Chlorovirus E3 Ligase-mediated Viral Host Interaction

In eukaryotic cells,protein synthesis and degradation are important means to maintain normal cell function.Degradation of intracellular proteins is mainly accomplished by the ubiquitin-proteasome system,which can quickly convert dysfunctional proteins in cells and short-term The degradation of regulatory proteins that control cell life activity plays an important role in the regulation of a variety of cellular functions.Because ubiquitin ligase E3 has specificity for the recognition of substrate proteins and the function of transferring ubiquitin molecules to substrates,it has become the most complex family of enzymes in this system,and SCF(Skp1-Cul1-F-Box The complex is the largest and most studied group.The chlorella virus model strain PBCV-1(Paramecium bursaria chlorella virus-1)is the first full-length sequencing member of the algae DNA virus family.Genome sequencing showed that it encodes both FBox and Skp1 proteins,and its host is small.Chlorella variablis genome has also been sequenced in full length,so the host infection process of chlorella virus is an important experimental system for studying virus-host protein interaction.This study uses yeast two-hybrid technology and mammalian cell heterologous expression methods to study virus-host protein interactions,and uses pull-down technology to screen host cell proteins that interact with the virus Skp1 in order to better understand chlorella Viral E3 ligase-mediated viral host interaction mechanism.This study not only provides an important theoretical basis for elucidating the protein-protein interaction between the E3 component of the chlorella virus and the host,but also provides the possibility for establishing a virus-cell mixed SCF complex dominated by viruses,thereby further improving it.Ubiquitin-Proteasome Pathway for Protein Degradation in Eukaryotic Cells.In this study,yeast codon optimization was performed using five F-Box protein genes provided by the laboratory.The optimized fragment was used as a template for full-length gene amplification and inserted into the pGBKT7 vector to construct the pGBKT7-F-Box bait plasmid.The same method was used to insert two Skp1 protein genes into the pGADT7 vector to construct the pGADT7-Skp1 prey plasmid.The Gold Yest Two-Hybrid System was used to transfer the bait plasmid into Y2 HGold yeast for toxicity and self-activation verification tests.The results showed that the insertion of F-Box fragments had no toxicity or self-activation effect on yeast growth.The two plasmids obtained were co-transformed into Y2 HGold yeast for detection of AbAr,HIS3,and MEL1 reporter genes,and transformed into Y187 yeast for detection of Lacz reporter gene.The results showed that five F-Box proteins interacted with two Skp1 proteins,and the effects were different.This experiment uses mammalian cell heterologous expression methods to continue the study of the interaction between the virus Skp1 and the host SCF components.An attempt was made to establish a complete model of virus-cell mixed SCF complex that may be dominated by viruses.The virus Skp1 was selected.Human Skp1 was fused with GST-tagged protein through gene synthesis.Virus F-box A682 L and human F-box Skp2 were fused with Flagtagged protein through gene synthesis and inserted into eukaryotic expression vector pcDNA3.1(-),Co-transfection of two Skp1 with different F-box into 293 T cells respectively,SDS-PAGE and Western blot detection of the target protein obtained by magnetic bead immunoprecipitation technology,the results showed that two Skp1 proteins and 2 There are interactions among all F-box proteins.Provides the possibility to establish a virus-cell mixed SCF complex with viral participation.In our laboratory,the Skp1 protein gene fragment of PBCV-1 was inserted into the expression vector pEGX-6P-1 to express a soluble protein,which was bound to Sepharose 4B resin and Pulldown with the infected chlorella NC64 A cell lysate In the experiment,the results of silver staining were analyzed by mass spectrometry.The results showed that 19 kinds of affinity proteins related to chlorella NC64 A were screened.The results of this study are helpful for the laboratory to further construct a virus-cell mixed SCF ubiquitin ligase complex model,and also provide a theoretical basis for mining the protein interaction between chlorella virus and host E3 components and its potential functions.