041] According to a preferred embodiment of the present invention, the composition contains an antiviral polypeptide having an amino acid sequence as shown in at least one of SEQ ID NOs: 1 to 5. That is, the polypeptide contained in the composition may include any one of the amino acid sequences in SEQ ID NOs: 1 to 5, or may include a combination of several of them.

[0042] Preferably, the composition contains an antiviral polypeptide represented by any one of the amino acid sequences SEQ ID NOs: 1-5.

[0043] According to some preferred embodiments of the present invention, the composition further comprises a pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" refers to an excipient that has substantially no effect on the effect of the active ingredient in the composition (such as the antiviral polypeptide provided by the present invention, etc.), and has substantially no effect on the health status and normal physiological activities of the subject (such as a human). For example, it may be a buffer, a preservative, a stabilizer, etc.

[0044] According to some preferred embodiments of the present invention, the composition further comprises other ingredients with antiviral activity. "Other ingredients with antiviral activity" may be any existing ingredient in the art that has a preventive and / or therapeutic effect on Zika virus and / or influenza virus and has no adverse effect on the action and effect of the antiviral polypeptide provided by the present invention, such as vaccine ingredients, antiviral drug ingredients, etc.

[0045] The third aspect of the present invention provides use of the antiviral polypeptide described in the first aspect, or the composition described in the second aspect, in the preparation of a medicament for preventing and / or treating diseases caused by viral infection.

[0046] Preferably, the virus is selected from Zika virus and / or influenza virus.

[0047] More preferably, the influenza virus is influenza A virus (IAV).

[0048] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0049] In the following examples, unless otherwise specified, all reagents used were commercial products purchased from regular chemical or biological reagent suppliers, and all were analytically pure.

[0050] The antiviral peptides used in the following examples were all commissioned to be synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd., and their amino acid sequences are shown in Table 1.

[0051] Table 1

[0052] Peptide name serial number sequence ARF4TP-1 SEQ ID NO:1 QVSPA ARF4TP-2 SEQ ID NO:2 SVSTSQVSPA ARF4TP-3 SEQ ID NO:3 NVTPV ARF4TP-4 SEQ ID NO:4 QVY ARF4TP-5 SEQ ID NO:5 NVTPI

[0053] Example 1

[0054] This example is used to illustrate the in vitro inhibitory effect of the antiviral polypeptide provided by the present invention on Zika virus infection.

[0055] Zika virus infection inhibition test was performed using Zika virus susceptible Vero cells (purchased from ATCC) according to the following steps:

[0056] 1. The Vero cells were cultured at 1×10 4 The inoculum size of pcs / well was inoculated in a 96-well plate;

[0057] 2. On the second day, after the cells attached and covered 80%-90% of the bottom area of ​​the well, the five antiviral peptides in Table 1 (hereinafter referred to as "ARF4TP") were used to prepare ARF4TP dilutions in OptiMEM medium, and the concentration gradients of 0, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM (the total amount of medium in each well was 100 μL) were contacted with the cells and incubated at 37°C for 4 hours for pretreatment;

[0058] 3. After pretreatment, the culture medium was removed and the cells were infected with Zika virus (GZ01) (MOI = 0.6);

[0059] 4. After 1 hour of Zika virus infection (shake the experimental plate slightly every 20 minutes for better infection), wash twice with PBS, and then add 100 μL / well of ARF4TP dilution prepared in OptiMEM medium to contact the cells with the same concentration gradient as in step 2 (Note: Due to the large difference between the cell experimental environment and the body, in order to avoid the influence of polypeptide degradation on the virus inhibition effect, in this step, ARF4TP is used to treat the cells again after infection);

[0060] 5. After culturing in a 37°C incubator for 1 day, centrifuge (2000 rpm, 5 min) to collect 50 μL of cell supernatant to extract RNA, then perform virus titration by qPCR and calculate IC 50 , the experimental results are shown in Table 2.

[0061] Table 2

[0062] Peptide name <![CDATA[IC 50 (μM)]]> ARF4TP-1 12.42 ARF4TP-2 64.65 ARF4TP-3 43.4 ARF4TP-4 3.25 ARF4TP-5 107.7

[0063] Example 2

[0064] This example is used to illustrate the in vivo inhibitory effect of the antiviral polypeptide provided by the present invention on Zika virus infection.

[0065] according to Figure 1 The experimental process shown in the figure uses 4-5 week old wild-type C57BL / 6JGpt mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The experimental group is intraperitoneally injected with ARF4TP-4 at a dose of 20 mg / kg for three consecutive days, and the control group is injected with PBS buffer at the same volume; the experimental group and the control group mice are intraperitoneally injected with interferon IFNλ antibody at a dose of 2 mg / mouse one day before infection.

[0066] According to 5×10 5 The experimental and control mice were inoculated with Zika virus (GZ01) at a dose of 1.0 PFU / mouse by intraperitoneal injection. Four hours after inoculation, the experimental and control mice were intraperitoneally injected with ARF4TP-4 (20 mg / kg) and PBS (same volume as the experimental group), respectively.

[0067] The Zika virus infection of mice in the experimental and control groups was tested as follows:

[0068] 1. Viremia detection

[0069] Mice were randomly selected from the treatment group and the control group, with 5 mice in each group. Blood was collected from the eye sockets on days 2, 4, and 6 after infection, and 50 ul of serum was used to extract total RNA. PrimeScript TMPLUS RT-PCR kit (RR096A), using total RNA as template and Zika virus-specific primers (ZIKVASF: 5'-GGTCAGCGTCCTCTCTAATAAACG-3', ZIKV-ASR: 5'-GCACCCTAGTGTCCACTTTTTCC-3'), detected the Zika virus genome copy number by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR), thereby quantifying the viral load and comparing the therapeutic effect of ARF4TP-4.

[0070] The results are as follows Figure 2 As shown in the figure, it can be seen that 2, 4, and 6 days after the virus attack, the Zika virus load detected in the serum of the experimental group was significantly lower than the viral load in the serum of the control group mice, indicating that ARF4TP-4 can effectively inhibit the viremia caused by Zika virus infection.

[0071] 2. Viral load in tissues and organs

[0072] The mice in the treatment group and the control group were randomly selected, with 6 mice in each group. Half of the mice were marked and killed 2 and 6 days after the virus attack, and then dissected. The testicles of the Zika-susceptible organs were taken, and 1 mL of sterile PBS was added to fully grind them. After centrifugation at 12,000 RPM for 2 minutes, 100 μl of supernatant was taken to extract total RNA. Similar to the method for detecting viral load in serum, the number of Zika virus genome copies in mouse testis was detected by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR), so as to quantify the viral load and compare the therapeutic effect of ARF4TP-4.

[0073] The results are as follows Figure 3 As shown in the figure, it can be seen that in the testicular samples of mice 2 and 6 days after infection, the Zika virus load detected in the experimental group was significantly lower than that in the control group, indicating that ARF4TP-4 can effectively inhibit the infection of Zika virus to susceptible organs.

[0074] 3. Pathological lesion detection

[0075] Mice were randomly selected from the treatment group and the control group, with 3 mice in each group. They were marked and killed 6 days after the virus attack, and then dissected. The testicles and brain, which are susceptible to Zika, were taken and fixed with 4% PFA and then stained with HE to observe and compare the tissue lesions.

[0076] Five fields of view were randomly selected for observation from each sample slice, with a magnification of 100 times. Figure 4The figure shows a photograph of a field of view of a sample slice of brain tissue and testicular tissue of the experimental group and the control group. It can be seen from the figure that compared with the mice in the control group infected with Zika virus, the inflammatory cell infiltration in the brain of the experimental group mice injected with ARF4TP-4 was alleviated; at the same time, pathological changes such as seminiferous tubule necrosis, intracavitary hemorrhage and exudate deposition in the testicle caused by viral infection were also effectively eliminated.

[0077] The same method was used to detect the in vivo anti-Zika virus infection effects of the remaining four ARF4TPs in Table 1. The results showed that they all showed anti-Zika virus infection effects, but the effects were different.

[0078] Example 3

[0079] This example is used to illustrate the in vivo inhibitory effect of the antiviral polypeptide provided by the present invention on influenza virus infection.

[0080] according to Figure 5 The experimental procedure shown uses 8-9 week old wild-type C57BL / 6JGpt mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The experimental group was intraperitoneally injected with ARF4TP-4 at a dose of 80 mg / kg one day before infection, and the control group was injected with PBS buffer at the same volume.

[0081] According to 1.5×10 4 The dose of PFU / each was challenged with influenza A virus (H1N1 WSN) in the following manner:

[0082] After anesthesia, nasal drops were administered to one nostril at a volume of 20 μL per mouse. Mice in the control group were nasally dripped with PBS in the same manner.

[0083] Four hours after challenge and for two consecutive days after infection, the experimental group was intraperitoneally injected with ARF4TP-4 at a dose of 80 mg / kg, and the control group was intraperitoneally injected with the same volume of PBS.

[0084] The Zika virus infection of mice in the experimental and control groups was tested as follows:

[0085] 1. Survival rate detection

[0086] After the challenge, body weight was monitored for two weeks (mice were considered dead when their body weight dropped by more than 25%). Figure 6 As shown in the figure, it can be seen that the survival rate of the experimental group mice administered with ARF4TP-4 after influenza infection was significantly improved compared with the control group mice injected with PBS.

[0087] 2. Detection of peptide drugs inhibiting influenza virus lung infection in vivo

[0088] 1) Viral load in tissues and organs

[0089] The experimental group and the control group of mice were randomly selected, with 6 mice in each group. Half of the mice were marked and killed 2 and 6 days after the infection. The lung, which is susceptible to influenza A, was taken and fully ground with 1 mL of sterile PBS. The cells were centrifuged at 12,000 rpm for 2 minutes, and 100 μL of the supernatant was taken to extract total RNA. The One Step TB reverse transcription kit from Takara was used. PrimeScript TM PLUS RT-PCR kit (RR064A), using total RNA as a template and influenza A virus-specific primers (FluA-F: 5'-GACCRATCCTGTCACCTCTGAC-3', FluA-R: 5'-GGGCATTYTGGACAAAKCGTCTACG-3', FluA-P: 5'-TGCAGTCCTCGCTCACTGGGCACG-3'), was used to detect the influenza virus genome copy number by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR), thereby quantifying the viral load.

[0090] The results are as follows Figure 7 As shown, in the lung samples 2 and 6 days after infection, the influenza A virus load detected in the experimental group was significantly lower than the viral load in the lungs of mice in the control group, indicating that ARF4TP-4 can effectively inhibit the infection of influenza A virus to susceptible organs.

[0091] 2) Pathological lesion detection

[0092] Mice were randomly selected from the treatment group and the control group, with 3 mice in each group. They were marked and killed 6 days after the challenge and then dissected. The lung, an organ susceptible to influenza A, was taken and fixed with 4% PFA and then stained with HE to observe and compare tissue lesions. The specific method was the same as in Example 2.

[0093] Five fields of view were randomly selected for observation from each sample slice, with a magnification of 10 times. Figure 8 exemplarily shows a photograph of a field of view of a sample slice of each of the experimental group and the control group, Fig. 9 The results of lung tissue pathology of mice in the experimental and control groups are shown in the figure. As can be seen from the figure, compared with the control group of mice infected with influenza A virus, the inflammatory cell infiltration in the lungs of the experimental group injected with ARF4TP-4 was alleviated; at the same time, the pathological changes such as thickening of the alveolar septum and congestion of some blood vessels in the lung tissue caused by virus infection were also effectively eliminated.

[0094] The same method was used to detect the in vivo anti-influenza virus infection effects of the remaining four ARF4TPs in Table 1. The results showed that they all exhibited anti-influenza virus infection effects, but the effects were different.

[0095] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An antiviral polypeptide, It is characterized in that The antiviral polypeptide has an amino acid sequence represented by the general formula (1): Z-VX 1 PX 2 Formula (1); Among them, V is valine, P is proline, and X is 1 and X 2 Each represents an arbitrary amino acid, and Z represents an arbitrary amino acid or a polypeptide composed of multiple arbitrary amino acids.

2. The antiviral polypeptide according to claim 1, in, X 1 Any one selected from serine, threonine and tyrosine.

3. The antiviral polypeptide according to claim 1, in, X 2 Any one selected from alanine, valine, leucine and isoleucine.

4. The antiviral polypeptide according to claim 1, in, Z comprises at least asparagine and / or glutamine.

5. The antiviral polypeptide according to claim 4, in, Z is selected from any one of asparagine, glutamine and a polypeptide with an amino acid sequence as shown in SEQ ID NO:

6.

6. The antiviral polypeptide according to any one of claims 1 to 5, in, The antiviral polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-5; Preferably, the amino acid sequence of the antiviral polypeptide is as shown in any one of SEQ ID NOs: 1-5.

7. An antiviral infection composition, It is characterized in that The composition comprises the antiviral polypeptide according to any one of claims 1 to 6.

8. The composition according to claim 7, in, The composition contains an antiviral polypeptide with an amino acid sequence as shown in at least one of SEQ ID NOs: 1-5; Preferably, the composition contains an antiviral polypeptide represented by any one of the amino acid sequences SEQ ID NOs: 1-5.

9. The composition according to claim 7 or 8, in, The composition further comprises a pharmaceutically acceptable excipient; And / or, the composition further comprises other ingredients with antiviral activity.

10. Use of the antiviral polypeptide according to any one of claims 1 to 6, or the composition according to any one of claims 7 to 9 in the preparation of a medicament for preventing and / or treating diseases caused by viral infection; preferably, the virus is selected from Zika virus and / or influenza virus, and preferably, the influenza virus is influenza A virus.

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