Screening And Identification Of Host Protein That Interacts With Foamy Virus Bet Protein

Foamy viruses are the only members of the Spumavirus genus,Spumaretroviruses,one of two families of the Retroviridae.Simian foamy viruses(SFV)are highly prevalent in all nonhuman primate species and can infect humans following exposure to infected animals and their tissues,blood or body fluids.PFV in humans currently have no obvious pathological damage to human and the transmission from human to human can not be achieved,while PFV infection of animal cells in vitro can cause cytopathic effec.In addition to the characteristics of the infection,the life cycle and regulation of gene expression of foamy virus are also very different from other retroviruses.Bet,one of PFV accessory proteins tend to be expressed at the early stage of viral infection with high level,while its function is unclear.Researches showed that Bet reduced transactivity of Tas by inhibiting the IP activity,thereby inhibiting viral replication;foamy virus tend to express Bet during chronic infection,suggesting that the Bet play an important role in the establishment of chronic infections.In addition,Bet can interact with cellular defense factors APOBEC3 resulting in preventing the latter's packaging to protect the replication of PFV.To further study on Bet,especially its interaction with the host cell factors,yeast two-hybrid screening of Bet and human universal cDNA library were performed.Co-immunoprecipitation experiments were conducted to confirm the candidate positive interactions between SETDB1 and Bet,as well as that between ZNF350 and Bet.Moreover,the impacts of SETDB1 and ZNF350 on foamy virus replication were detected.On the other hand,shRNA vectors targeting on SETDB1 and ZNF350 were contrasted,respectively.In detail,experiments were performed as follows:1.Screening of host proteins interacting with Bet by yeast two-hybrid system.Recombinant plasmid pGBKT7-Bet was successfully constructed and transfected into yeast strain Y2H.Bet protein was successfully detected with Myc-tagged antibody by Western Blot after obtaining high level of protein samples.The growth of Y2H[pGBKT7-Bet]on selective medium indicated that Bet can not autoactivate promoters and is non-toxic as a bait protein.Two-hybrid yeast mating between Y2H[pGBKT7-Bet]and universal cDNA library were performed,followed by screening on lower stringency as well as high stringency selective medium,result in that 44 candidate positive clones were obtained.After sequencing and searching in NCBI,13 candidate positive protein were identified.And then the confirmation of positive interactions were finished by specific turning yeast mating,which showed that all the 13 candidates were genuine positive.Among them,we selected SETDB1 and ZNF350 to be further studied.2.The interaction of SETDB1 and Bet in 293T cells.The respective expression vectors of Bet and SETDB1 were co-transfected into 293T cells for 48h,followed by co-immunoprecipitation with Myc-tagged antibody.Cell lysates and Co-IP product were separately detected by Western Blot with Myc-tagged and His-tagged antibodies.The results showed that the SETDB1 interaction with Bet in 293T cells.The expression vector of SETDB1 and pHSRV13 plasmid containing the full-length PFV genome were co-transfected into 293T cells for 48 h,followed by co-cultured with the indicator cell line LTR-Luc,to detect viral titers by measuring luciferase activities.The results indicated that overexpression of SETDB1 inhibits foamy virus replication.3.The interaction of ZNF350 and Bet in 293T cells.The respective expression vectors of Bet and ZNF350 were co-transfected into 293T cells for 48h,followed by co-immunoprecipitation with Myc-tagged antibody.Cell lysates and Co-IP product were separately detected by Western Blot with Myc-tagged and His-tagged antibodies.The results showed that the ZNF350 interaction with Bet in 293T cells.The expression vector of ZNF350 and pHSRV 13 plasmid containing the full-length PFV genome were co-transfected into 293T cells for 48h,followed by co-cultured with the indicator cell line LTR-Luc,to detect viral titers by measuring luciferase activities.The results indicated that overexpression of ZNF350 inhibits foamy virus replication.4.Construction of shRNA vectors targeting on SETDB1 and ZNF350,respectively.Three pairs of siRNA sequences were designed by on-line analysis software,which could specifically inhibit the expression of SETDB1 and ZNF350.Then the constructed double strain shRNA were cloned into the pLsunny plasmid which has the human U6 promoter.To sum up,we identified 13 kinds host proteins interacting with PFV Bet using yeast two-hybrid system,and then confirmed the interaction between SETDB1 and Bet as well as ZNF350 and Bet by co-immunoprecipitation.On this basis,we explored that overexpression of SETDB1 and ZNF350 inhibited PFV replication,respectively.Moreover,shRNA expression vector was constructed to provide a basis for further functional studies.Meanwhile,in-depth analysis of the research results suggested that Bet may play important roles in PFV replication,in multiple ways.