Role Of MiR-711 And Its Targeted Notch/NF-?B Signaling In The Regulation Of Kaposi's Sarcoma-associated Herpesvirus Life Cycle And Host Cytikine Expression By Human Immunodeficiency Virus Type 1 Vrial Protein R

Background: Kaposi's sarcoma-associated Herpesvirus(KSHV)is the causative agent of Kaposi's sarcoma(KS)and is closely related to the occurrence of primary effusion lymphoma(PEL)and multicentric Castleman's disease(MCD).The interaction between human immunodeficiency virus type 1(HIV-1)and KSHV plays an important role in the development of AIDS-related KS(AIDS-KS).Previous studies of our group confirmed that HIV-1 encoded viral protein R(Vpr)targeted the I?B?(inhibitor of ?B?)through up-regulating micro RNAs(mi RNAs)in host cells.mi R-942-5p activated the nuclear factor-?B(NF-?B)signaling pathway,thereby promoting KSHV latentcy.Further research suggested that Notch signaling pathway is involved in Vpr regulated KSHV life cycle and some of the host cell cytokine release processes.Nevertheless,the underlying mechanism remains unclear.Objective: To investigate the role and mechanism of Vpr in regulating KSHV life cycle and the expression of some host cytokines through the Notch signaling pathway.Methods: mi RNA microarray technology was used to identify a set of mi RNAs whose expression was differently regulated by Vpr.Bioinformatics,luciferase reporter analysis and western blot showed that mi R-711 was upregulated and repressed the expression of Notch1.Point mutation analysis was performed to validate the putative binding site of mi R-711 in Notch1 3' untranslated region(UTR).Subsequently,gain-of-function and loss-of-fuction assays were applied to determine the mi R-711 role in Vpr regulated KSHV lytic gene m RNA and protein production and release of viral particle.Real-time quantitative polymerase chain reaction(RT-q PCR)experiment was used to examine the role of mi R-711 in the release of host cytokines by Vpr protein.Finally,western blot,luciferase reporter assay and RT-q PCR were applied to identify the mechanismof mi R-711 targeted Notch signaling pathway and its downstream NF-?B signaling pathway in the regulation of KSHV life cycle and host cytokine release by Vpr protein.Results: mi RNA microarray result showed a series of mi RNAs were upregulated by Vpr;bioinformatics analysis,luciferase reporter assays and western blot confirmed that Vpr significantly upregulated mi R-711,which directly targeted the 3'UTR of Notch1 and repressed the production of Notch1 protein,a key protein of Notch pathway.Repression of mi R-711 not only activated Notch signaling pathway which was inhibited by Vpr,but also significantly promoted KSHV lytic replication and effectively restored the expression of some host cytokines.Conversely,overexpression of mi R-711 significantly enhanced Vpr-mediated regulation of KSHV life cycle and some host cytokines.Mechanically,mi R-711 targeted Notch1 could act on the I?B? promoter,thereby inhibiting the NF-?B signaling pathway by affecting the expression of I?B? gene.Conclusion: HIV-1 Vpr inhibits Notch signaling pathway by upregulating mi R-711,which activates NF-?B signaling pathway,thereby promoting KSHV latent infection and affecting the release of some host cytokines.