Mapping Of The Proteins Interacting Domains Of Chicken Anemia Virus

As one of the immunosuppressive diseases, Chicken infectious anemia (CIA) is caused by chicken anemia virus (CAV) and characterized by anemia in 2~3 weeks age chickens. The immunosuppression caused by CAV leads a higher susceptibility to IBD, MD and ND in young chickens.CAV is comprised of 3 proteins, VP1 is the structural protein, while VP2 and VP3 are the nonstructural ones. The protein-protein interactions and the complicated networks are the key factors replication and pathogenicity of this virus. Therefore conducting on the researches of the interactions of CAV proteins will benefit to further understand the biological functions of the viral proteins and the replication process. In this study, the technologies of studying the protein-protein interactions, such as the yeast two-hybrid system (H2Y), coimmunoprecipitation (Co-IP) and confocal assays were applied to investigate the potential interactions of the three viral proteins. The results were shown as follows: The rabbit antiserums against the three viral proteins were firstly prepared to meet the needs of antibodies of different species used in the Co-IP and confocal assays. The 962 bp VP1 gene and the whole of VP2 and VP3 genes were amplified by PCR from the genome DNA of CAV strain M9905 and cloned into expression vectors pET28a or pET30a respectively. After identified by PCR amplification, restriction endonucleases digestion and sequencing, the recombinant pET28a-VP1 (962bp), pET28a-VP2 and pET30a-VP3 were obtained. The expressions in E.coli DE3 were induced by IPTG. Proteins were immunized rabbits to prepare the antibodies. Anti-VP1, VP2 and VP3 serums were tested for reactivity in indirect ELISA, IFA and Western blot. The indirect ELISA titers were high, and the antiserum respectively reacted well with the recombinat proteins VP1, VP2 and VP3 through IFA and Western blot assays.To characterize the proteins physical interaction (s) of the virus, firstly, the ProQuest GAL 4 two-hybird system was used."Bait"vectors and"Prey"vectors were constructed and cotransformed into yeast competent cells. The interaction between these two fusion proteins was measured by the reconstitution of a functional transcriptional activator that triggers the expression of reporter genes (His, LacZ and URA). Two positive colonies were screened. They are pDEST32-VP2+pDEST22-VP1 and pDEST32-VP22+pDEST22-VP3, so we verified the interactions between VP1 and VP2, and demonstrated firstly the interaction of VP2 and VP3. Further the specific interaction between VP1 and VP2 was confirmed by coimmunoprecipitation and confocal assays, also did VP2 and VP3.To identify the interaction domains of the proteins after detecting the interaction components, we furthermore constructed the deletion mutations vectors of VP1, VP2 and VP3 according to the hydrophilicity, antigenicity and surface probability. Through yeast two-hybrid system screen, the results showed that the deletion of the N-terminal residue of VP1 gene(59, 86, 117 and 167 aa)did not prevent formation of the VP1-VP2 complexes; the C-terminal deletion of 36 and 95 aa also had no effect on the interaction to VP2, so the interaction domain located in 168~354 aa of VP1; VP2 N-terminal deletion of 30 aa could also interact with the total VP1, but not its C-terminal trunctions, so the interaction domain located in 31~217 aa of VP2. Carboxy-terminal domain of VP3 (truncated of the NES) was sufficient to bind VP2, so the interaction domain of VP2 and VP3 located in 46~122 aa of VP3. VP2 C-terminal deletion of 17 aa did not contribute the formation of this complexes, so the domain located in 1~199 aa of VP2.