Screening Of Host Proteins That Interact With CVS-M Protein Using Yeast Two-hybrid System

Rabies is an acute zoonotic disease caused by the rabies virus.Rabies virus belongs to therabies virus genus, Rhabdoviridae family. It is a kind of neurotropic virus, nearly100%mortality after infection. Matrix protein is an important part of the rabies virus. The mainfunction is involved in virus assembly and budding, inducing apoptosis, inhibition geneexpression and so on. Those functions show that it may play an important role in the infectioncycle of the rabies virus. But the molecular mechanism of the interaction with the host proteinwas unknown. Yeast two-hybrid system is an important tool for researching protein interactionsand functions and screening new proteins. In order to study the interaction between the hostprotein and matrix protein, we first get CVS11-M gene by PCR. Then construct a yeastexpression vector pGBKT7-CVS11-M, translated the recombinant plasmid and the human braincDNA library into Y2HGold yeast competent cells. Positive clones were screened by nutritionaldeficiencies culture. Positive clones were further identified by restriction enzyme digestion andyeast two-hybrid cotransformation and so on. Using BLAST to analysis the homology of thepositive clones, finally find that there are four kinds of protein interact with the M protein. Theyare Homo sapiens prolyl4-hydroxylase, alpha polypeptide II (P4HA2)， Homo sapiensreticulocalbin1, EF-hand calcium binding domain (RCN1)，Homo sapiens TGF-beta activatedkinase1/MAP3K7binding protein2(TAB2)and Homo sapiens HCLS1associated protein X-1(HAX1)。HAX-1is a multifunctional protein relative with cell apoptosis, cell migration and calciumhomeostasis. Its structure is similar with the Bcl-2family, contain BH1, BH2domains. It isassociated with liver disease, cancer, systemic lupus erythematosus, avian flu and many otherdiseases. HAX-1gene has homology domain with Bcl-2families. This may explain why itsfunction is closely related with apoptosis. When the HAX-1over express in human Tlymphocytes, the cell were able to induce apoptosis cased byγ-ray and serumdeprivation-induced stimulation. When over expressed in HeLa cells, it inhibited the express ofpro-apoptotic protein Bax. Apoptosis has been the focus of life science research. Research onHAX-1is increasing. However, what role of HAX-1play in rabies virus infection has not beenreported. We chose HAX-1protein for further research. Using GST pull-down andco-immunoprecipitation technology to verified the interactions of the two proteins both in vitro and in vivo. The study of HAX-1protein interaction with CVS11-M is to explore the newfeatures of the M protein at the molecular level. The result also provides new ideas anddirection for further research of NF-κB pathway. In addition, it provides further help to find amethod for treating rabies.