Latency-associated Nuclear Antigen Inhibits Lytic Replication Of Kaposi's Sarcoma-associated Herpesvirus By Regulating Let-7a/RBPJ Signaling

Background: Kaposi's sarcoma-associated herpesvirus(KSHV)is the etiology of Kaposi's sarcoma(KS).KSHV has a dual-phase life cycle,latent and lytic modes during its infection of host cells.Latent associated nuclear antigen(LANA)is a key factor in the establishment and maintenance of latency,and the transcription and transcriptional activation(RTA)controls the switch from latency to lytic replication.Inhibition of RTA expression is an important mechanism for LANA to maintain latent infection.In host cell,recombination signal sequence binding protein J kappa(RBPJ)binds with LANA,which plays an important role in inhibiting the expression of RTA.However,whether RBPJ is regulated by KSHV and the relationship between RBPJ and lytic replication of KSHV is unclear.Objective: To explore the influence of LANA on the let-7a/RBPJ signaling pathway,the mechanism of LANA regulating let-7a expression and the direct relationship between let-7a and RBPJ,and the influence of let-7a and RBPJ on lytic replication of KSHV.Methods: We transfected 293 T cells with the plasmid pCAGGS-LANA or the empty vector pCAGGS,determined let-7a mRNA with northern blotting,the levels of let-7a?pri-let-7afd?LIN28B?NF-?B and RBPJ mRNA with qPCR assays.RBPJ protein was determined with a western blotting assay.iSLK.219 cells were transfected with si-RNA directed against LANA or nonspecific siRNA,Test the above indicators.and than overexpression and RNA Interference techniques to alter LIN28 B and NICD expression levels,to detection of let-7a,pri-let-7afd,RBPJ expression levels,Analyze the molecular mechanism of LANA regulation let-7a.We used a luciferase assay to explore the association between let-7a and its potential binding site in the RBPJ 3?-UTR.To further investigate the effects of let-7a on RBPJ expression,RBPJ mRNA and protein were analyzed in 293 T cells let-7a overexpressing or silencing,Let-7a inhibits the expression of RBPJ at the mRNA and protein levels of iSLK.219 cells.overexpress let-7a and downregulate RBPJ,respectively,The green fluorescent protein(GFP)-and red fluorescent protein(RFP)-positive cells were observed with an inverted fluorescence microscope in iSLK.219 cells,The intracellular and extracellular KSHV genome copy numbers were determined with qPCR assays,and the ability of the culture fluid to infect 293 T cells analyzes virus production and infectivity.Result:1.LANA increased the expression of let-7a and pri-let-7afd,and reduced the RBPJ mRNA and protein levels in the 293 T cell.2.LANA could down-regulate the expression of NF-?B and LIN28 B in 293 T cells;let-7a could be up-and down-regulated after silencing Lin28 B and Lin28 and LANA in iSLK.219 cells,respectively.3.LANA increased the expression of NICD.The overexpression of NICD increased let-7a and pri-let-7afd;4.let-7a suppressed the luciferase activity from the wild-type RBPJ 3?-UTR in a dose-dependent manner,but had no effect on the mutated RBPJ 3?-UTR.let-7a decrease the expression of RBPJ;5.Overexpression let-7a inhibited the lytic reactivation of KSHV,and downregulate viral replication levels and infectious virus production in a dose-and time-dependent manner.6.RBPJ knockdown inhibited lytic reactivation of KSHV and downregulated viral replication levels and infectious virus production in a dose-and time-dependent manner..Conclusion: 1.LANA reducing RBPJ through upregulating let-7a.2.LANA could induce the primary transcripts of let-7a by increasing cellularNICD and promote the processing of the let-7a primary transcripts by suppressing NF-?B/LIN28 B signaling.3.Regulation of let-7a/RBPJ signaling might play an important role in LANA inhibition of lytic replication of KSHV.