Identification Of Host Proteins Interacting With Rabies Virus M Protein By Yeast Two-hybrid System

Rabies is a kind of epidemic disease since its seriously endangers in public health and safety.Rabies is a type of lethal neurological disease caused by rabies virus(RABV).From the initial knowledge and development of rabies,it has affected humans for several thousands years and it is one of the oldest zoonotic diseases known.However,through the long-term researches on RABV by researchers,many achievements have been made so far.The RABV is composed of five proteins: N,P,M,G,and L respectively.The five proteins have different functions,but these proteins can collaboratively exert their functions together.Among them,the M protein is the smallest one containing 20 k Da of molecular weight.M protein plays a major role in the morphological maintenance,transcription and replication.The previous research results of our group showed that the amino acids at positions 44 and 46 of M protein were substituted by the corresponding amino acids of virulent GX01 isolate induced a great influence on the replication and transcription of RABV.Therefore,we thought that the amino acids at position 44 and 46 of M protein have greatly research value and decided to further screen its interacting proteins in the host.In this study,yeast two-hybrid system was used to screen the proteins which interact with the M protein of RABV(rRC-HL-M(F44L/S46G)).The yeast two-hybrid system become a widely tool for studying protein-protein interaction because of its simple principle,sensitivity to protein-protein interaction,high efficiency,and high degree of simulation etc.Through molecular cloning and primers designing,the M gene was amplified by PCR.Then the M gene was ligated to p MD18-T plasmid to construct a p MD18-T-M vector.The p MD18-T-M vector was extracted and purified,and then the p MD18-T-M vector was identified by double enzymes digestion,the digested product was ligated with the vector p GBKT7(BD)used in the yeast two-hybrid system with T4 ligase to construct a successful bait expression vector BD-M.The bait protein was then co-cultured with the genetic hybrid library(Mate &Plate ? Library-Mouse Brain(Normalized))and screened by auxotrophic medium and drug,and identified by blue and white spots.The blue positive clones were picked out for sequencing.BLAST sequences with NCBI library were used.Finally,three interacting proteins: Lamtor5,GTF2E2 and PIAS1 were obtained.Since all three are host cellular proteins,their expression can be detected directly in the host cells.NA cells were infected with RABV.Then the NA cells samples were collected and prepared to cells’ lysates.Using Western Blot and real-time quantitative PCR,the changes of m RNA level and the protein level of Lamtor5,GTF2E2 and PIAS1 in the NA cells infected with rRC-HL and rRC-HL(M44/46)were detected respectively during 24,48 and 72 hours post-infection.The results showed that the m RNA level of Lamtor5 and GTF2E2 genes has slightly increased and their protein levels were similarly identified slight up-regulated.However,The m RNA level and protein-expressing level of PIAS1 gene showed a slightly down-regulation.These results were consistence with that in the mouse brain.In this study,three host cellular proteins interacting with M protein of RABV were screened.PIAS1 is functions as a SUMO E3 ligase,relating to the antiviral signaling pathway such as STAT and NF-κB in innate immunity.The Pias1 is functions as a key down-stream regulator of the antiviral signaling pathway in vivo during RABV invasion and participates in the body’s antiviral defense.These results have important practical significance for further exploring the structural and functional correlation of M protein.