Antiviral Activity And Mechanism Of The Peptide Ev37 From The Venom Of Euscorpiops Validus

Viruses are widespread in nature and can infect various organisms,including animals,plants,bacteria,fungi.Virus,especially that can infect humans and animals,not only brings huge economic losses to people,but also seriously endangers human health and life safety.Dengue virus(DENV)belongs to the Flavivirus,and its genome is positive single-strand RNA.DENV can cause dengue fever,dengue hemorrhagic fever,dengue shock syndrome.The mortality rate and the incidence rate are extremely high.With the expansion of the geographical distribution of Aedes aegypti,its incidence has increased 30-fold in recent decades and is one of the most dangerous tropical viruses in the world.Zika virus(ZIKV),like DENV,belongs to the Flavivirus,and is also an arbovirus.ZIKV can cause congenital malformations in the fetus,including microcephaly,craniofacial deformity,paralysis,ocular malformations and miscarriage.ZIKV infection in adults can cause autoimmune disease,Guillain-Barre syndrome,which makes the immune system attack the peripheral nerves,causing muscle weakness and severe paralysis.In recent years,ZIKV has broken out in many regions of America.Hepatitis C virus(HCV),which has 7 genotypes and 67 gene subtypes,belongs to the Hepatovirus of the Flaviviridae.The genome is similar to DENV and is a positivestrand RNA virus with a size of 9.6 kb.Under natural conditions,HCV can only infect humans and chimpanzees.Continuous infection with the HCV can lead to chronic hepatitis and other life-threatening complications,including liver failure and hepatocellular carcinoma.About 170 million people worldwide are infected with HCV,but there is currently no effective vaccine to prevent HCV infection.Although clinically available direct viricidal drugs(DAAs)can be used to treat patients with HCV infection,new drugs need to be developed due to its high cost and resistance to drugs.There is currently no effective vaccine or drugs for DENV and ZIKV,and cross-infection between viruses can cause more serious consequences,so it is necessary to find a drug with multiple antiviral functions.Scorpion is an ancient creature with a history of more than 400 million years of evolution.During the long-term evolution,its tail has a venom gland that can spray venom.The components of scorpion venom are complex and multifunctional,including ion channel modulators,proteases,protease inhibitors,antimicrobial peptides and antiviral peptides.Most antiviral scorpion venom peptides inhibit viral infection by directly killing viral particles or damaging the envelope of the virus.At the same time,scorpion is a rare and precious medicinal material.It has a medicinal history of thousands of years in traditional Chinese medicine.Scorpion,especially the tail venom gland,can treat many diseases.For example,in the Ming Dynasty,Li Shizhen clearly recorded in the "Compendium of Materia Medica".Therefore,the peptides in the scorpion venom are a valuable drug resource.In this paper,the scorpion venom peptide Ev37,which was screened from the venom cDNA library of Euscorpiops validus collected from Mengzi,Yunnan,was cloned into the recombinant expression vector pET-32 a and expressed in E.coli BL21(DE3)by prokaryotic expression.The recombinant peptide Ev37 was purified and identified by nickel column affinity chromatography,enzyme digestion,high performance liquid chromatography and MOLDI-TOF MS analysis,and the purity was more than 95%.Furthermore,the structural analysis of scorpion venom peptide Ev37 was carried out by SWISS-MODEL with the homologous scorpion venom peptide Hge36(56%)as template.It was found to have a typical CSαβ composed of α-helix and β-sheet.The secondary structure of the recombinant scorpion venom peptide Ev37 was analyzed by circular dichroism,which contains α-helix and β-sheet structure.The scorpion venom peptide Ev37 has 50% homolog with Scorpine derived from Pandinus imperator,which has been reported that could inhibit DENV-2 replication.So,we have detected the antiviral activity of the scorpion venom peptide Ev37 against DENV-2.The scorpion venom peptide Ev37 can reduce DENV-2 RNA and protein level in cells and viral particles in supernatant in a concentration-dependent way at noncytotoxic concentrations.Moreover,we found that the scorpion venom peptide Ev37 can inhibit the infection of DENV-2 within a certain range of MOIs,and the effect is similar.The step-by-step analysis of the DENV-2 infection process by qRT-PCR showed that the scorpion venom peptide Ev37 had no effect on the adsorption and nucleic acid replication process of DENV-2 infection,and the infectivity of DENV-2 virus particles was not affected.Finally,it mainly inhibited the process of DENV-2 entering the host cell.Since DENV-2 mainly enters host cells through clathrin-mediated endocytosis,it is tested whether the scorpion venom peptide Ev37 has an effect on clathrin expression.The results showed that the scorpion venom peptide Ev37 had no effect on clathrin expression,indicating that scorpion venom peptide Ev37 inhibited the entry of DENV-2 into host cells not by affecting the expression of clathrin.Next,confocal technique was used to intuitively detect that the scorpion venom peptide Ev37 had no effect on the attachment of DENV-2.DENV-2 enters the host cell by first reaching the early endosomes by clathrin-mediated endocytosis,and then the fusion between the viral membrane and the endocytic membrane being occured in the late endosomes and releasing its genome to the cytoplasm under low pH conditions.For example,we obtained the N-terminal HA-tagged scorpion venom peptide Ev37-NHA by molecular cloning,prokaryotic expression and purification,and found that Ev37-N-HA peptide can enter cells and aggregate by confocal and can co-localize well with early and late endosomes.H3 is a known peptide that enters cells by endocytosis.The acidification of the organelles affects the endocytosis escape and distribution of H3 in the cytoplasm.For example,the acidification inhibitor Bafilomycin A1(BAF)can make distributed H3 peptide became aggregated in the cytoplasm.Then,the experimental results showed that scorpion venom peptide Ev37 could also aggregate the H3 peptide in the cytoplasm,and the effect was stronger than BAF.It indicates that peptide Ev37 is capable of affecting the acidification of organelles,like endosomes.It was then found that the peptide Ev37 could increase the colocalization of DENV-2 envelope protein E with the late endosome marker Rab7,suggesting that the peptide Ev37 may inhibit DENV-2 by inhibiting endocytic acidification,which blocks viral particles in late endosomes.Peptide Ev37 and BAF could shift the fluorescence value to the left compared with the control by flow cytometry with acid-sensitive dye Lysosensor Green,confirming that peptide Ev37 inhibited acidification of intracellular acidic organelles.Further,it was found that both peptide Ev37 and acidification inhibitor BAF can increase the volume of acidic organelles and have no effect on nonacidic organelles by confocal.It is once again proved that peptide Ev37 inhibits acidification of the acidic organelles,thereby blocking the fusion of the viral membrane with the endocytic membrane and release of DENV-2 genome into the cytoplasm,and finally suppresses DENV-2 infection.Then,the antiviral activity of the peptide Ev37 against other viruses was examined to detect its broad spectrum and specificity of antiviral function,such as HCV,ZIKV,herpes simplex virus type 1(HSV-1),Sendai virus(SeV)and adenovirus(Ad V).The broad spectrum of antiviral activity of peptide Ev37 is reflected that peptide Ev37 can inhibit the infection of HCV,ZIKV,HSV-1 and other viruses the way enter the cells similar to the DENV-2(through a low pH-dependent membrane fusion pathway),although HSV-1 is DNA virus rather than a positive-strand RNA virus.The antiviral specificity of peptide Ev37 is that it can not inhibit SeV and Ad V whose entry way differs from DENV-2 in a concentration-dependent pathway,and the acidification inhibitor BAF has a similar conclusion with peptide Ev37.Once again,it was demonstrated that peptide Ev37,which is both broad-spectrum and specific,inhibited viral infection by inhibiting endosome acidification.Finally,the antiviral activity of peptide Ev37 was detected in vivo in the A129 mouse model with type I interferon receptor knockdown that ZIKV can infect.As a result,peptide Ev37 can reduce ZIKV virus titer in the blood,liver,spleen,kidney and brain of infected mice.It indicates that peptide Ev37 still has antiviral activity in vivo.In summary,this paper first expressed the recombinant peptide Ev37 derived from Euscorpiops validus by prokaryotic expression,and found that it is different from most scorpion venom peptides in antiviral mode when studied its antiviral function.Instead of directly action on virus,peptide Ev37 inhibits the infection of DENV-2,HCV,ZIKV,HSV-1 which release their genome into host cells by membrane fusion process in low pH-dependent way via inhibiting endosome acidification.There is no inhibitory activity against SeV whose membrane fusion ocurred under neutral pH condition and Ad V which enters the cytoplasm by destroying the endosome membrane by self-protein.These studies provide new strategies and ideas for the prevention and control of DENV-2,HCV,ZIKV,HSV-1 and other viruses which enter the cells by low pH-dependent membrane fusion.