Study On The Structure And Functions Of An Antiflavivirus Peptide From Spider Alopecosa Nagpag

Spiders belong to the order arachnida in the phylum arthropoda,which originated from the pre-paleozoic devonian period and is currently one of the most diverse predator groups on land.As more and more spider venom components are discovered,it is recognized that spider venoms are one of the new tools for pharmacological research and disease treatment.Like many venomous animals such as Marine cone-snails,scorpions,and snakes,spider venom is a highly complex cocktail of toxins rich in salts,nucleotides,polyamines,polypeptides,free amino acids,neurotransmitters,proteins,and enzymes that target multiple receptors.This study focused on Alopecosa nagpag from Shangri-la,Yunnan province.This type of spider has been discovered for nearly 20 years,but its venom components have not been studied,let alone the antiviral active peptides.Although some animal-derived antiviral peptides are available,there is still a lack of in vivo toxicology studies,most of which have been screened only at the cellular level.Flavivirus is a single-stranded RNA virus transmitted mainly by the natural vectors Aedes aegypti and Aedes albopictus.The increasing geographical distribution of mosquito vectors and the lack of effective vector control methods,vaccines or antiviral treatments have resulted in the spread of flavivirus infection,placing a heavy health and economic burden on many tropical and subtropical areas.At the same time,the complex evolution of flavivirus and the lack of vaccination strategies have made it urgent to develop new antiviral drugs as an alternative to treating them.Dengue virus and zika virus belong to the flavivirus family,encoding three structural proteins and seven non-structural proteins.The complex of unstructured proteins NS2B and NS3 is necessary for the viral protease to remain active.In this study,we collected A nagpag spider venom,through a series of purification methods such as dextran gel chromatography and reversed-phase high performance liquid chromatography(HPLC)separation,we identified an Antiviral peptide,and according to the reasonable naming method named it Antiviral-Ly cotoxin-An la(Av-LCTX-An1a).Amino acid sequence of An la was determined by matrix-assisted laser analytical tandem time-of-flight mass spectrometry.This polypeptide is composed of 36 amino acids and contains 6 cysteines.The theoretical molecular weight of the first-order sequence is 4193.77 Da,which is 6 Da higher than the molecular weight of the natural peptide,indicating that three pairs of intramolecular disulfide bonds are formed in the natural peptide.After successful renaturation,it was found by cell proliferation and hemolytic activity detection that An1a had no cytotoxicity and hemolytic activity at concentrations less than 20 M,which could be used in subsequent experiments.Then,the anti-DENV2 and ZIKV activities of An la were verified in vitro on HUVEC and A549 cells by fluorescence quantitative nucleic acid amplification,plaques and immunofluorescence detection,and we established that 10μM Anla could inhibit 80%virus replication.We continued to explore the antiviral mechanism of An1a,and constructed and successfully expressed DENV and ZIKV NS2B-NS3 protease expression vectors,respectively.Real-time fluorescence inhibition experiment and Dixon analysis proved that Anla could inhibit NS2B-NS3 protein to inhibit DENV2 and ZIKV replication.As a competitive inhibitor of DENV NS2B-NS3 protease,the Ki value was 9.47±1.23 M.As a competitive inhibitor of ZIKV NS2B-NS3 protease,the Ki value was 12.54±1.88 M.This study revealed the interaction between Anla and the NS2B-NS3 active protease of DENV2 and ZIKV,indicating that An1a may be a candidate drug against flavivirus infection.Although antiviral peptides were first found in the venom of A.nagpag spiders,other components of the venom are still largely unknown.Considering the complexity ofA.nagpag spider venom,more work is needed to study its bioactive peptides.In summary,our findings not only confirm An1a as an anti-flavivirus candidate,but also suggest that spider venom is a potential resource rich in antiviral precursor molecules.