Role Of MicroRNAs And Cellular Signaling Pathways In Human Immunodeficiency Virus Type 1 Negative Factor Protein (Nef) Regulation Of Kaposi's Sarcoma-associated Herpesvirus Latency

Background: Kaposi's sarcoma-associated herpesvirus(KSHV)is causally linked to several acquired immunodeficiency syndrome(AIDS)-related malignancies,including KS,primary effusion lymphoma(PEL)and multicentric Castleman's disease.The interaction of human immunodeficiency virus type 1(HIV-1)and KSHV has a central role in promoting the aggressive manifestations of KS in AIDS patients.We have previously shown that HIV-1 Tat triggers KSHV reactivation from latency by modulating the JAK/STAT pathway.Accessory negative factor(Nef)is another early and secretory HIV-1-encoded protein,which may also influence KSHV latency.Objective: To explore the regulation of KSHV latency by HIV-1 Nef and investigate the possible molecular mechanisms involved in this process,including micro RNAs(mi RNAs)and signaling pathways.Methods: Initially,we utilized soluble protein or eukaryotic expression plasmid to express HIV-1 Nef in PEL cells.Real-time PCR(real-time quantitative polymerase chain reaction),Western blot,IFA(immunofluorescence assay)and virion release assay were performed to detecte the expression of KSHV transcripts and proteins,and production of infectious viral particles.To explore the molecular mechanisms involved in this process,on one hand,we employed microarray analysis to identify the mi RNAs regulated by Nef,luciferase reporter analyses to select the mi RNAs directly targeted the 3'UTR(untranslated region)of KSHV lytic switch protein RTA(replication and transcriptional activator),bioinformatics and point mutation analyses to valiad the putative binding sites in the RTA 3'UTR.Subsequently,gain-of-function and loss-of-fuction were applied to determine the mi RNAs mediated Nef regulation of KSHV latency.On the other hand,we examined the influence of Nef on NF-?B(nuclear factor ?B)pathway by IFA,ELISA(enzyme linked immunosorbent assay),luciferase reporter analyses and Western blot.Afterwards,overexpression of IKK2(I?B kinase 2)and suppression of phosphorylating I?B?(inhibitor of ?B ?,I?B?)were used to determine the effect of NF-?B pathway on Nef-mediated inhibition of KSHV lytic replication.Results: Exogenous Nef appeared to penetrate KSHV-infected PEL cells.Internalized and ectopic expression of Nef in PEL cells suppressed the expression of KSHV viral lytic phase m RNA transcripts and proteins,and production of infectious viral particles.Mechanically,on the one hand,microarray analysis identified a set of mi RNAs whose expression was differentially regulated by Nef.Bioinformatics and luciferase reporter analyses showed that two of the Nef-upregulated mi RNAs,a cellular mi RNA 1258(mi R-1258)and a KSHV mi RNA K12-3*(mi R-K3*),directly targeted the 3'UTR of KSHV lytic switch protein RTA.As a result,overexpression of these two mi RNAs significantly enhanced Nef-mediated KSHV inhibition,whereas repression of these mi RNAs with specific suppressors relieved Nef-mediated inhibition in PEL cell lines.On the other hand,Nef inhibited KSHV replication by modulating NF-?B pathway,which exhibited an inhibitory effect on KSHV replication.Conclusion: These novel findings demonstrate that,by regulating cellular/viral mi RNAs and signaling pathways,Nef may promote KSHV latency,and contribute to the pathogenesis of AIDS-related malignancies.