Effect Of Calpain-2 On Chikungunya Virus Replication And Its Mechanism

Background and ObjectiveChikungunya fever,a vector-borne disease transmitted by Aedes aegypti and Aedes albopictusis,is caused by Chikungunya virus infection.As early as 1987,China reported the first case of Chikungunya fever.To date,it has been estimated that 39%of the world’s population lives in countries and regions,where CHIKV is endemic,at risk of infection.With the changes in the ecological environment and climatic conditions,Chikungunya virus has been spreading faster and faster worldwide,likely to threat to more new countries and regions,when the trend of global warming has become more pronounced,the activities of Aedes aegypti and Aedes albopictus have gradually expanded,and international economic,trade,and cultural exchanges have become more frequent.However,the cellular receptors of Chikungunya virus have been not clear at present,and the pathogenic mechanism of joint symptoms caused by Chikungunya virus infection remains unknown.At the same time,there are no specific drugs and vaccines for Chikungunya fever.Therefore,this study is planned to analyze the expression and activity changes of calpain-2 protein after Chikungunya virus infection,and determine whether calpain-2 protein affects the replication of Chikungunya virus and then explore its specific role.The mechanism is expected to lay a theoretical foundation for exploring the pathogenic mechanism of Chikungunya virus and provide scientific basis for prevention and control of Chikungunya fever.Methods1.Cells and mice were infected with Chikungunya virus respectively,and the expression and activity level of calpain-2 protein in cells and mice were analyzed by real-time fluorescent quantitative PCR and calpain activity detection kit.2.Hela cells applied with inhibitor III were infected with Chikungunya virus.The expression of Chikungunya virus E1 gene in the cells was detected by real-time quantitative PCR and the virus titer in the supernatant was tested by plaque formation experiments.3.Hela cells transfected with calpain-2 protein specific small interfering RNA were infected with Chikungunya virus.The expression of Chikungunya virus E1 gene in the cells was detected by real-time quantitative PCR and the virus titer in the supernatant wes tested by plaque formation experiments.4.Hela cells applied with inhibitors III and calpain-2 protein specific small interfering RNA were infected with Chikungunya virus.The expression levels of vimentin and tubulin in the cells were detected by Western blot.5.Hela cells applied with inhibitor III were infected with Chikungunya virus.The distribution of vimentin and tubulin in the cells were observed by immunofluorescence technology.Results1.Chikungunya virus infection did not significantly affect the expression and activity of calpain-2 protein in cells and joints,muscle tissues of mice.2.Inhibitor III could significantly reduce the replication level of Chikungunya virus in Hela cells and the virus titer in the supernatant.3.Calpain-2 protein specific small interfering RNA reducing the expression levels of calpain-2 protein could significantly decrease the replication level of Chikungunya virus in Hela cells and the virus titer in the supernatant.4.The inhibitor III and calpain-2 protein specific small interfering RNA did not affect the expression levels of vimentin and tubulin in Hela cells.5.After Chikungunya virus infecting Hela cells,vimentin lost its usual filamentous structures and retracted from the plasmamembrane,which could be inhibited by inhibitor Ⅲ.6.After Chikungunya virus infecting Hela cells,tubulin converged around cell nucleus slightly,which could not be inhibited by inhibitor Ⅲ.ConclusionsThis study found that chikungunya virus infection had no significant effect on the expression and activity of calpain-2 protein,but the inhibitor Ⅲ and calpain-2 protein specific small interfering RNA significantly inhibited the replication and virus titer of Chikungunya virus in Hela cells,which indicated that calpain-2 protein played an important role on the replication of Chikungunya virus.At the same time,the inhibitor Ⅲ and calpain-2 protein specific small interfering RNA could neither significantly affect the expression levels of vimentin and tubulin in the cells,nor change the phenomenon that tubulin converged around cell nucleus slightly.However,inhibitor Ⅲ could inhibit the rearrangement of vimentin losing its usual filamentous structures and retracting from the plasmamembrane due to Chikungunya virus infection,which might be one of the mechanisms by which calpain-2 protein affected Chikungunya virus replication.It is important to understand the biological significance of Chikungunya virus replication and develop antivirus strategies.