Study Of The Entry Mechanism Of Protein Transduction Domain Originated From Nervous Necrosis Virus

Viral nervous necrosis(VNN)is a disease that great threat to the marine aquaculture industry,especially larvae and juveniles.VNN causes high mortalities(80 %-100 %)and has been responsible for heavy financial losses.The causal agent of the VNN is the nervous necrosis virus(NNV),which is an non-enveloped RNA virus.In recent years,several kinds of treatments against VNN have been developed,including immune therapy drugs such as recombinant protein and DNA vaccine,or anti-NNV biological macromolecules such as siRNA and antiviral peptide.However,the therapeutic effects of these drugs have been weakened by the bad delivery,especially the cytomembrane barrier when the drugs are delivered in vivo.Thus it is weathy to study how to make these drugs cross the cell membrane barrier,reach the target,and exert the antiviral effect on effective concentration.Previous study shown that a short peptide containing 14 amino acids was found to be protein transduction domain(PTD),which locates in N-domain of virus-like particle(VLP)of orange-spotted grouper nervous necrosis virus(OGNNV).This short peptide is named as NNV-PTD.NNV-PTD was fused with exogenous protein(GFP),expressed prokaryotically,and found to facilitate GFP enter Asian sea bass cell(SB).In this study,NNV-PTD was determined as not a key factor affecting the formation of OGNNV-VLP spatial structure,as CP can still form VLP spatial structure and VLP can still enter SB cell when NNV-PTD was deletion from CP or translocated from N-domain to C-domain.Next,we further studied the entry mechanism of NNV-PTD and found that NNV-PTD entered SB cell via endocytosis pathway,which can be demonstrated by the fact that NNV-PTD can not enter SB cell when the temperature is reduced to 4°C.Furthermore,a number of chemical inhibitors were used in the entry experiments and it was confirmed that NNV-PTD enter SB cell via clathrin-mediated endocytosis(CME)and micropinocytosis in a cytoskeleton-,pH-,dynamin-,lipid-,cholesterol-dependent manner.Meanwhile,the formation of micropinocytosis is myosin ?-dependent.Interestingly,NNV-PTD enters SB cell via CME and micropinocytosis,which is different from that of OGNNV-VLP.Since VLP was confirmed to enter SB cell via CME but not dependent of micropinocytosis.By cell line screening using entry experiment,we found that,besides SB cell line,NNV-PTD can enter many cell lines,such as insect cells S2 and SF9,fish cells GB,SSN-1 and MFF-1,and mammalian cell Hela.It illustrated that NNV-PTD,as a novel PTD with great application potential,constitutes a promising tool to deliver the antiviral macromolecules or drugs into various cells across the cell membrane barrier.At the same time,the study of NNV-PTD entry mechanism and dependent factors provide not only valuable basis for NNV-PTD transduction application,but also a new breakthrough for VNN prevention or therapy.