Interactions Of The MCMV Neurotropic Proteins M122 And M112-113 With Host Protiens From Brain

Objectives:To investigate the biological functions of M122 and M112-113 proteins of murine cytomegalovirus (MCMV) in pathogenesis of developmental brain disorders and brain damage caused by MCMV, the screening for mouse brain cDNA library was performed by a yeast two-hybrid system for looking for the potential mouse brain proteins interacting with M122 or M112-113 proteins.Methods:1) Constructions of bait plasmid containing MCMV M122 or M112-113 gene:The DNA fragment of coding M122 or M112-113 protein was amplified by RT-PCR, and then inserted into pMD18-T simple vector. After verified with restriction endonuclease digestion of EcoRI and SaiI, the right fragments of M122 and M112-113 genes determined by sequencing were subcloned into pGBKT7-BD vector. Then recombinant plasmid pGBKT7-M122 and pGBKT7-M112-113 were verified by both the same endonuclease and sequence analysis.2) The test of bait plasmids for autoactivation and toxicity:The right fragment of recombinant plasmid pGBKT7-M122 and pGBKT7-M112-113 were independently transformed into the yeast cell AH 109 by small-scale lithium acetate method. The transformed yeast cells were plated on nutrient deficiency medium SD/-Trp and SD/-Trp containing X-α-gal, and the color, number and size of colonies on the plates were observed.3) Verification of fusion protein expression:The expression of pGBKT7-M122 and pGBKT7-M112-113 fusion proteins in yeast cells were confirmed by western blot analysis.4) Yeast two-hybrid screening:The yeast strain AH109 containing pGBKT7-M122 or pGBKT7-M112-113 was mated with the yeast strain Y187 containing mouse brain cDNA library, respectively. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade or SD/-Trp/-Leu/-His for screening the positive clones.5) Isolation of plasmid DNA from putatively positive yeast clones and bioinformatic analysis:Positive yeast colonies were harvested and repeatedly streaked for 3 times on SD/-Trp/-Leu/-His/-Ade plates containing X-a-gal to separate the positive interactors from non-interactors. The plasmids were isolated from blue yeast colonies, and transformed into E. coli strain JM109. The plasmids were then isolated from E. coli, and subjected to sequencing and analysis by BLAST on the NCBI GenBank database.6) Verification of the interactions between candidate proteins and M122 or M112-113 protein in yeast:Each candidated prey plasmid and bait plasmid were co-transformed into yeast strain AH109 one for one to verify the interactions of candidated prey proteins with M122 or M112-113. The candidated prey proteins were tested for autoactivations by co-transforming into AH109 yeast with an empty pGBKT7-BD, respectively.7) Confirmation of the interactions between candidate proteins and M122 protein in mammalian cells:To test whether M122 protein of murine cytomegalovirus can bind to Stx8, Atp1b3, Kcnab1, Ankrd17 or Myst4 in mammalian cells capable of supporting MCMV infection, chemiluminescent co-immunoprecipitation experiment was performed.8) Mapping of the M122 domains involved in the interaction with Stx8, Atp1b3, Kcnab1, Ankrd17 or Myst4:Seven Plasmids encoding various truncated M122 were constructed and were transformed respectively into yeast strain AH109 together with candidate plasmids PGADT7-Stx8, pGADT7-Atp1b3, pGADT7-Kcnab1, pGADT7-Ankrd17 or pGADT7-Myst4. The M122 domains involved in the interaction were identified by auxotrophic selection.Results:1) The reconstructed bait plasmids pGBKT7-M122 and pGBKT7-M112-113 were successfully constructed. The size of the inserted DNA fragments in pGBKT7-BD vector were correct. The sequencing results of recombinant bait plasmids were compared with MCMV M122 and M112-113 sequence by BLAST assay, which showed that they had the homology in nucleotide sequences.2) The reconstructed bait plasmids were transformed into yeast cells AH109 successfully. They had no autoactivation and toxicity for yeast strain AH109.3) The reconstructed bait plasmids pGBKT7-M122 and pGBKT7-M112-113 could stably express their proteins in the yeast cells AH 109.4) 24 proteins interacting with MCMV M122 were screened, and three of them were unknown proteins.3 proteins interacting with M112-113 were obtained.5) Interaction of candidate proteins and M122 or M112-113 in yeast was further confirmed by using yeast-hybrid assay. But the autoactivation of Aplg1, Cul1 and Pias2 candidate proteins were observed.6) This interactions of M122 with Stx8, Atp1b3, Kcnab1, Ankrd17 and Myst4 were verified by chemiluminescent co-immunoprecipitaion assay.7) Mapping analysis suggested that the 1-148 residues of M122 are responsible for the interactions of M122 with Stx8, Atp1b3, Kcnab1, Ankrd17 and Myst4.Conclusions:1) The recombinated bait plasmids pGBKT7-M122 and pGBKT7-M112-113 had no autoactivation and toxicity and were proved to be suitable for the further research on the interactors with M122 and M112-113 in the yeast two-hybrid sestem.2) A class of proteins in mouse brain interacting with M122 or M112-113 had been obtained, providing the clues for clarifying the roles of M122 or M112-113 in neuropathogenesis of the brain disorders caused by MCMV.3) This interactions of M122 with Stx8, Atp1b3, Kcnab1, Ankrd17 and Myst4 were verified, and the binding site was mapped to the 1-148 residues within M122. It provides an experimental basis for further studying the actions of M122 in molecular neuropathogenesis of the brain disorders caused by MCMV.