Identification And Preliminary Functional Studies Of CVS-P-interacting Cellular Proteins

Rabies is an important zoonosisaffecting the central nervous systemcaused byrabies virus (RABV), and the infection is almost always fatal. RABVbelongs to thegenus Lyssavirus in the family Rhabdoviridae and consists of a non-segmentednegative strand RNA genome. It encodes fivestructureproteins, nucleoprotein(N),phosphoprotein (P), matrix protein (M), glycoprotein(G), and polymerase (L),in theorder of3’-N-P-M-G-L-5’.The RABV P is a polymerase cofactor and plays anessential role in viral replication and transcription. P protein can interact with the hostproteins such as STAT1, STAT2and STAT3, leading to the inhibition of IFNsignaling.It makes the virus to escape from the host immune system and contribute tothe spread of virus in the host. In addition, the interaction between P and LC8mightcontribute to the retrograde axoplasmic transport of RABV in neurons. Therefore,LC8will be an important target protein to explore the pathogenic mechanism ofRABV. However, the molecular mechanism of the interactions between P and the hostproteins on the transcription and replication of RABV is still unknown.CVS is the challenge standard virus of RABV, and it is a more valuable target toresearch than others. Yeast two-hybrid system is an effective tool for the study ofprotein-protein interactions. In order to explore the novel host proteins that interactwith P, we first amplified the full-length of CVS-P gene by PCR and then cloned itinto pGBKT7vector to construct plasmid pGBKT7-CVS-P as a bait used in yeasttwo-hybrid screening. The recombinant bait plasmid was transformed into Y2HGoldcell to detect self-activation and toxicity.The transformants were plated on nutrientdeficiency medium after the Y2HGold yeast cells expressing P and the Y187yeastcells expressing the human brain cDNA library were mated. Twenty four positiveclones were identified as the candidate interacting proteins of CVS-P. Finally, twelvepositive clones were obtained after identified by PCR and yeast two-hybridco-transformation assay. The results of sequencing analysis showed that three positiveproteins were encoded by these positive clones, which were SNAP-associated protein(Snapin), zinc finger protein350(ZNF350) and COP9signal some subunit5(COPS5). Snapin is a multifunctional protein associated with synapses and involved inmany diseases, for examplepsittacosis, rheumatoid arthritis, Pakinson’s disease,prostate cancer and many viral diseases. Snapin exists in the cytoplasm and is animportant component of SNARE complex. It can regulate the pre-synaptichomeostasis and the transport of BRCE1in vivo. It can also modulate the cellulardistribution of human cytomegalovirus helicase and positively affect the replication ofHIV in host T cells.Although Snapin has so many functions, there was no research onthe role of Snapin in the pathogenesis of Rabies virus.So, we choose Snapin as the target protein for further research. The interactionwas further verified by GST pull-down and co-immunoprecipitation assays. Then weconfirmed the co-localization of the two proteins by double immunofluorescentstaining. Finally,we study the preliminary effects of Snapin on the replication ofRABV. The levels of mRNA and protein of Snapin in the brain of mice inoculatedwith RABV were both up-regulated. The levels of mRNA and protein of P in the cellsoverexpressing Snapin were also up-regulated. Our research verified the specificinteraction of P and Snapin for the first time. Snapin may promote the formation ofvial RNP and contribute to the replication and transcription of RABV throughup-regulating the P protein. Furthermore, Snapin is a vital protein that mediatessynaptic transmission, so it might promote the spread of virus particles via synapsesand increase viralpathogenicity. In the future, we will further explore the molecularmechanism underlying the fuctional interaction between RABV P and Snapin. It willprovide a new direction for prevention and treatment of RABV.