Human Immunodeficiency Virus Type 1 Vrial Protein R(Vpr) Inhibits The Lytic Cycle Replication Of Kaposi's Sarcoma-Associated Herpesvirus:Role Of Notch/NF-?B Signaling Pathway

Background: Kaposi's sarcoma-associated herpesvirus(KSHV)is etiologically associated with Kaposi's sarcoma(KS)and is also closely related to two other lymphoproliferative diseases named asprimary effusion lymphoma(PEL)and multicentric Castleman's disease(MCD).Human immunodeficiency virus type 1(HIV-1)has taken a great part in promoting the aggressive manifestations of AIDS-KS.We have previously shown that HIV-1 Tat triggers KSHV reactivation from latency by modulating the JAK/STAT pathway,while HIV-1 Nef may inhibit KSHV replication via a cellular micro RNA(hsa-mi R-1258)which directly targets the lytic switch protein replication and transcriptional activator(RTA).As another secretory protein encoded by HIV-1,Vpr may as well have an impact on KSHV life cycle.Objective: To evaluate the effect of HIV-1 Vpr on the lytic cycle replication of KSHV and to identify the pivotal signaling pathways involved.Methods: Firstly,recombinant plasmid containing HIV-1 Vpr gene was constructed and then expressed via an Escherichia coli overexpression system.After purification and identification,the soluble Vpr protein was obtained.Secondly,recombinant HIV-1 Vpr protein was added to cells latently infected with KSHV,followed by immunofluorescence assay(IFA)to confirm whether soluble Vpr could be taken up.Next,the m RNA transcription levels and protein expression of KSHV lytic genes as well as a variety of cytokines in both PEL cell lines and TIVE-LTC after coculture with soluble Vpr were detected by real-time quantitative PCR(RT-q PCR)and Western blot,respectively.Then the supernatants of the KSHV positive cells were collected to measurethe production of infectious viral particles.Furthermore,to explore the molecular mechanism involved,we evaluated the influence of soluble Vpr on Notch signaling and nuclear factor ?B(NF-?B)signaling pathways by RT-q PCR,Western blot,IFA and luciferase reporter analysis.Afterwards,overexpression of intracellar Notch(ICN)and suppression of NF-?B activity by I?B-DN were used to determine the effect of Notch and NF-?B signaling pathways,respectively,on Vpr-mediated inhibition of KSHV lytic replication.Results: The recombinant plasmid carrying Vpr gene was constructed and expressed successfully.The purificated Vpr protein appeared to penetrate KSHV-infected PEL and TIVE-LTC cells.Internalized Vpr suppressed the expression of KSHV viral lytic m RNA transcripts and proteins as well as the production of infectious viral particles.Moreover,soluble Vpr changed the expression of some cytokines.Mechanically,Vpr exerted an inhibitory effect on KSHV replication via downregulation of Notch signaling and activation of NF-?B signaling pathway.Furthermore,overexpression of ICN inhibited NF-?B activity while suppression of NF-?B activation did not show any impact on Notch signaling.Conclusions: Exogenous Vpr could be efficiently taken up by KSHV-infected cells and exhibited a negative effect on KSHV replication while elevated the expression of some cytokines as well.Moreover,Vpr might exert its inhibition on KSHV replication through suppression of Notch signaling,which in turn activated the downstream NF-?B signaling pathway.These data suggested that in AIDS-related KS(AIDS-KS)patients,Vpr protein might help KSHV escape from the immune clearance by inhibiting KSHV lytic replication,which in turn facilitated the establishment of latent infection and tumor formation.