Screening And Efficacy Of The Novelsmall-molecule Compounds For Promoting Thereactivation Of KSHV

Kaposi’s sarcoma associated herpesvirus（KSHV）,is aγ-herpesvirus with a linear double-stranded DNA genome.As a herpesvirus,KSHV has latent and lytic phases of the viral life cycle,and both contribute to viral pathogenesis.Epidemiological studies have shown that KSHV is the etiologic agent of Kaposi’s sarcoma（KS）,Primary effusion lymphoma（PEL）and Multicentric cattleman’s disease（MCD）.Kaposi’s sarcoma is a common complication in AIDS patients and transplantation recipients.Hence,KSHV infection has been a serious public health threat.Currently,there is no cure for KS,PEL and MCD.The limited therapeutic drugs mostly aim to suppress the development of tumors by inhibiting the lytic replication of virus.One problem for these drugs is that they cannot eliminate the latently infected virus and tumor continue to grow.Given this,researchers has proposed the"lytic induction"strategy to eliminate KSHV in tumor cells,which involves drugs that reactivate latent viruses in combination with antiviral drugs.The study aims to screen new compounds that can promote KSHV lytic reactivation to enrich the potential therapeutic drug library for KSHV-related diseases.The main results are as follows:First of all,we used existing platforms of the laboratory to establish a drug screening method to induce KSHV lytic reactivation.The 4000 compounds in the preliminary screening were selected from FDA approved drug library（L1300）and the library of drug-active compounds（LO2219）and bought from Selleck and Sigma respectively.The results showed that 10 kinds of compounds including Doxycycline,RG2833,Nexturastat A and Rocilinostat can induce the expressions of GFP in iSLK.RGB and have the potentials to induce the lytic reactivation of KSHV.We further investigated the ability of these compounds to induce RTA expressions in iSLK.RGB,promote transcription of lytic genes and promote KSHV genome replication.4 out of the 10 compounds are particularly effective in inducing KSHV lytic reactivation of iSLK.RGB cell:Arbidol Hydrochloride,RG2833,Nexturastat A and Rocilinostat.Experiments further proceeded in BCBL-1 cells demonstrate that Arbidol Hydrochloride fail to induce lytic activation of KSHV in BCBL-1 cells.This implies that Arbidol Hydrochloride might induce lytic reactivation of KSHV in iSLK.RGB cells with aid of tetracycline-induced exogenous expression of RTA.Drug incubation experiment in 293T.219 cells showed that RG2833,Nexturastat A and Rocilinostat can significantly induce KSHV lytic reactivation of 293T.219 cells,with Rocilinostat being the most efficient one.In the cytotoxicity test,the above compounds start to show damage to 293T.219 cells at a concentration of 2.5μM.Among which,Nexturastat A has the lowest cytotoxicity to 293T.219 cells and half toxic concentration(CC₅₀)of 10μM.Cytotoxicity experiments showed that the overall cytotoxicity among RG2833,Nexturastat A and Rocilinostat has little difference,with Nexturastat A being the least toxic one.Further drug incubation experiment indicated that RG2833,Nexturastat A and Rocilinostat can all induce gene expression,genome replication and the release of virions in BCBL-1 cell,among which,Rocilinostat has the highest ability in inducing the lytic reactivation of KSHV on BCBL-1 cells.Finally,this research explored the mechanism used by Rocilinostat in its inducing KSHV lytic reactivation and found that the addition of Rocilinostat to iSLK.RGB cells with silence of RTA can rescue the expression RTA.This means that Rocilinostat can directly increase the expression of RTA.Further dual-luciferase reporter gene assay showed that the Rocilinostat can inhibit NF-κB promoter activity activated by vFLIP.This preliminarily suggests that Rocilinostat might induce KSHV lytic reactivation parts by inhibiting NF-κB signal transduction pathway activated by vFLIP,which further activate expressions of RTA and lytic reactivation of KSHV.In conclusion,we firstly established an efficient drug screening method which can feasibly induce KSHV lytic reactivation and screen 4000 compounds.Finally,this work identfied that RG2833,Nexturastat A and Rocilinostat can induce KSHV lytic reactivation,which enriches alternative drug libraries for the treatment of KSHV.