TREA

Broad-spectrum polypeptide against enterovirus and application thereof

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Information

  • Patent Grant
  • 11999806
  • Patent Number
    11,999,806
  • Date Filed
    Monday, January 21, 2019
    6 years ago
  • Date Issued
    Tuesday, June 4, 2024
    8 months ago
Information
Abstract
Provided are a series of polypeptides with antiviral activity. The present invention provides a new strategy for preventing and controlling Enterovirus such as EV71, CVA16, CVA6, CVB3, and CVB5 viruses and provides a new theoretical basis for accelerating the research and development of a polypeptide small molecule drug against Enterovirus such as EV71, CVA16, CVA6, CVB3, and CVB5 viruses.
Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of Application No. PCT/CN2019/072455, filed Jan. 21, 2019, which claims the priority of Chinese patent application No. 2018100562971 filed with the Chinese Patent Office on Jan. 20, 2018, entitled “INHIBITOR OF ENTEROVIRUS 71 AND APPLICATION THEREOF”, each of which is incorporated herein by reference in its entirety.


SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 19, 2021, is named 46761US_sequencelisting.txt, and is 4,889 bytes in size.


BACKGROUND

The invention relates to the field of biomedicine, and in particular to a polypeptide against various enterovirus and applications thereof.


SUMMARY

Enterovirus is a positive-sense single-stranded RNA virus, and belongs to the Enterovirus genus of the Picornaviridae family, mainly including human Enterovirus (EV), Coxsackie A virus (CVA), Coxsackie B virus (CVB), Echovirus, Rhinovirus, and Poliovirus. Enterovirus infections are widely distributed around the world and exhibit complex and diverse clinical manifestations, ranging from mild fever, fatigue, respiratory diseases, to herpes angina, hand-foot-and-mouth disease, and to severe aseptic meningitis, myocarditis, encephalitis, poliomyelitis, etc. At present, there is a lack of specific drugs that can effectively treat or prevent enterovirus infections.


Herpetic angina is mainly caused by Coxsackievirus Group A type 2 (CVA2), CVA4, CVA6, CVA9, CVA16, CVA22, and Coxsackievirus group B type 1 (CVB1), CVB2, CVB3, CVB4, or CVB5. Herpetic angina often causes fever, and the temperature is mostly not high or moderate. Occasionally, the fever is as high as 40° C. or even causing convulsions. The fever may last about 2 to 4 days. Older children may complain of sore throat, which can affect swallowing. Infants and young children may show salivation, refusal to eat, and restlessness. Sometimes it is accompanied by headache, abdominal pain or myalgia. About 25% of children under 5 years old with herpetic angina may be accompanied by vomiting. The patient may show typical symptoms in the pharynx, including pharyngeal hyperemia, and several (from 1 to 2 up to more than 10) small (1-2 mm in diameter) gray-white herpes surrounded by redness in the oral mucosa within 2 days of onset. After 2 to 3 days, the redness increases and the herpes rupture to form a yellow ulcer. This mucosal rash is more common in the anterior column of the tonsils, but also in the soft palate, uvula, and tonsils, but does not involve the gums and buccal mucosa. The course of the disease is usually 4 to 6 days, occasionally extended to 2 weeks.


Hand-foot-and-mouth disease is mainly caused by enterovirus 71 (EV71), CVA6, CVA8, CVA10, CVA16, CVB3 and CVB5. The common clinical manifestations of hand-foot-and-mouth disease include acute fever, mouth pain, anorexia, and scattered herpes or ulcers in the oral mucosa, mostly on the tongue, buccal mucosa, and hard palate, and also affecting soft palate, gums, tonsils and pharynx. Maculopapular rashes appear in the hands, feet, buttocks, arms and legs, and then turn into herpes which are surrounded by inflammatory redness and contains less fluid in the blister. Typically, there are more rashes on the hands and feet, both on the dorsum and the vola, from a few to dozens, and after subsided, they leaves no traces and no pigmentation. Some children with hand-foot-and-mouth disease have herpetic angina as the first symptom, and then red rashes in the palm, sole, buttocks and other parts. When the course of the disease develops rapidly, a small number of children can develop from hand-foot-and-mouth disease to severe aseptic meningitis and encephalitis, manifested as fever, headache, nausea, vomiting, and then meningeal irritation, as well as body temperature fluctuations with low-grade fever in the most case and sometimes with fever up to 40° C. or more, often bimodal fever in the course of the disease. Other symptoms include such as sore throat, muscle aches, skin rash, photophobia, diarrhea, swollen lymph nodes, and sometimes mild paralysis.


Myocarditis is mainly caused by CVB1-61 and Echovirus. Depend on the extent and location of the disease, the clinical manifestations of patients with viral myocarditis include asymptomatic case in mild case to heart failure, cardiogenic shock and sudden death in severe case. Patients often have a history of upper respiratory or intestinal infections 1 to 3 weeks before the onset of symptoms, manifested as fever, body aches, sore throat, fatigue, nausea, vomiting, diarrhea and other symptoms, and then palpitations, chest tightness, chest pain or precordial pain, dizziness, dyspnea, edema, and even Adams-Stokes syndrome, and in some patients, heart failure or cardiogenic shock.


Enterovirus is a positive-sense single-stranded RNA virus, which has a genome of about 7.5 kb, containing a large ORF encoding a polyprotein. The polyprotein can be further hydrolyzed into 4 structural proteins (VP1-VP4) and 7 nonstructural proteins (2A-2C and 3A-3D). In enteroviruses (including EV71, CVA and CVB), protein 3A is an extremely conserved nonstructural protein, which exists in the form of homodimers in the intracellular membrane and plays an important role in the replication of viruses and the regulation of host innate immunity.


After being infected by the virus, the host cell will activate a series of natural immune mechanisms, including RNA interference (RNAi)-mediated antiviral immunity. After the virus infects the host cell, due to the structure of the virus genome or replication intermediates, long double-stranded RNA (dsRNA) derived from the virus will be produced. These dsRNAs are recognized by the host Dicer protein and cleaved into viral small interfering RNA (vsiRNA). Then, vsiRNA binds with Argonaute (AGO) protein to form RNA-induced silencing complex (RISC), which ultimately mediates the degradation of the viral target gene RNA, thereby inhibiting the replication of the virus to eliminate the virus.


BRIEF DESCRIPTION OF THE INVENTION

In view of this, the present invention provides a polypeptide and application thereof.


In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions.


The present invention provides use of enterovirus RNA suppressing protein (ERSP) as a target in the preparation of a medicine for preventing and/or treating viral diseases.


The present invention also provides use of a polypeptide in the preparation of an inhibitor of ERSP, wherein the function of ERSP is inhibited by the polypeptide, and the viral nucleic acid is cut by Dicer (endoribonuclease Dicer) to produce viral small interfering RNA (vsiRNA).


The invention also provides use of a polypeptide in the preparation of a medicine for preventing and treating viral diseases.


In some specific embodiments of the present invention, the ERSP is enterovirus nonstructural protein 3A.


In some specific embodiments of the present invention, the enterovirus belongs to the Picornaviridae enterovirus genus, including human Enterovirus (EV), Coxsackie A virus (CVA), Coxsackie B virus (CVB), Echovirus, Rhinovirus, and Poliovirus.


In some specific embodiments of the present invention, the viral diseases include hand-foot-and-mouth disease, myocarditis, herpes angina, aseptic meningitis, encephalitis and viral cold.


In some specific embodiments of the present invention, the amino acid sequence of the polypeptide comprises CR, CK and/or DLL.


In some specific embodiments of the present invention, the polypeptide has a sequence selected from:

    • I. (X1) (X2)DLL, (X2)DLL(X3), DLL(X3) (X4), (X5)YC(X6), C(X6),
    • wherein,
    • X1 is isoleucine (I),
    • X2 is selected from serine (S) or alanine (A),
    • X3 is selected from alanine (A) or lysine (K) or glutamine (Q) or arginine (R) or serine (S) or cysteine (C),
    • X4 is selected from serine (S) or alanine (A),
    • X5 is selected from glutamic acid (E) or glutamine (Q),
    • X6 is selected from arginine (R) or lysine (K); or
    • II. a sequence with deletion, addition or substitution of at least one amino acid in the sequence in I; or
    • III. a sequence inhibiting the activity of ERSP and having at least 50% homology to the sequence in I or II; or
    • IV. the complementary sequence of the sequence in I or II or III.


The “amino acid” in the present invention includes natural amino acids or unnatural amino acids. Amino acid types well known to those skilled in the art are within the scope of the present invention.


In some specific embodiments of the present invention, the sequences of I is as shown in any one of SEQ ID NOs: 1-14, without the sequence of cell-penetrating peptide and the sequence of peptide linker.


The present invention also provides a polypeptide capable of inhibiting the activity of ERSP.


In some specific embodiments of the present invention, the amino acid sequence of the polypeptide comprises CR, CK and/or DLL.


In some specific embodiments of the present invention, the amino acid sequence of the polypeptide comprises YCR and/or YCK.


In some specific embodiments of the present invention, the polypeptide has a sequence selected from:

    • I. (X1) (X2)DLL, (X2)DLL(X3), DLL(X3) (X4), (X5)YC(X6), C(X6),
    • wherein,
    • X1 is isoleucine (I),
    • X2 is selected from serine (S) or alanine (A),
    • X3 is selected from alanine (A) or lysine (K) or glutamine (Q) or arginine (R) or serine (S) or cysteine (C),
    • X4 is selected from serine (S) or alanine (A),
    • X5 is selected from glutamic acid (E) or glutamine (Q),
    • X6 is selected from arginine (R) or lysine (K); or
    • II. a sequence with deletion, addition or substitution of at least one amino acid in the sequence in I; or
    • III. a sequence inhibiting the activity of ERSP and having at least 50% homology to the sequence in I or II; or
    • IV. the complementary sequence of the sequence described in I or II or III.


In some specific embodiments of the present invention, the sequence of the polypeptide in I is as shown in any one of SEQ ID NOs: 1-14, without the sequence of penetrating peptide and the sequence of peptide linker.


On this basis, the present invention also provides a nucleic acid having a nucleotide sequence encoding the polypeptide.


The present invention also provides a recombinant vector comprising the nucleic acid.


On this basis, the present invention also provides a host cell comprising the recombinant vector.


The present invention also provides a medicine comprising the polypeptide and pharmaceutically acceptable excipients.


The present invention also provides a vaccine comprising the polypeptide and pharmaceutically acceptable excipients.


The invention also provides a method for treating enterovirus infections comprising oral administration or injection of the medicine to a subject in need thereof, wherein the injection is intramuscular injection, intraperitoneal injection or intravenous injection.


The present invention also provides a method for preventing enterovirus infections comprising administration of the vaccine to a subject in need thereof.


In the present invention, the term “prevent”, “preventing” or “prevention” means that various methods or measures for preventing the occurrence or development of diseases, including medical, physical or chemical methods for preventing or reducing the occurrence or development of various symptoms of diseases are performed before the occurrence of diseases confirmed by clinical standards.


In the present invention, the term “treat”, “treating” or “treatment” means that various methods or measures are performed to prevent and reduce the occurrence or development of the disease, inhibit, suppress, reduce, improve, slow down, stop, delay or reverse the development or aggravation of the disease course, alleviate or reduce various indicators, including symptoms or complications of diseases, disorders or conditions, or cure or eliminate diseases, disorders or conditions.


In the present invention, the term “medicine” refers to a single compound, a composition of multiple compounds, or a composition or formulation with a single compound as the main active ingredient, or a composition or formulation with multiple compounds as active ingredients, which can be used to prevent or treat a certain disease. A “medicine” should be understood as not only to refer to a product approved for production by the administrative agency in accordance with the laws and regulations of a country, but also to refer to various material forms formed with a single compound as the active ingredient in order to achieve the approval for production. “Formed” should be understood as obtaining through chemical synthesis, biological transformation or purchase.


The present invention also provides an inhibitor of enterovirus 71, wherein the inhibitor is polypeptide P2 with the amino acid sequence shown in SEQ ID NO: 2.


The present invention also provides variants of the inhibitor, wherein the variant is 3A-TAT-EP with the amino acid sequence as shown in SEQ ID NO: 3, 3A-EP-DRI with the amino acid sequence as shown in SEQ ID NO: 4, or 3A-EP-PEG4-PA with the amino acid sequence as shown in SEQ ID NO: 5.


In addition, the present invention also provides use of the inhibitor or the variants in the preparation of an inhibitor of enterovirus.


RNA interference is an antiviral immune mechanism. In the antiviral process with RNA interference, the double-stranded RNA produced during viral RNA replication is recognized by the host Dicer protein and cleaved into small interfering RNA (siRNA). These virus-derived small interfering RNAs (vsiRNAs) are transferred and assembled into RNA-induced silencing complexes, which mediate the degradation of homologous viral RNA to achieve antiviral goals. For example, the nonstructural protein 3A of EV71 can bind to the viral double-stranded RNA to prevent it from being cut by Dicer, and thus inhibit the production of virus-derived small interfering RNAs (vsiRNAs), so as to achieve the purpose of escaping from the host anti-viral mechanism via RNA interference.


The present invention provides a series of polypeptides with antiviral activity. The present invention provides a new strategy for preventing and controlling enterovirus such as EV71, CVA16, CVA6, CVB3, and CVB5 viruses and provides a new theoretical basis for accelerating the research and development of a polypeptide small molecule drug against enterovirus such as EV71, CVA16, CVA6, CVB3, and CVB5 viruses.





BRIEF DESCRIPTION OF DRAWINGS


FIGS. 1A-1B show the detection of the ability of polypeptide P2 to penetrate the cell membrane and enter the cytoplasm by fluorescent labeling.



FIG. 2 shows the result of detecting the cytotoxicity of polypeptide P2 by CCK-8.



FIG. 3 shows the results of detecting the antiviral efficacy of polypeptide P2 in RD cells.



FIGS. 4A-4C show the results of detecting the antiviral efficacy of polypeptide P2 in various cells.



FIG. 5 shows the results of detecting the antiviral efficacy of the variants of polypeptide P2.



FIG. 6 shows the results of detecting the antiviral efficacy of polypeptides 3A-EP-DRI and 3A-TAT-EP.



FIG. 7 shows the results of detecting the antiviral activity of polypeptide P1 against EV71 in mice.



FIG. 8 shows the results of detecting the antiviral activity of polypeptide P1 against CVA16 in mice.



FIG. 9 shows the results of detecting the anti-EV71 effect of polypeptide CR in RD cells.



FIG. 10 shows the results of detecting the antiviral activity of polypeptide CR against EV71 in mice.



FIG. 11 shows the results of detecting the antiviral activity of polypeptide CR against CVA16 in mice.



FIG. 12 shows the results of mouse body weight measurement in toxicity evaluation of polypeptide ER-DRI.



FIG. 13 shows the results of HE staining for toxicity evaluation of polypeptide ER-DRI in mice.



FIG. 14 shows the results of detecting the membrane penetration efficiency of polypeptide P1 in RD cells.



FIG. 15 shows the results of cytotoxicity experiments of polypeptide P1.



FIG. 16 shows the results of cytotoxicity experiments of polypeptide 3A-TAT-EP.



FIG. 17 shows the results of cytotoxicity experiments of polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA.



FIG. 18 shows the results of detecting the antiviral effects of polypeptides EP-PA, EP-CHOL, 3A-EP-DRI and 3A-EP-PEG4-PA.



FIG. 19 shows the results of toxicity experiments of ER-DRI and ER in RD cells.



FIG. 20 shows the results of detecting the anti-EV71 effects of ER-DRI and ER in RD cells.



FIG. 21 shows the results of detecting the antiviral activity of polypeptide ER-DRI against EV71 in mice.



FIG. 22 shows the results of detecting the anti-CVA16 effects of polypeptides P2, ER, ER-DRI, R8 and TAT.



FIG. 23 shows the results of toxicity experiments of polypeptides BP8, BP10 and BP15 in RD cells.



FIG. 24 shows the results of toxicity experiments of polypeptides BP8, BP10 and BP15 in Vero cells.



FIG. 25 shows the results of detecting the anti-CVB5 effect of polypeptides BP8, BP10 and BP15 in RD cells.



FIG. 26 shows the results of detecting the anti-CVB3 effect of polypeptide BP8 in Vero cells.



FIG. 27 shows the results of detecting the anti-CVB5 effect of polypeptide BP8 in mice.



FIG. 28 shows the results of detecting the anti-CVA6 effect of polypeptide ER-DRI in Vero cells.





DETAILED DESCRIPTION

The present invention discloses polypeptides and uses thereof. Those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described according to the preferred embodiments, and it is obvious that the method and application described herein may be changed or appropriately modified and combined without departing from the content, spirit and scope of the present invention.


The EV71 3A protein contains 86 amino acid residues. Protein 3A has the ability to form dimer, and its dimerization plays a key role in the proper functioning of 3A. Based on the composition and sequence characteristics of the two helical structures of the dimerization domain of protein 3A, the applicant designed polypeptide EP (shown in SEQ ID NO: 1), which can bind to the EV71 nonstructural protein 3A and interfere with the dimer formation of protein 3A, thereby inhibiting virus replication and infection.


In order to allow the inhibitor to penetrate the cell membrane and enter the cell to inhibit viruses, polypeptide P2 (SEQ ID NO: 2) is designed using polypeptide EP as the core sequence. P2 polypeptide is capable of competing with al, thereby preventing dimerization of 3A, inhibiting the function of 3A in anti-innate immunity, and finally achieving anti-viral purposes.


Based on P2, a series of variants are constructed according to the modification of P2. It is shown by experiments that the variants of P2 have improved ability to inhibit viruses, which gives great significance to the prevention and treatment of diseases caused by enteroviruses.


The variants include 3A-TAT-EP with the amino acid sequence as shown in SEQ ID NO: 3, 3A-EP-DRI with the amino acid sequence as shown in SEQ ID NO: 4, and 3A-EP-PEG4-PA with the amino acid sequence as shown in SEQ ID NO: 5.


The present invention also provides uses of the inhibitor of enterovirus 71, comprising preparing the inhibitor of enterovirus 71 using polypeptide P2 or the variants of polypeptide P2, or preparing the inhibitor of enterovirus 71 using polypeptide P2 or the variants of polypeptide P2 together with other effective ingredients.


The present invention also includes an inhibitor with the activity of inhibiting EV71, which is obtained by using different cell-penetrating sequence, by modifying the sequence of or by using unnatural amino acids in polypeptide P2.


Enterovirus 3A protein is a high-efficiency ERSP which can bind to viral dsRNA to prevent it from being cut by Dicer, and thus inhibit the production of virus-derived vsiRNA, thereby avoiding the antiviral immunity of the host by RNAi pathway.


The polypeptide and the derivatives thereof according to the present invention are capable of inhibiting the function of enterovirus protein 3A, and may be an emerging therapeutic drug for EV71, which targets new targets and is of great significance for avoiding antiviral drug resistance.


Compared with the prior art, the present invention has the following advantages:


The P2 polypeptide and variants thereof have potent antiviral activity. This will provide a new strategy for the prevention and treatment of enterovirus, also provide a new theoretical basis for accelerating the development of anti-enterovirus polypeptide small molecule drugs. In addition, the clear antiviral mechanism of the P2 series polypeptides can ensure the safety for their application and the clarity for the optimization approach, which is convenient for further development in the future.


The polypeptides provided by the present invention are shown in Table 1.



















SEQ ID







Peptide
NO:
Sequence
Tested
Antiviral(s)
IC50/EC50
CC50





















P1
6
RRRRRRRRAISDLLAS
In
EV71/CVA-
7.038 μM
>100 μM





vivo
16







P2
2
RRRRRRRREEVRQYCRDQ
in
EV71/CVA-
 1.208 μM/
>100 μM





vitro
16
1.533 μM






CR
7
YGRKKRRQRRRGSGCR
In
EV71
IC50/EC50
CC50





vivo

  1.7 μM






3A-TAT-EP
3
YGRKKRRQRRRGSGEEVRQY
In
EV71
 4.36 μM
>100 μM




CREQGWIIP
vitro








EP-PA
8
EEVRQYCREQGWIIP-βAK-C16
In
EV71
8.175 μM
/




(palmitic acid (C16:0))
vitro








EP-CHOL
9
EEVRQYCREQGWIIP-AK-
In
EV71
11.35 μM
/




cholesterol (Chol)
vitro








EP
1
EEVRQYCREQGWIIP
/
/
/
/





3A-EP-DRI
4
Acetylated (Ac): Ac-
In
EV71
5.242 μM
>100 μM




piiwgqercyqrveepprrrqrr
vitro







kkrgy-NH2








(all amino acids are 








D-amino acid)









3A-EP-
5
EEVRQYCREQGWIIP-AK-
In
EV71
4.912 μM
>100 μM


PEG4-PA

Polyethylene glycol 4 
vitro







(PEG4)-K-C16









ER
10
YGRKKRRQRRRGSGEEVRQY
In
EV71/CVA-
  1.26 μM/
 290 μM




CR
vitro
16
3.211 μM






ER-DRI
11
Acetylated (Ac):
In
EV71/CVA-
  0.64 μM/
 117 μM




Ac-rcyqrveepprrrqrrkkrgy-NH2
vivo
16/CVA-
0.856 μM





(all amino acids are 

6/CVA-8






D-amino acid)









BP-8
12
YGRKKRRQRRRGSGEAVREY
In
CVB5/CVB3
   1.1 μM/
>200 μM




CK
vitro

  3.2 μM






BP-10
13
YGRKKRRQRRRGSGEAVREY
In
CVB5
   1.5 μM/
>200 μM




CKEK
vitro








BP-15
14
YGRKKRRQRRRGSGEAVREY
In
CVB5
  6.25 μM/
>100 μM




CKEKGWLVP
vitro









In the present invention, polypeptides RRRRRRRR (R8) and YGRKKRRQRRR (TAT) are penetrating peptides, GSG is a peptide linker, and the amino acid sequences without the penetrating peptide and the peptide linker are the core sequences. In each example of the present invention, a negative control polypeptide is provided to show that the core sequences of the polypeptides in the present invention have good antiviral effects.


The materials and reagents used in the present invention are commercially available.


The present invention will be further explained below in conjunction with examples.


Example 1

Detection of Membrane Penetration Efficiency of Polypeptide P2


1. Material:


MEM medium (Thermo), serum (Gibco), immunofluorescence plate (NEST), PBS, DAPI, and paraformaldehyde were purchased in the market.


Polypeptide P2 was synthesized by Nanjing GenScript Company, and its sequence is shown in SEQ ID NO: 2.


2. Experimental Procedure


The experiment was performed in two groups in order to observe whether the addition of EV71 virus affect the entry of polypeptides into cells. In one group, EV71 virus and then polypeptide P2 were added, and in the other group, only the polypeptide was added. Negative control was set in each group.


Immunofluorescence experiment was performed as follows.

    • (1) 1 ml of RD cells in a special dish for immunofluorescence were treated and then collected at 30% confluence.
    • (2) The medium in the dish was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual medium.
    • (3) 4% paraformaldehyde solution was prepared by dissolving 4 g paraformaldehyde solid in 100 ml PBS. Then 1 ml of the prepared 4% paraformaldehyde solution was added to each dish and incubated for 5 minutes to fix the cells.
    • (4) The 4% paraformaldehyde solution was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual paraformaldehyde.
    • (5) 1 mg/ml DAPI solution was diluted with PBS to 100 ng/ml, and the diluted DAPI solution was added to the dish and incubated for 15 minutes.
    • (6) The solution was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual solution.
    • (7) The dish was observed under a fluorescence microscope.


Polypeptides were labeled with fluorescence and tested for their membrane penetration efficiency in RD cells. Two groups of experiments were performed. One group was control group without virus infection, in which R8 and P2 were added respectively; the other group was EV71 infection group, in which cells were infected with EV71 and then R8 and P2 were added respectively. The two groups of experiment were carried out at the same time, with virus MOI=0.1, and a polypeptide concentration of 1 μM. The cells were fixed 12 h after the addition of the polypeptide, immunofluorescence experiments were performed, and the nuclei were stained with DAPI. The results show that the polypeptides can enter cells infected with or without virus, showing a good ability to penetrate cell membranes.


As shown in FIGS. 1A-1B, with or without virus, the polypeptides can be observed in both cells infected with or without virus, indicating that polypeptide P2 has a good ability to penetrate cell membrane.


Example 2

Detection of Cytotoxicity of Polypeptide P2


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptide P2 was desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide P2 was added to generate gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM (final concentration), respectively.
    • (3) The test was performed 24 hours after the addition of the polypeptide, and 10 μl of viable cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was incubated at 37° C. for 2 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 2 and Table 2. Taking the cell viability of untreated group as 100%, there is no significant difference between the cell viability of the cells added with 100 μM polypeptide and that of the control group (untreated cells), which indicates that polypeptide P2 used in this study is not toxic to cells within 100 μM.











TABLE 2







Significance


Concentration of

analysis


polypeptide P2

(vs. without


(μM)
Cell viability (%)
treatment)



















R8
104.243900
103.446800
 96.068770
P = 0.4378


0
 98.040830
104.606000
102.740100
/


0.01
 98.040830
104.606000
102.740100
P = 0.5000


0.1
 94.404550
103.995900
100.622500
P = 0.2844


1
105.193200
105.370600
 98.812520
P = 0.3357


10
 96.602830
101.132500
 99.031580
P = 0.1444


100
 90.146030
106.773400
 94.068210
P = 0.2116









Example 3

Detection of Antiviral Efficiency of Polypeptide


1. Materials


Total RNA extraction kit (Omega), 24-well plate, 100 mm dish, and 50 ml syringe, 0.22 μm filter membrane (Millipore), one step qRT-PCR kit (Takara) were purchased in the market. The water used in the RNA extraction and qRT-PCR was DEPC-treated water, and the entire experiment was carried out in an RNase free environment.


2. Amplification Virus






    • (1) RD cells were plated in five 100 mm dishes.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, 1 μl EV71 virus, CVA16 virus, CVB3 virus, or CVB5 virus at 10{circumflex over ( )}7 PFU/ml were added, respectively.

    • (3) After 2 days, the cells were observed to see if the cytopathic effect (CPE) phenomenon occurred. When the changes were obvious, samples were collected as follows.

    • (4) The supernatant in the 100 mm dish was transferred into a 15 ml centrifuge tube, and centrifuged at 500 g for 5 min.

    • (5) The supernatant was transferred into a new 15 ml centrifuge tube, and filtered through 0.22 μm filter membrane with a 50 ml syringe.

    • (6) RNA was extracted from 100 μl of the supernatant obtained in step (5) and subjected to one step qRT-PCR to measure the virus titer, and the previously extracted virus RNA with known titer was handled together as a control.


      3. Measure Antiviral Efficiency of Polypeptide in RD Cells.

    • (1) Different cells were plated in 24-well cell plates.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum (0.5 ml/well), and 5 μl of 10{circumflex over ( )}6 PFU/mL EV71 viruses per well were added.

    • (3) After 1 h, different polypeptides were added at a final concentration of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with total RNA extraction kit as follows.

    • (5) The supernatant in the wells was discarded, then 350 μl of TRK Lysis Buffer was added to each well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12,000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12,000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The column was centrifuged at 12000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12,000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





RD cells added with R8 penetrating peptide (sequence: RRRRRRRR) were used as a negative control.


The results are shown in FIG. 3 and Table 3. The amount of viral RNA in the experimental group added with R8 and infected with EV71 virus was set as 100%. After the addition of polypeptide P2, the amount of virus decreased with the increase of the peptide concentration, and when the peptide concentration is 50 μM, the amount of virus is reduced to about 4%, demonstrating that polypeptide P2 has a good antiviral effect. The virus titer was detected by qRT-PCR, and the IC50 value of P2 was 6.372 μM. As seen in FIG. 2, polypeptide P2 has no cytotoxicity within 100 μM. Therefore, it is shown that the polypeptide has a good antiviral effect and it is safe.









TABLE 3







Results of 3 experiments










Concentration




of polypeptide




(μM)
Relative Percentage of viral RNA (%)
















0.01
85.112880
75.771370
106.099300



0.1
77.353810
74.103940
 77.353810



1
46.972400
46.319070
 78.617520



10
17.463790
22.758900
 20.427540



50
 2.891023
 2.288333
 10.245860










RD cells were used to test the antiviral effect of polypeptide P2. EV71 (MOI=0.1) was added to RD cells at 80% confluence. After 1 hour of infection, polypeptide P2 was added at concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively. The samples were collected 24 hours later, and the total RNA was extracted. The level of viral genomic RNA was detected by qRT-PCR. The cells infected with the same type virus and added with penetrating peptide R8 were used as a control group. The results showed that with the increase of the polypeptide concentration, the viral RNA level decreased significantly, demonstrating that polypeptide P2 has obvious anti-EV71 activity.


Example 4

Test Antiviral Efficiency of Polypeptide P2 in Various Cells


1. Materials


HEK293T, Vero, and Huh7.5 cells


2. Determination of Antiviral Efficiency of Polypeptide P2 in Various Cells






    • (1) 293T cells, Vero cells and Huh7.5 cells were plated in 24-well plates respectively.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum (0.5 ml/well), and 5 μl of 10{circumflex over ( )}6 PFU/mL EV71 virus per well was added.

    • (3) After 1 h, polypeptide P2 was added to different cells at a final concentration of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with a total RNA extraction kit as follows.

    • (5) The supernatant in each well was discarded, then 350 μl of TRK Lysis Buffer was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12,000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12,000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The empty column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12,000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





It can be seen in FIGS. 4A-4C that whether in RD cells, 293T cells, Vero cells or huh7.5 cells, polypeptide P2 can play a significant antiviral effect.


The IC50 values measured in 293T cells, Vero cells and Huh7.5 cells were 9.677 μM, 1.958 μM and 1.842 μM, respectively.


Example 5 Test Antiviral Efficiency of Polypeptide P2 Variants

1. Materials


Polypeptides 3A-TAT-EP (shown in SEQ ID NO: 3), 3A-EP-DRI (shown in SEQ ID NO: 4) and 3A-EP-PEG4-PA (shown in SEQ ID NO: 5) were used and were all synthesized by company.


2. Determination of Antiviral Efficiency of the Variants of Polypeptide P2






    • (1) 293T cells were plated in a 24-well cell plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum (0.5 ml/well), and 5 μl of 10{circumflex over ( )}6 PFU/mL EV71 virus per well was added.

    • (3) After 1 hour, polypeptides P2, 3A-TAT-EP, 3A-EP-DRI and 3A-EP-PEG-PA were added at a final concentration of 0.01 μM, 0.1 μM, 1 μM, and 10 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with a total RNA extraction kit.

    • (5) The supernatant in each well was discarded, then 350 μl of TRK Lysis Buffer was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12,000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The empty column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated water was added to the column and centrifuge at 12000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.


      3. Detection of Virus Inhibitory Activity of 3A-TAT-EP and 3A-EP-DRI in RD Cells by CCK8 Method

    • (1) RD cells in good growth state were plated in a 96-well plate at 1×104 cells per well and cultured in 5% CO2 at 37° C. for 24 h.

    • (2) polypeptides (3A-TAT-EP, 3A-EP-DRI) were diluted in 2-fold series with MEM containing 2% FBS to obtain 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM concentrations at a final volume of 100 μl in a new 96-well plate. Each concentration was tested in triplicate.

    • (3) The diluted virus solution was added to the plate of step (1), 100 μL per well with the final virus concentration was 0.1 MOI. Wells without polypeptide and virus (well without treatment), wells with virus only were used as controls.

    • (4) The mixture of step (2) was added to the 96-well plate plated with cells in step (1) and incubated in 5% CO2 at 37° C. for 24 h. Then CCK8 kit was used to determine the inhibitory effect of the polypeptide on virus.

    • (5) The inhibition rate of different concentrations of polypeptides on viral infection was calculated using the following formula:

      OD means the value of OD450-OD630, inhibition rate=(OD well with treatment−OD well with virus only)×100%/(OD well without treatment−OD well with virus only)





The results are shown in FIG. 5. It is shown than the polypeptide P2 variants 3A-TAT-EP (shown in SEQ ID NO: 3), 3A-EP-DRI (shown in SEQ ID NO: 4) and 3A-EP-PEG4-PA (shown in SEQ ID NO: 5) can also be used as an EV71 inhibitor, and have improved antiviral effect. The IC50 of 3A-EP-PEG4-PA measured by qRT-PCR is 3.25 μM.


In FIG. 6, the determination of the viral inhibitory activity of the polypeptides by the CCK8 method shows that the IC50 of 3A-TAT-EP is 4.36 μM, and the IC50 of 3A-EP-DRI is 3.56 μM.


Example 6

Detection of Membrane Penetration Efficiency of Polypeptide P1


1. Material:


MEM medium (Thermo), serum (Gibco), immunofluorescence plate (NEST), PBS, DAPI, and paraformaldehyde were purchased from markets.


Polypeptide P1 was synthesized by Nanjing GenScript Company, and its sequence is shown in SEQ ID NO: 6.


2. Experimental Procedure


The experiment was performed in two groups in order to observe whether the addition of EV71 virus affects the entry of polypeptides into cells. In one group, EV71 virus and then polypeptide P2 were added, and in the other group, only the polypeptide was added. Negative control was set in each group.


Immunofluorescence experiment was performed as follows.

    • (1) 1 ml of RD cells in a special dish for immunofluorescence were treated and then collected at 30% confluence.
    • (2) The medium in the dish was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual medium.
    • (3) 4% paraformaldehyde solution was prepared by dissolving 4 g paraformaldehyde solid in 100 ml PBS. Then 1 ml of the prepared 4% paraformaldehyde solution was added to each dish and incubated for 5 minutes to fix the cells.
    • (4) The 4% paraformaldehyde solution was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual paraformaldehyde.
    • (5) 1 mg/ml DAPI solution was diluted with PBS to 100 ng/ml, and the diluted DAPI solution was added to the dish and incubated for 15 minutes.
    • (6) The solution was aspirated, and the dish was washed three times (5 minutes for each wash) with 1 ml of 0.01 mol/L PBS (pH 7.4) to remove residual solution.
    • (7) The dish was observed under a fluorescence microscope.


Polypeptides were labeled with fluorescence and tested for their membrane penetration efficiency in RD cells. Two groups of experiment were performed. One group was control group without virus infection, in which blank, R8, P1 and P2 were added respectively; the other group was EV71 infection group, in which cells were infected with EV71 and then blank, R8, P1 and P2 were added respectively. The two groups of experiment were carried out at the same time, with virus MOI=0.1, and a polypeptide concentration of 1 μM. The cells were fixed 12 h after the addition of the polypeptide, immunofluorescence experiments were performed, and the nuclei were stained with DAPI. The results show that the polypeptides can enter cells infected with or without virus, showing a good ability to penetrate cell membranes.


As shown in FIG. 14, the polypeptides can be observed in both cells infected with or without virus, indicating that polypeptide P1 has a good ability to penetrate cell membrane.


Example 7

Determination of Cytotoxicity of Polypeptide P1


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptide P1 was desired to be able to inhibit viruses, also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide P1 was added to generate gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM (final concentrations), respectively.
    • (3) The test was performed 24 hours after the addition of the polypeptide, and 10 μl of viable cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was incubated at 37° C. for 2 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 15 and Table 4. Taking the cell viability of untreated group as 100%, there is no significant difference between the cell viability of the cells added with 100 μM polypeptide and that of the control group (untreated cells), which indicates that polypeptide P1 used in this study is not toxic to cells within 100 μM.











TABLE 4







Significance


Concentration

analysis


of polypeptide

(vs. 0 μM


(μM)
Cell viability (%)
group)



















0
104.243900
103.446800
 96.068770



0.01
102.884300
 98.822470
106.317300
P = 0.3481


0.1
105.509100
104.853800
 99.136560
P = 0.2964


1
102.718800
105.463300
102.899100
P = 0.2124


10
102.687200
107.144700
 98.611670
P = 0.3428


100
100.233100
 97.060650
 98.376590
P = 0.1920









RD cells were used to test the antiviral effect of polypeptide P1. When RD cells reached 80% confluence, polypeptide P1 was added at concentrations of 0 μM (control group), 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM, respectively. Three parallel wells were used for each concentration. The test was carried out 24 hours later, and the cell viability was detected using the CCK-8 kit. The results showed that there was no significant difference in the cell survival rate between the group added with 100 μM polypeptide P1 and the control group, indicating that polypeptide P1 is not toxic to cells within 100 μM.


Example 8

Determination of Cytotoxicity of Polypeptide 3A-TAT-EP


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptide 3A-TAT-EP was desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide 3A-TAT-EP was added to generate gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM (final concentrations), respectively.
    • (3) The test was performed 24 hours after the addition of the polypeptide, and 10 μl of viable cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was incubated at 37° C. for 2 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 16 and Table 5. Taking the cell viability of untreated group as 100%, there is no significant difference between the cell viability of the cells added with 100 μM polypeptide and that of the control group (untreated cells), which indicates that polypeptide 3A-TAT-EP used in this study is not toxic to cells within 100 μM.











TABLE 5







Significance


Concentration

analysis


of polypeptide

(vs. 0 μM


(μM)
Cell viability (%)
group)



















0
104.243900
103.446800
 96.068770



0.01
102.884300
 98.822470
106.317300
P = 0.3481


0.1
105.509100
104.853800
 99.136560
P = 0.2964


1
102.718800
105.463300
102.899100
P = 0.2124


10
102.687200
107.144700
 98.611670
P = 0.3428


100
100.233100
 97.060650
 98.376590
P = 0.1920









RD cells were used to test the antiviral effect of polypeptide 3A-TAT-EP. When RD cells reached 80% confluence, polypeptide 3A-TAT-EP was added at concentrations of 0 μM (control group), 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM, respectively. Three parallel wells were used for each concentration. The test was carried out 24 hours later, and the cell viability was detected using the CCK-8 kit. The results showed that there was no significant difference in the cell survival rate between the group added with 100 μM polypeptide 3A-TAT-EP and the control group, indicating that polypeptide P1 is not toxic to cells within 100 μM.


Example 9

Determination of Cytotoxicity of Polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA were desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, 3A-EP-DRI or 3A-EP-PEG4-PA was added to generate gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM (final concentrations), respectively.
    • (3) The test was performed 24 hours after the addition of the polypeptide, and 10 μl of viable cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was incubated at 37° C. for 2 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 17 and Table 6. Taking the cell viability of untreated group as 100%, there is no significant difference between the cell viability of the cells added with 50 μM polypeptide and that of the control group (untreated cells), which indicates that polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA used in this study are not toxic to cells within 50 μM.










TABLE 6







Concentration



of polypeptide
Cell viability (%)









(μM)
3A-EP-DRI
3A-EP-PEG4-PA












0.01
105.545100
106.594900


0.1
95.854640
94.885600


1
91.332440
89.878870


10
95.693140
95.289370


20
100.942100
98.358010


30
99.004040
99.488560


40
88.990580
99.327050


50
78.492600
94.804850









RD cells were used to test the antiviral effect of polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA. When RD cells reached 80% confluence, polypeptide 3A-EP-DRI or 3A-EP-PEG4-PA was added at concentrations of 0 μM (control group), 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively. Three parallel wells were used for each concentration. The test was carried out 24 hours later, and the cell viability was detected using the CCK-8 kit. The results showed that there was no significant difference in the cell survival rate between the group added with 50 μM polypeptide 3A-EP-DRI or 3A-EP-PEG4-PA and the control group, indicating that polypeptides 3A-EP-DRI and 3A-EP-PEG4-PA are not toxic to cells within 50 μM.


Example 10

Detection of Antiviral Efficiency of Polypeptide


1. Materials


Total RNA extraction kit (Omega), 24-well plate, 100 mm dish, and 50 ml syringe, 0.22 μm filter membrane (Millipore), one step qRT-PCR kit (Takara) were purchased in the market. The water used in the RNA extraction and qRT-PCR was DEPC-treated water, and the entire experiment was carried out in an RNase-free environment.


2. Amplification of Virus






    • (1) RD cells were plated in five 100 mm dishes.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, 1 μl EV71 virus at 10{circumflex over ( )}7 PFU/ml was added, respectively.

    • (3) After 2 days, the cells were observed to see if the CPE phenomenon occurred. When the changes were obvious, samples were collected as follows.

    • (4) The supernatant in the 100 mm dish was transferred into a 15 ml centrifuge tube, and centrifuged at 500 g for 5 min.

    • (5) The supernatant was transferred into a new 15 ml centrifuge tube, and filtered through 0.22 μm filter membrane with a 50 ml syringe.

    • (6) RNA was extracted from 100 μl of the supernatant obtained in step (5) and subjected to one step qRT-PCR to measure the virus titer, and the previously extracted virus RNA with known titer was handled together as a control.


      3. Measure Antiviral Efficiency of Polypeptide in RD Cells

    • (1) Different cells were plated in 24-well cell plates.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum (0.5 ml/well), and 5 μl of 10{circumflex over ( )}6 PFU/mL EV71 virus per well were added.

    • (3) After 1 h, different polypeptides were added at a final concentration of 0.01 μM, 0.1 μM, 1 μM and 10 μM, respectively. Wells without treatment were used as control.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with total RNA extraction kit as follows.

    • (5) The supernatant in the wells was discarded, then 350 μl of TRK Lysis Buffer was added to each well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12,000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12,000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





The test results of polypeptides EP-PA, EP-CHOL, 3A-EP-DRI and 3A-EP-PEG4-PA are shown in FIG. 18 and tables 7 to 10.











TABLE 7





Concentration

Significance


of polypeptide

analysis


EP-PA

(vs. 0 μM


(μM)
Relative viral RNA amount (%)
group)



















0
100.569000
80.164300
99.430990
/


0.01
 83.066650
76.474690
56.742880
P = 0.0539


0.1
 79.012950
67.554600
53.702980
P = 0.0271


1
 86.289260
75.980030
56.914250
P = 0.0672


10
 43.712254
61.850070
27.037620
P = 0.0075


















TABLE 8





Concentration

Significance


of polypeptide

analysis


EP-CHOL

(vs. 0 μM


(μM)
Relative viral RNA amount (%)
group)



















0
100.569000
80.164300
99.430990
/


0.01
 75.485760
59.539330
59.747630
P = 0.0141


0.1
 75.761750
70.988640
60.975830
P = 0.0191


1
 96.783520
76.068370
59.554410
P = 0.1382


10
 39.120456
66.179340
37.087750
P = 0.0081


















TABLE 9





Concentration

Significance


of polypeptide

analysis


3A-EP-DRI

(vs. 0 μM


(μM)
Relative viral RNA amount (%)
group)



















0
100.569000
80.164300
99.430990
/


0.01
 75.931920
73.357810
72.892740
P = 0.0222


0.1
 72.523670
70.587060
61.379300
P = 0.0139


1
100.475300
95.841450
90.712620
P = 0.3832


10
 29.812460
19.585250
 7.211936
P = 0.0007


















TABLE 10





Concentration

Significance


of polypeptide

analysis


3A-EP-PEG4-PA

(vs. 0 μM


(μM)
Relative viral RNA amount (%)
group)



















0
100.569000
80.164300
99.430990
/


0.01
 72.500240
68.123620
59.095570
P = 0.0127


0.1
 69.964290
68.685590
67.809440
P = 0.0105


1
107.086800
89.256320
75.236370
P = 0.4607


10
 24.576220
24.508320
11.301410
P = 0.0004









RD cells were used to test the antiviral effect of polypeptides EP-PA, EP-CHOL, 3A-EP-DRI and 3A-EP-PEG4-PA. EV71 virus was added to RD cells at 80% confluence (MOI=0.1). After 1 hour of infection, polypeptides EP-PA, EP-CHOL, 3A-EP-DRI or 3A-EP-PEG4-PA was added at concentrations of 0.01 μM, 0.1 μM, 1 μM, and 10 μM, respectively. The samples were collected 24 hours later, and the total cell RNA was extracted. The level of viral genomic RNA was measured by qRT-PCR. The cells infected with virus but without polypeptide treatment were used as a control group. The results show that all polypeptides have anti-EV71 activity, and increasing the polypeptide concentration significantly inhibits the expression level of viral RNA.


The results of anti-CVA16 virus effect of polypeptides P2, ER and ER-DRI are shown in FIG. 22 and tables 11-15.












TABLE 11







Concentration




of polypeptide




P2




(μM)
Virus inhibition rate (%)





















0.15625
 19.070900
16.870410
 2.933985



0.3125
 27.139360
12.836190
 7.885086



0.625
 24.388750
24.572130
15.220050



1.25
 42.542790
30.990220
38.325180



2.5
 71.149150
60.696820
55.745720



5
101.039100
86.552570
93.154040




















TABLE 12







Concentration




of polypeptide




ER




(μM)
Virus inhibition rate (%)





















0.15625
11.002450
 6.418093
 5.134474



0.3125
 8.618582
15.220050
11.002450



0.625
19.437650
 6.784841
 8.618582



1.25
15.953550
21.271390
 8.985330



2.5
44.376530
24.755500
34.474330



5
71.699260
73.716380
62.530560




















TABLE 13







Concentration




of polypeptide




ER-DRI




(μM)
Virus inhibition rate (%)





















0.15625
 7.885086
 3.667482
 8.801956



0.3125
 12.102690
 11.185820
 21.454770



0.625
 15.770170
 13.754320
 27.139360



1.25
 59.046460
 58.863080
 50.794620



2.5
 93.520780
 84.168700
 77.933980



5
104.706600
107.273800
106.173600




















TABLE 14







Concentration




of polypeptide




R8




(μM)
Virus inhibition rate (%)





















0.15625
−5.187778
10.914710
9.456473



0.3125
−9.961808
7.600530
1.900133



0.625
0.5410566
6.009722
5.744587



1.25
5.612020
−0.8911521
5.479452



2.5
4.949183
−1.941439
3.500955



5
−1.654997
0.8274984
7.733098


















TABLE 15





Concentration



of polypeptide



TAT



(μM)
Virus inhibition rate (%)


















0.15625
−3.004861
5.877154
0.1767565


0.3125
2.960672
−0.8837826
−2.076889


0.625
−3.946531
0.5744587
1.104728


1.25
4.021211
−0.4860804
−3.535130


2.5
1.237296
−2.607159
−1.654997


5
−1.654997
1.782304
−2.800764









RD cells were used to test the antiviral effect of polypeptides P2, ER and ER-DRI, and penetrating peptide R8 and TAT were used as controls. EV71 was added to RD cells at 80% confluence (MOI=0.1). After 1 hour of infection, polypeptides P2, ER, ER-DRI, R8 or TAT was added at concentrations of 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM, respectively. The CCK-8 kit was used to test the inhibitory activity of the polypeptide against the virus 24 hours after infection. Inhibition rate of polypeptide=(OD well with treatment−OD well with virus only)×100%/(OD well without treatment−OD well with virus only), OD refers to the value of OD450-OD630. The results show that the peptides P2, ER, and ER-DRI can significantly inhibit CVA16, while the penetrating peptide R8 and TAT have no effect on the virus. The IC50 of P2 is 1.533 μM, the IC50 of ER is 3.211 μM, and the IC50 of ER-DRI is 0.856 μM.


Example 11

Test Antiviral Activity of Polypeptide P1 against EV71 and CVA16 in Mice


1. Materials


P1 (sequence: RRRRRRRRAISDLLAS) was commercially synthesized. Newborn 2-day-old ICR suckling mice were used in the experiment.


2. Antiviral Activity of Polypeptide P1 in Mice






    • (1) 8 2-day-old ICR mice were randomly divided into two groups, 4 in each group. The 8 mice were administered EV71 by intraperitoneal injection at a dose of 108 PFU/ml.

    • (2) At the same time, one group was intraperitoneally injected with 10 mg/kg of polypeptide P1 as a treatment group, and the other group was intraperitoneally injected with an equal amount of PBS as a control group.

    • (3) Polypeptide and PBS were injected every 12 h for 5 days.

    • (4) At the fifth day, the mice were euthanized, and their hind limb muscle tissues were collected and triturated with Trizol (Invitrogen) to extract total RNA.

    • (5) Fluorescence quantification experiments were performed using a one step qRT-PCR kit.





The results are shown in FIG. 7 and Table 16. It can be seen that P1 can significantly reduce the EV71 viral load in mice.


3. The Procedure for Detecting the Antiviral Activity of P1 Against CVA16 in Mice is Similar as the EV71 Experiment.


The results are shown in FIG. 8 and Table 17. It can be seen that P1 can significantly reduce the viral load of CVA16 in mice.











TABLE 16







Significance




analysis




(treatment


Sample
Number of EV71 virus copy in muscle tissue (Copies/g)
vs. control)




















PBS (control)
1.206809e+009
1.122504e+009
8.221773e+008
2.381125e+008
P = 0.0286


P1 (treatment)
9.934962e+007
9.702361e+007
4072066.000
1.202581e+007


















TABLE 17







Significance




analysis




(treatment


Sample
Number of CVA16 virus copys in muscle tissue (Copies/g)
vs. control)




















PBS (control)
1.606804e+007
3.814315e+007
4.760443e+007
4.175508e+007
P = 0.0286


P1 (drug)
3538852
5293714
5875037
6810972









Example 12

Detection of Antiviral Activity of Polypeptide CR against EV71 in RD Cells






    • (1) RD cells in good growth state were plated in a 96-well plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Group without polypeptide and virus, and group with virus but without polypeptide were included as controls. The final virus concentration was 0.1 MOI.

    • (3) 1 h later, polypeptide CR at a final concentration of 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0.15625 μM was added, respectively. Group without polypeptide and virus, and group with virus but without polypeptide were set as controls.

    • (4) 24 hours after virus infection, when the change in the control group with only virus was obvious, 10 μl of viable cell detection agent CCK-8 was added to each well and mixed well.

    • (5) The plate was incubated at 37° C. for 2 h.

    • (6) The absorbance value at OD450 was measured with a microplate reader. Inhibition rate of polypeptide=(OD well with treatment−OD well with virus only)×100%/(OD well without treatment−OD well with virus only), OD refers to the value of OD450-OD630.





The results are shown in FIG. 9 and Table 18. The IC50 of CR inhibiting EV71 is 1.7 μM.










TABLE 18





Concentration



of polypeptide



CR



(μM)
Virus inhibition rate (%)


















0.15625
−4.345738
−7.142856
−8.595442


0.3125
13.289320
2.833130
3.745496


0.625
16.422570
7.118847
16.770710


1.25
50.804320
47.503000
45.630250


2.5
78.259310
70.504210
64.429770


5
74.465790
71.500600
69.255700









Example 13

Antiviral Activity of Polypeptide CR against EV71 and CVA16 in Mice


1. Material


Polypeptide CR (sequence: YGRKKRRQRRRGSGCR) was commercially synthesized. Newborn 2-day-old ICR suckling mice were used in the experiment.


2. Antiviral Activity of Polypeptide CR in Mice






    • (1) 8 2-day-old ICR mice were randomly divided into two groups, 4 in each group. The 8 mice were administered EV71 by intraperitoneal injection at a dose of 108 PFU/ml.

    • (2) At the same time, one group was intraperitoneally injected with 10 mg/kg of polypeptide CR as a treatment group, and the other group was intraperitoneally injected with an equal amount of PBS as a control group.

    • (3) Polypeptide and PBS were injected every 12 h for 5 days.

    • (4) At the fifth day, the mice were euthanized, and their hind limb muscle tissues were collected and triturated with Trizol (Invitrogen) to extract total RNA.

    • (5) Fluorescence quantification experiments were performed using a one step qRT-PCR kit.





The results are shown in FIG. 7 and Table 16. It can be seen that CR can significantly reduce the EV71 viral load in mice.


3. The Procedure for Detecting the Antiviral Activity of CR Against CVA16 in Mice is Similar to the EV71 Experiment.


The results are shown in FIG. 11 and Table 20. It can be seen that CR can significantly reduce the viral load of CVA16 in mice.











TABLE 19







Significance




analysis




(treatment


Sample
Number of virus copy in muscle tissue (Copies/g)
vs. control)




















PBS (control)
3.131500e+007
7.089894e+007
5.287820e+007
7.465289e+007
P = 0.0240


CR (treatment)
1.879620e+007
1.122332e+007
1.738292e+007
9852432.0000


















TABLE 20







Significance




analysis



Number of virus copies in muscle tissue
(treatment


Sample
(Copies/g)
vs. control)




















PBS (control)
3310458
2870615
1076559
1.899580e+007
P = 0.2628


P1 (treatment)
 228883
1832418
 71627
1061937










Example 14

Toxicity Evaluation of ER-DRI in Mice


1. Material


Polypeptide ER-DRI was commercially synthesized. Newborn 2-day-old ICR suckling mice were used.


2. Toxicity Evaluation of ER-DRI in Mice






    • (1) A total of 12 10-day-old suckling mice were randomly divided into two groups, 6 in each group. One group was intraperitoneally injected with 20 mg/kg ER-DRI once daily for 3 consecutive days, and the other group was injected with an equal amount of PBS as a control.

    • (2) The body weight of the mice was recorded daily for a total of 15 days.

    • (3) Four weeks after the administration, the mice were euthanized and dissected, and the brain, liver, lung, and kidney were collected for HE staining.





The results are shown in FIG. 12. There is no significant difference in body weight between the 20 mg/kg polypeptide injection group and the PBS group. The HE staining results in FIG. 13 showed that there is no significant pathological change in the brain, liver, lung and kidney of the 20 mg/kg peptide injection group and the PBS group, and there was no significant difference between groups.


Example 15

Toxicity Detection of ER and ER-DRI in RD Cells


1. Experimental Materials


CCK-8 reagent was purchased from MCE.


2. Experiment Procedure


Polypeptides ER and ER-DRI were desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide ER or ER-DRI was added to generate gradient concentrations of 0.46 μM, 2.34 μM, 4.68 μM, 9.37 μM, 18.75 μM, 37.5 μM, 75 μM, 150 μM, 300 μM (final concentrations), respectively.
    • (3) The test was performed 24 hours after the addition of the polypeptide, and 10 μl of viable cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was incubated at 37° C. for 2 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 17 and Table 6. Taking the cell viability of untreated cells as 100%, the 50% cytotoxic concentration (CC50) of TAT-ER-DRI is calculated to be 117 μM, and the CC50 of TAT-ER is calculated to be 290 Therefore, polypeptides ER and ER-DRI have very low toxicity based on the comparison of the 50% cytotoxic concentration (290 μM, 117 μM) and the half-inhibitory activity (IC50 1.26 μM, 0.64 μM).












TABLE 21







Concentration




of polypeptide




ER-DRI




(μM)
Cell viability (%)





















0.46875
102.759000
100.188600
99.276570



2.34375
104.410400
 98.834360
96.326180



4.6875
101.204300
 98.509610
97.632100



9.375
 96.049800
 96.789120
94.149670



18.75
 95.835600
 91.144020
89.008960



37.5
 86.003300
 86.770260
86.970640



75
 68.902140
 70.415330
71.949260



150
 34.001950
 34.616900
34.084860



300
 18.206690
 17.577920
18.690360




















TABLE 22









Concentration













of ER













(μM)
Cell viability (%)
















0.46875
99.932980
 99.338760
100.043500



2.34375
98.551070
 98.074300
102.662300



4.6875
99.145290
100.810500
103.132100



9.375
98.530330
 98.475060
102.406600



18.75
98.993280
 96.588750
 98.468150



37.5
94.847540
 95.690510
 92.235730



75
89.693010
 89.789740
 90.356330



150
72.923510
 74.319240
 76.716850



300
42.210510
 43.392040
 48.408390










Example 16

Detection of Antiviral Activity of ER and ER-DRI against EV71 in RD Cells


1. Material


Total RNA extraction kit (Omega), 24-well plate, 100 mm dish, and 50 ml syringe, 0.22 μm filter membrane (Millipore), one step qRT-PCR kit (Takara) were purchased in the market. The water used in the RNA extraction and qRT-PCR was DEPC-treated water, and the entire experiment was carried out in an RNase free environment.


2. Detect Polypeptide Antiviral Efficiency in Cells






    • (1) RD cells in good growth state were plated in a 96-well plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Group without polypeptide and virus, and group with virus but without polypeptide were included as controls. The final virus concentration was 0.1 MOI.

    • (3) 1 h later, polypeptide CR at a final concentration of 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0.15625 μM was added, respectively. Group without polypeptide and virus, and group with virus but without polypeptide were set as controls.

    • (4) 24 hours after virus infection, when the change in the control group with only virus was obvious, 10 μl of viable cell detection agent CCK-8 was added to each well and mixed well.

    • (5) The plate was incubated at 37° C. for 2 h.

    • (6) The absorbance value at OD450 was measured with a microplate reader. Inhibition rate of polypeptide=(OD well with treatment−OD well with virus only)×100%/(OD well without treatment−OD well with virus only), OD refers to the value of OD450-OD630.





The results are shown in FIG. 20 and Tables 23 to 25. It can be seen that the IC50 of ER against EV71 in RD cells is 1.26 μM, and the IC50 of ER-DRI against EV71 in RD cells is 0.64












TABLE 23







Concentration




of polypeptide




ER-DRI




(μM)
Virus inhibition rate (%)





















0.15625
18.061620
 2.991017
−1.026955



0.3125
20.924260
 2.092426
−2.130934



0.625
37.804870
40.474960
37.946090



1.25
75.853660
72.426190
77.522470



2.5
78.677790
85.288830
79.756090


















TABLE 24





Concentration



of polypeptide



ER



(μM)
Virus inhibition rate (%)


















0.15625
1.360719
0.5905035
16.611040


0.3125
−1.591784
−0.1797201
15.763800


0.625
8.177150
7.432604
21.912710


1.25
37.599480
40.038510
48.844680


2.5
83.453140
79.756090
80.924260



















TABLE 25







Concentration




of polypeptide




TAT




(μM)
Virus inhibition rate (%)





















0.15625
4.465894
4.749035
17.426000



0.3125
5.997427
8.301158
11.634490



0.625
−2.908619
6.216215
12.728440



1.25
−0.733587
2.162163
11.879020



2.5
7.014158
7.657658
20.115830










Example 17

Detection of Antiviral Activity of ER-DRI against EV71 in Mice


1. Experimental Materials


Polypeptide ER-DRI was commercially synthesized. 10 2-day-old suckling mice were used.


2. Experiment Procedure






    • (1) 10 2-day-old ICR mice were randomly divided into two groups, 5 in each group. The 10 mice were administered with EV71 by intraperitoneal injection at a dose of 108 PFU/ml.

    • (2) At the same time, one group was intraperitoneally injected with 10 mg/kg of polypeptide ER-DRI as a treatment group, and the other group was intraperitoneally injected with an equal amount of PBS as a control group.

    • (3) Polypeptide and PBS were injected every 12 h for 5 days.

    • (4) At the fifth day, the mice were euthanized, and their lung tissues were collected and triturated with Trizol (Invitrogen) to extract total RNA.

    • (5) Fluorescence quantification experiments were performed using a one step qRT-PCR kit.





The results are shown in FIG. 21 and Table 26. It can be seen that ER-DRI can significantly reduce the viral load of EV71 in the mouse lung.











TABLE 26







Significance




analysis




(treatment


Sample
Number of virus copy in lung tissue (Copies/g)
vs. control)





















PBS
2.229596e+007
1.550789e+007
2.656590e+007
2.644002e+007
3.103317e+007
P = 0.0079


(control)


ER-DRI
294534
484192
2956937
549759
116302


(treatment)









Example 18

Toxicity of Polypeptides BP8, BP10 and BP15 in RD Cells


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptides BP8, BP10 and BP15 were desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) RD cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide BP8, BP10 or BP15 was added to generate gradient concentrations of 3.0625 μM, 6.125 M, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM (final concentrations), respectively.
    • (3) The samples were collected 24 hours after the addition of the polypeptide, and 10 μl of live cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was placed at 37° C. for 4 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 23 and Tables 27 to 29. Taking the cell viability of untreated cells as 100%, the cell viability of cells added with 200 μM BP8 and BP10 are substantially the same as that of the control group (untreated cells), the cell viability of cells added with 100 μM BP15 are substantially the same as that of the control group (untreated cells), and the cell viability is decreased to 20% after the addition of 200 μM polypeptide. These results indicate that BP8 and BP10 are not toxic to cells within 200 μM, and BP15 is not toxic to cells within 100 μM.












TABLE 27







Concentration




of polypeptide




BP8




(μM)
Cell viability (%)





















3.0625
102.1895
103.1369
104.4



6.125
101.1158
103.5158
101.1158



12.5
94.86318
99.34739
104.8421



25
93.41055
97.26318
99.85265



50
95.17897
94.16844
97.64213



100
93.09476
93.60002
96.25265



200
88.92633
93.09476
92.08423




















TABLE 28







Concentration




of polypeptide




BP10




(μM)
Cell viability (%)





















3.0625
97.73014
98.99117
103.5309



6.125
98.54981
93.25347
92.62295



12.5
94.82976
97.35183
100.8827



25
109.4578
99.55864
96.2169



50
109.1425
115.4477
116.3934



100
106.8726
112.232
114.3758



200
79.69735
85.18285
97.54098




















TABLE 29







Concentration




of polypeptide




BP15




(μM)
Cell viability (%)





















3.0625
90.96416
85.76992
91.09402



6.125
103.6901
105.7678
97.71668



12.5
96.67783
98.75552
95.24941



25
96.67783
92.912
95.05463



50
107.391
103.6251
100.2489



100
96.28826
99.4048
104.9237



200
9.674276
18.37463
12.59604










Example 19

Toxicity of Polypeptides BP8, BP10 and BP15 in Vero Cells


1. Experimental Materials


CCK-8 reagent was from MCE.


2. Experimental Procedure


Polypeptides BP8, BP10 and BP15 were desired to be able to inhibit viruses, and also to be non-toxic to cells. Therefore, the cytotoxicity test was used for detection, and untreated cells were used as the control group.


The experiment was performed as follows.

    • (1) Vero cells were plated in a 96-well plate at 100 μl per well.
    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum. Then, polypeptide BP8, BP10 or BP15 was added to generate gradient concentrations of 3.0625 μM, 6.125 M, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM (final concentrations), respectively.
    • (3) The samples were collected 24 hours after the addition of the polypeptide, and 10 μl of live cell detection reagent CCK-8 was added to each well and mixed well.
    • (4) The plate was placed at 37° C. for 4 h.
    • (5) The absorbance value at OD450 was measured with a microplate reader.


The results are shown in FIG. 24 and Tables 30, 31 and 32. Taking the cell viability of untreated cells as 100%, the cell viability of cells added with 200 μM BP8 and BP10 are substantially the same as that of the control group (untreated cells), the cell viability of cells added with 100 μM BP15 are substantially the same as that of the control group (untreated cells), and the cell viability is decreased to 20% after the addition of 200 μM polypeptide. It indicates that BP8 and BP10 are not toxic to cells within 200 μM, and BP15 is not toxic to cells within 100 μM. The toxicities of the polypeptides in RD cells and Vero cells are relatively consistent.












TABLE 30







Concentration




of polypeptide




BP8




(μM)
Cell viability (%)





















3.0625
103.2048
105.2007
108.7417



6.125
101.4665
111.3814
119.3004



12.5
95.54333
106.8102
109.1924



25
96.63783
99.47065
113.6348



50
94.44883
108.2266
112.4759



100
99.6638
96.70221
97.34603



200
87.23801
92.0023
92.51736




















TABLE 31







Concentration




of polypeptide




BP10




(μM)
Cell viability (%)





















3.0625
106.5527
106.2952
107.1965



6.125
118.8497
119.236
118.9785



12.5
121.7469
120.8456
123.9359



25
116.7895
117.3689
116.0169



50
109.1924
118.399
120.0086



100
92.77489
92.32421
94.06254



200
83.05316
86.72295
82.21619




















TABLE 32







Concentration




of polypeptide




BP15




(μM)
Cell viability (%)





















3.0625
107.6472
105.9733
109.128



6.125
125.3523
127.0907
124.0647



12.5
121.5538
120.3949
122.777



25
116.2744
111.0595
118.2059



50
111.5101
109.4499
102.9473



100
75.77796
87.10925
76.80808



200
13.64905
13.13399
13.4559










Example 20

Test Antiviral Effect of Polypeptides BP8, BP10 and BP15 against CVB5 in RD Cells


1. Materials


Polypeptides BP8 (shown as SEQ ID NO: 12), BP10 (shown as SEQ ID NO: 13), and BP15 (shown as SEQ ID NO: 14) were all commercially synthesized.


2. Antiviral Efficiency of Polypeptides BP8, BP10 and BP15






    • (1) DR cells were plated in a 24-well cell plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum (0.5 ml/well). CVB5 viruses were added at MOI=0.01, and the group without CVB5 as control.

    • (3) After 1 h, polypeptides BP8, BP10 or BP15 was added at a final concentration of 0.78 μM, 1.56 μM, 3.13 μM and 6.25 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with total RNA extraction kit as follows.

    • (5) The supernatant in the wells was discarded, then 350 μl of TRK Lysis Buffer was added to each well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12,000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





The results are shown in FIG. 25 and Tables 33, 34 and 35. It can be seen that the polypeptides BP8, BP10 and BP15 all can significantly inhibit CVB5. The IC50 of BP8 is 1.545 μM, the IC50 of BP10 is 1.335 μM, and the IC50 of BP15 is 6.758 μM.












TABLE 33







Concentration




of polypeptide




BP8




(μM)
Virus inhibition rate (%)





















0.78
12.95326
11.39885
10.25664



1.56
55.69942
47.92739
42.01265



3.13
51.42481
41.70977
45.98439



6.25
50.21543
54.14502
49.8704




















TABLE 34







Concentration




of polypeptide




BP10




(μM)
Virus inhibition rate (%)





















0.78
6.347029
6.12571
5.45871



1.56
58.80824
54.23212
56.01254



3.13
65.56288
66.96887
67.23652



6.25
78.62692
77.07251
76.12545




















TABLE 35







Concentration




of polypeptide




BP15




(μM)
Virus inhibition rate (%)





















0.78
−1.81361
0.13541
−0.25921



1.56
30.95842
35.10354
33.44521



3.13
43.26418
29.66312
37.15236



6.25
56.08803
38.98956
46.59892










Example 21

Test Antiviral Effect of Polypeptide BP8 against CVB3 in Vero Cells


1. Materials


Polypeptides BP8 (shown as SEQ ID NO: 12) was commercially synthesized.


2. Anti-CVB3 Effect of Polypeptide BP8






    • (1) Vero cells were plated in a 24-well cell plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum and CVB3 virus, 0.5 ml per well. MOI of the virus was 0.01. Wells without CVB3 virus was set as control group.

    • (3) After 1 h, polypeptide BP8 was added at a final concentration of 0.25 μM, 0.5 μM and 10 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with total RNA extraction kit as follows.

    • (5) The supernatant in the wells was discarded, then 350 μl of TRK Lysis Buffer was added to each well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





The results are shown in FIG. 26 and Table 36. It can be seen that polypeptide BP8 is able to inhibit the replication of CVB3. The IC50 of BP8 is 4.125 μM.












TABLE 36







Concentration




of polypeptide




BP8




(μM)
Virus inhibition rate (%)





















2.5
40.9035
25.50707
36.01721



5
64.04425
53.44192
54.17947



10
87.83036
86.81623
93.6386










Example 22

Test Antiviral Effect of Polypeptide BP8 against CVB5 in Mice


1. Materials: Polypeptide BP8 (shown as SEQ ID NO: 12) was commercially synthesized. 10 2-day-old ICR suckling mice were used.


2. Antiviral activity of peptide BP8 in mice






    • (1) 10 2-day-old ICR suckling mice were randomly divided into two groups, 5 in each group. The 10 suckling mice were challenged by intraperitoneal injection of CVB5 at a dose of 108 PFU/ml.

    • (2) At the same time, one group was intraperitoneally injected with 10 mg/kg of polypeptide BP8 as a treatment group, and the other group was intraperitoneally injected with an equal amount of PBS as a control group.

    • (3) Polypeptide and PBS were injected every 12 h for 5 days.

    • (4) At the fifth day, the mice were euthanized, and their hind limb muscle tissues were collected and triturated with Trizol (Invitrogen) to extract total RNA.

    • (5) Fluorescence quantification experiments were performed using a one step qRT-PCR kit.





The results are shown in FIG. 27 and Table 37. It can be seen that virus copy number in the treatment group (BP8) is significantly lower than that of the blank control group (PBS) by nearly 80 times.











TABLE 37







Significance




analysis




(treatment


Sample
Number of virus copy in muscle tissue (Copies/g)
vs. control)





















Vehicle
2.15e+008
3.16e+008 
5.78e+008
  4e+008
7.41e+008
P = 0.0015


(control)


BP8
3.43e+006
7.00 + 006
8.53e+006
7.03e+006
1.24e+006


(treatment)









Example 23

Detection of Antiviral Effect of Polypeptide ER-DRI against CVA6 in Vero Cells


1. Materials


Polypeptide ER-DEI (SEQ ID NO: 11) was commercially synthesized.


2. Anti-CVA6 Effect of Polypeptide ER-DEI






    • (1) Vero cells were plated in a 24-well cell plate.

    • (2) After the cells reached 70%-80% confluence, the MEM medium containing 10% serum was replaced with a MEM medium containing 2% serum and CVA6 virus, 0.5 ml per well. MOI of the virus was 0.01. The wells without CVA6 virus was set as control group.

    • (3) After 1 h, polypeptide ER-DRI was added at a final concentration of 2.5 μM, 5 μM and 10 μM, respectively.

    • (4) Samples were collected 24 hours after virus infection, and RNA was extracted with total RNA extraction kit as follows.

    • (5) The supernatant in the wells was discarded, then 350 μl of TRK Lysis Buffer was added to each well and the plate was shaken on the shaker for 5 minutes.

    • (6) 350 μl of 70% ethanol (DEPC-treated) was added to the well and the plate was shaken on the shaker for 5 minutes.

    • (7) The mixture in the well was transferred to a RNA extraction column and centrifuged at 12000 g for 1 min.

    • (8) The solution collected in the recovery tube was added back to the column and centrifuged at 12000 g for 1 min.

    • (9) RNA washing buffer 1 was added to the column and centrifuged at 12,000 g for 30 s.

    • (10) RNA washing buffer 2 was added to the column and centrifuged at 12,000 g for 1 min.

    • (11) Step (10) was repeated once.

    • (12) The column was centrifuged at 12,000 g for 2 min to completely remove residual RNA washing buffer.

    • (13) 50 μl DEPC-treated H2O was added to the column and centrifuge at 12,000 g for 2 min.

    • (14) 2 μl RNA sample was used according to one step qRT-PCR kit for fluorescence quantification experiment.





The results are shown in FIG. 28 and Table 38. It can be seen that polypeptide ER-DRI is able to inhibit the replication of CVA6. The IC50 of ER-DRI is less than 0.625 μM.












TABLE 38







Concentration




of polypeptide




ER-DRI




(μM)
Virus inhibition rate (%)





















0.625
71.8
74.9
67.5



2.15
72.6
85.3
90.4



2.5
91.3
93.4
91.7



5
94.3
94.9
96.4



10
91.4
97.8
97.9










The polypeptides and application thereof provided by the present invention are described in detail above. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and the core idea thereof. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims
  • 1. A polypeptide capable of inhibiting the activity of enterovirus protein 3A, wherein the polypeptide consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 14.
  • 2. A nucleic acid encoding the polypeptide of claim 1.
  • 3. A medicine comprising the polypeptide according to claim 1 and pharmaceutically acceptable excipients.
  • 4. An inhibitor of enterovirus, wherein the inhibitor is polypeptide P2 with the amino acid sequence shown in SEQ ID NO: 2.
  • 5. A variant of an inhibitor of enterovirus, wherein the variant is 3A-TAT-EP with the amino acid sequence as shown in SEQ ID NO: 3, 3A-EP-DRI with the amino acid sequence as shown in SEQ ID NO: 4, or 3A-EP-PEG4-PA with the amino acid sequence as shown in SEQ ID NO: 5.
  • 6. A method of treating a disease caused by enterovirus, comprising administering the polypeptide according to claim 1 to a subject in need thereof; wherein the enterovirus belongs to the Picornaviridae enterovirus genus, including human Enterovirus (EV), Coxsackie A virus (CVA), Coxsackie B virus (CVB), Echovirus, Rhinovirus and Poliovirus;and the disease is selected from hand-foot-and-mouth disease, myocarditis, herpes angina, aseptic meningitis, encephalitis and viral cold.
  • 7. The polypeptide of claim 1, which is polypeptide P2 with the amino acid sequence shown in SEQ ID NO: 2.
  • 8. The polypeptide of claim 1, which is 3A-TAT-EP with the amino acid sequence as shown in SEQ ID NO: 3, 3A-EP-DRI with the amino acid sequence as shown in SEQ ID NO: 4, or 3A-EP-PEG4-PA with the amino acid sequence as shown in SEQ ID NO: 5.
  • 9. The polypeptide of claim 1, wherein the polypeptide is set forth in SEQ ID NO: 10.
Priority Claims (1)
Number Date Country Kind
201810056297.1 Jan 2018 CN national
PCT Information
Filing Document Filing Date Country Kind
PCT/CN2019/072455 1/21/2019 WO
Publishing Document Publishing Date Country Kind
WO2019/141263 7/25/2019 WO A
US Referenced Citations (1)
Number Name Date Kind
20160376562 Venskus et al. Dec 2016 A1
Foreign Referenced Citations (3)
Number Date Country
105394045 Mar 2016 CN
105518128 Apr 2016 CN
106668832 May 2017 CN
Non-Patent Literature Citations (6)
Entry
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Related Publications (1)
Number Date Country
20230192769 A1 Jun 2023 US