Cell-penetrating Peptide TAT Mediated Anti-Porcine Reproductive And Respiratory Syndrome Virus Nsp9 Nanobody Nb6 Inhibition Of Viral Infection

Porcine reproductive and respiratory syndrome?PRRS?is a swine viral infectious disease characterized with reproductive and respiratory diseases,and is also one of the most important infectious diseases that causes significantly economic losses to the global swine industry.The disease is caused by porcine reproductive and respiratory syndrome virus?PRRSV?.The high frequency mutation,immunosuppression,persistent infection and antibody-dependent enhancement of PRRSV are the main factors that make the uneffective vaccination.In addition,no antiviral agent is currently available.PRRSV non-structural protein Nsp9 is an RNA-dependent RNA polymerase.Nsp9 gene is relatively conserved among different strains,which is considered to be a target for anti-viral drugs.In previous studies,our laboratory has successfully produced a nanobody,Nb6,specifically targeting Nsp9 and demonstrated that intracellularly expressed Nb6 can significantly inhibit the replication of PRRSV in M ARC-145 cells.However,the limitation of Nb6 enters the cells prevents its application to inhibit the viral infection inside the cells.Therefore,establishment of a safe,efficient,and nontoxic delivery system is critical to improve the application of Nb6to inhibit PRRSV infection of the cells.Cell penetrating peptides?CPPs?,short peptides containing about 5-30 amino acids,can enter cells via direct translocation or endocytosis without causing cytotoxicity.Currently,they have been used as tools for the delivery of various cargoes into cells,such as plasmid DNA,siRNA,proteins,viruses,imaging agents,and various other nanoparticles.A trans-activating transduction?TAT?peptide is a human immunodeficiency virus type 1?HIV-1?transactivator.Recently,a TAT-fused nanobody was reported shown to have significant anticancer effects and is expected to be a new approach for development of anticancer drug.However,no reports has been seen on the application of CPPs in PRRSV prevention and control.The objectives of this study were to generate TAT-Nb6 fusion protein,determine its activities to penetrate cell membrane and inhibit PRRSV infect ion of the cells,and understand its antiviral mechanism,which may provide a novel strategy to control PRRSV infection.The main works and results of this study were as follows:1.Detection of cellular uptake of TAT-Nb6TAT-Nb6 fusion protein was expressed using prokaryotic expression system.The gene of TAT-Nb6 was amplified by Overlap PCR,and cloned into pET-21b vector to construct the recombinant plasmid pET-21b-TAT-Nb6.TAT-Nb6 fusion protein,existed in the form of inclusion body,was expressed in BL21?DE3?cells transformed with pET-21b-TAT-Nb6plasmid after induced with IPTG,followed by purification using a Ni-NTA affinity chromatography column,renature and concentration.The interaction of TAT-Nb6 and PRRSV Nsp9 recombinant protein was detected using ELISA,and the result showed that TAT-Nb6 maintained strong binding ability to Nsp9.After incubated with MARC-145 cells and PAM s,TAT-Nb6 in both cells was detected by Western blot,indirect immunofluorescence and flow cytometry.The results showed that TAT delivered Nb6 into both cells in a time-and dose-dependent manner.Cell viability data showed that TAT-Nb6exhibited no toxicity at concentration up to 30?M.Furthermore,we demonstrated that TAT could deliver Nb6 into cultured cells as well as pig organs in vivo.These results indicated that the TAT-Nb6 fusion protein,expressed in E.coli,has the abilities to interact with the Nsp9 recombinant protein and to enter cells.2.Determine the antivial activity of TAT-Nb6Using IFA and pulldown assay,we proved that TAT-Nb6 could interact with PRRSV-encoded Nsp9.Then anti-PRRSV activity of TAT-Nb6 were analyzed.The results show that TAT-Nb6inhibited the replication of multiple strains of PRRSV,including genotyp e 1?GZ11-G1?and genotype 2?SD16,JX-A1,GD-HD,VR-2332?strains,on MARC-145 cell lines or primary PAM s in a dose-dependent manner.However,the suppression efficiencies by TAT-Nb6 were different among the tested PRRSV strains.In order to identify the binding regions within Nsp9 involved in the Nsp9-Nb6 interaction,yeast two-hybrid was performed.The results showed that the Nb6 could recognize two discontinuous regions in the C-terminus end of Nsp9,Nsp9_aa454-551 and Nsp9_aa599-646.Further analysis of the amino acid homology of Nsp9_aa454-646 revealed that the strains among genotype 1 or genotype 2 were highly conserved and the sequence identity reached 92.2%-100%and 92.7%-100%,respectively.Whereas,the sequence similarity between genotype 1 and 2 was only about 77.7-81.3%.In summary,TAT-Nb6 fusion protein could enter the cells and inhibit the replication of various PRRSV strains in M ARC-145 cells and PAM s,which indicated that TAT-Nb6 has the potential to be developed as a new anti-PRRSV drug.In addition,and the antiviral mechanism of Nb6 has also been elucidated.