RTA And LANA Co-regulate The Let-7a/RBPJ Signaling To Control KSHV Replication

Background: Kaposi's sarcoma-associated herpesvirus(KSHV),also called as human herpesvirus 8,is the etiologic agent of Kaposi,s sarcoma(KS).Like other herpesviruses,KSHV has a dual-phase life cycle,which includes latent infection and lytic reactivation.Viral protein replication and transcription activator(RTA)is a "switch" of lytic replication,expressed only in the early stage of de novo infection and lytic replication.Latency-associated nuclear antigen(LANA),encoded by KSHV ORF73 is a major latency protien and is continuously expressed in KSHV-infected cells.Recombination signal binding protein RBPJ has dual effect on the regulation of KSHV replication.RBPJ binds to RTA is essential to activate KSHV lytic replication,while RBPJ binds to LANA contributes the establishment and maintenance of latent status.However,the mechanisms of RBPJ promoting lytic replication and contributing latent maintenance still not cleally understood.Both the promoters of RTA and LANA contain RBPJ binding element.Our previous studies found that both RTA and LANA regulated let-7a,and RBPJ is directily targeted by let-7a.These results suggest that the let-7a/RBPJ signal is co-regulated by both RTA and LANA,and through let-7a/RBPJ signaling the transcription of RTA and LANA is mutually regulated.Objective: To clarify that RTA and LANA co-regulate let-7a/RBPJ signaling and RBPJ is involved in mutual transcription regulation of RTA and LANA.Methods: Firstly,overexpression and RNA interference were used to determine the effect of RTA on the let-7a/RBPJ signaling pathway.RTA expresson plasmids or empty vectors as control were transfected into 293 T cells using Lipofectamine reagent.siRNAs specific against RTA or non-specific siRNAs as control were transfected into iSLK.RTA ells using Lipofectamine reagent.Quantitative real-time PCR(qPCR)was used to detect the transcription levels of let-7a,Lin28 B,p65 and RBPJ,and western-blot was used to detect the protein levels of Lin28 B,p65 and RBPJ.Then,the fluctuation of expression levels of RTA,LANA,let-7a and RBPJ during de novo infection in HMEC-1 cells and lytic reactivation in iSLK.219 cells induced with Doxycycline(DOX)were assayed by qPCR and(or)western-blot.Further,the different ratios of RTA and LANA expression plasmids were co-transfected into 293 T cells to confirm the effect of RTA and LANA on let-7a/RBPJ signaling.Finally,the dual luciferase reporter assay system was used to detect the effect of RBPJ on the activity of LANA promoter induced by RTA and the effect of RBPJ on the activity of RTA promoter inhibited by LANA.Results: 1.Overexpression of RTA up-regulated p65,Lin28 B,RBPJ and down-regulated let-7a,while down-regulated RTA has opposite effects.2.From de novo infection to latency status establishment,the expression of RTA decreased while the expression of LANA gradually increased.The expression of let-7a increased shortly and then decreased.The expression level of RBPJ was opposite to that of let-7a.3.During lytic replication,the expression level of RTA increased dramatically,and the expression level of LANA also increased,while the expression of RBPJ increased and let-7a decreased.4.The direction of let-7a/RBPJ signaling pathway is controlled by relative amounts of RTA and LANA.5.Knockdown RBPJ attenuated the activation of the LANA promoter by RTA and attenuated the inhibitory effect of LANA promoter on the RTA promoter.Conclusion:1.The Let-7a/RBPJ signaling is dually regulated by RTA and LANA but with opposite direction.RTA decreases let-7a through NF-?B/Lin28 B signal.2.The transcription of RTA and LANA is mutually regulated through let-7a/RBPJ signaling pathway.3.RTA and LANA compete to regulate let-7a/RBPJ,and RBPJ in turn regulates the expression of RTA and LANA,thus forming a coupling feedback loop that plays an important role in the replication status transition of KSHV.The theoretical and practical significance of the study: The let-7a/RBPJ signal is an important signaling pathway for RTA and LANA co-regulation of KSHV replication,enriching the mechanism of RTA and LANA regulation of KSHV replication.