The Mechanism Research Of Hsp90 Inhibitors Regulating HSV-1 Genome Replication Phase

Herpes simplex virus type I?HSV-1?is one of the Alphaherpesvirinae,which could initiate labial herpes caused by the reactivation of HSV-1 primary infection,and genital herpes caused by secondary infection even causes herpes encephalitis to cause death.Currently,herpesvirus-induced diseases have high prevalence,strong recurrence and other characteristics leading to further waste of social resources and drug resistance,so it is necessary to further study the mechanism between host protein and virus to promote the discovery of new antiviral targets and the development of new antiviral drugs.Previous researches in our laboratory founded that Hsp90 inhibitor AT-533hindered HSV-1 nuclear egress and assembly,moreover Hsp90?maintained the stability of VP16 to guarantee the transactivation of?genes,which indicated Hsp90participated in most stages of HSV-1 development.Further,other research indicated Hsp90 inhibitor geldanamycin reduced viral titers and induced the abnormal location of HSV-1 DNA polymerase catalytic subunit p U_L30.However,the particular interaction between Hsp90 and HSV-1 DNA polymerase still unknown.Therefore,this study mainly focused on elucidating the role and specific mechanism of Hsp90 in DNA replication phase,thus providing more theoretical basis and experimental evidence for the Hsp90 of related pathways to tamper it as a target of novel anti-virus candidates.First,the present research identified CC₅₀,IC₅₀ and antiviral working concentrations of two Hsp90 inhibitors 17-AAG and AT-533 in human foreskin fibroblast?HFF?through cell activity assay and cytopathic effect experiments?CPE?.The results showed that Hsp90 inhibitors not affected the transcription level of virus gene and directly induced the decrease of virus DNA copy number.Secondly,this research identified the mechanism of Hsp90 direct regulation of HSV-1 DNA replication phase through protein simulation docking,western blotting and co-immunofluorescence experiments.The interaction between Hsp90 and HSV-1p U_L42 is obtained by protein simulation docking and protein co-immunoprecipitation experiments,indicating that Hsp90 inhibitor may directly through regulating p U_L42proteins affect HSV-1 DNA replication.Therefore,through western blotting and immunofluorescence assay,Hsp90 inhibitors induced the degradation of virus and plasmid-expressed p U_L42 via autophagy pathway,and overexpression of p U_L42reverted HSV-1 DNA copy number.Through western blotting and immunofluorescence experiments,Hsp90 inhibitors could not affect the expression of HSV-1 helicase-primerase,further highlighting the indispensable role of Hsp90 inhibitors regulating HSV-1 DNA polymerase processivity factor p U_L42 in DNA replication phase.Last but not the least,this research was the first to find that Hsp90 inhibitors did not affect the transcription level of virus gene and directly induced the decrease of virus DNA copy number,and it was clear that Hsp90 can interact with HSV-1 DNA polymerase processivity factor p U_L42,clarifying that Hsp90 inhibitors could destroy the stability of HSV-1 p U_L42 to degradation through autophagy pathway.Moreover,further studies and elucidation of the direct effects between Hsp90 inhibitors 17-AAG,AT-533 and HSV-1 DNA polymerase whole enzyme were manipulated.These two Hsp90 inhibitors directly docked in HSV-1 DNA polymerase holoenzyme and inhibited its complex formation.This research further elucidated the mechanism of Hsp90inhibitors on HSV-1 DNA replication and provided new research ideas and drug targets for drug development in the HSV-1 genome replication phase.