Comparative Analysis Of Structure And Enzyme Activity Verification In Vitro Of Thymidine Kinase Encoded By MDV And Other Alpha Herpesviruses

Marek’s Disease(MD)induced by Marek’s Disease Virus(MDV)is characterized by severe immunosuppression,neurological symptoms,and rapid onset lymphoma.MD can be used as a biomedical model of a natural tumor.MDV belongs to the Herpesviridae,a member of the alpha herpesvirus subfamily,and has a genomic structure similar to that of the other alpha herpesvirus subtypes,such as herpes simplex virus(HSV).In α-herpesviruses,the UL23 gene encoded thymidine kinase(TK)is a non-essential protein for replication in vitro but essential for virulence in vivo.Importantly,viral TK gene usually used as a therapeutic drug target for anti-herpesvirus infection and as a suicide gene for anti-tumor therapy,as well as an important site for genetically engineered vaccine development.As a homolog of HSV/TK,the function of MDV/TK remains unknown.In this project,the following studies were carried out to investigate the structure and function of MDV/TK:1.Structural prediction analysis of MDV/TK proteinThe amino acid sequences and tertiary structures of MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK were compared by Meg Align and Pymol softwares,respectively.Then,the tertiary structures of TK proteins were docked with interacted substrates of nucleosides and their analogues using Autodock software.Next,the five TK activity pockets were predicted using POCASA online software.The results showed that the amino acid sequence homology of these five TKs was low,but the spatial conformations were very similar.Especially,the enzymatic function relevant amino acid sites were highly conserved.The five TKs had intermolecular interactions with the substrate molecules thymidine nucleoside(TR)and acyclovir(ACV),and the action sites were also highly conserved.In addition,the comparative analysis with the activity pockets revealed that MDV/TK had potential active pocket that potentially wraps the substrate molecule of TR.Thus,we hypothesize that MDV/TK may perform similar biological functions as HSV/TK and PRV/TK.2.Expression and purification of MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK proteinsTo verify whether all five TKs possessing enzymatic activity,the expression of MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK proteins were induced in E.coli by constructing prokaryotic expression plasmids,respectively.After optimizing the expression temperature and IPTG concentration,SDS-PAGE results showed that MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK proteins were soluble expressed in the supernatant of lysed cells.After purification by GST-tag column and molecular sieve,the five high-purity TK proteins were successfully obtained.This provides the material basis for verifying their enzymatic activities in vitro.3.In vitro enzymatic activity validation of MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK proteinsTo further validate the enzymatic activities of the five TKs,the catalytic reaction conditions were first confirmed.Then,the MDV-1/TK,MDV-2/TK,HVT/TK,HSV-1/TK and PRV/TK proteins were added to the systems with ATP and TR as substrates and reacted at 37°C for 1 h,respectively.The catalytic reaction products were detected using high performance liquid chromatography(HPLC).The results showed that all five protein catalytic reactions produced ADP and d TMP.ACV was then used as a substrate to in the same reaction conditions as the TR substrate molecule.The catalytic reaction products were ADP and ACV-MP detected by HPLC.These results indicate that MDV-1/TK,MDV-2/TK and HVT/TK all exert thymidine kinase activity and are capable of catalyzing phosphorylation of nucleosides and their analogs,which is consistent with the HSV-1/TK and PRV/TK.In summary,we revealed the similarity of tertiary structures and enzymatic functional regions between MDV/TK and HSV-1/TK and PRV/TK through comparative structural and functional analysis.We further validated the catalytic activity of MDV/TK as a thymidine kinase in vitro.These results provide basis to further explore its role and mechanism in viral pathogenesis and vaccine development,and also provides a good basis for the construction of viral and tumor models using TK as a target in α herpesviruses.