The Regulatory Function And Mechanism Of RBBP4 In HIV Transcription And Latency

BackgroundThe acquired immune deficiency syndrome caused by human immunodeficiency virus is one of the most threatening infectious diseases. Although HAART (highly active anti-retroviral therapy) can effectively inhibit HIV replication and reduce viral load to the lower limit of detection, due to the presence of HIV latent reservoirs, HIV could not be completely removed, so latent HIV is considered to be the last barrier to completely clear HIV from the body. HIV latent state is essentially a reversible transcriptional silencing state, the establishment and maintenance of HIV latency involves a variety of host transcriptional inhibitory factors and epigenetic modification. The host transcriptional inhibitory factors binding to 5’LTR (long terminal repeat) of HIV proviral genome and recruitment of HDACs play an important role in HIV transcriptional silencing and establishment of latency. RBBP4 (Retinoblastoma binding protein 4), as a histone chaperone, can be involved in the transcriptional silencing of genes by associating with HDACs. As a molecule closely related with HDACs, RBBP4 has not been reported in the HIV transcription and latency. Therefore, we will study the role and mechanism of RBBP4 in HIV transcription and latency in this paper.Aim1. To study the expression of RBBP4 in peripheral blood PBMCs (peripheral blood mononuclear cells) from normal control and HIV infected patients, and verify the expression of RBBP4 in HIV infected CEM-ss and TZM-bl cells in the laboratory;2. To reveal the effect of RBBP4 on HIV, and study the regulation of HIV LTR mediated transcription by RBBP4, and explore the binding of RBBP4 on HIV LTR promoter.3. To further study the potential mechanism of RBBP4 on HIV transcription and latency, provide experimental data and scientific basis for the removal of HIV latent reservoir.Methods1. Real-time PCR was used to detect the expression of RBBP4 in PBMCs from HIV infected patients with no treatment (30 cases), HIV infected patients with effective treatment (30 cases) and healthy controls (30 cases). The expression of RBBP4 after HIV infection was qualitatively and quantitatively detected by real-time PCR、western blot and immunofluorescence in CEM-ss and TZM-bl cells.2. Firefly luciferase activity driven by HIV LTR promoter was detected by dual luciferase reporter system to study the transcriptional regulation of HIV LTR promoter by RBBP4. EMSA (electrophoretic mobility shift assay) was used to determine binding of RBBP4 to the NF-κB (-115～-75) and NR2F1 (-345～-319) binding sites on HIV LTR.3. In the model of 293T cells transfected with pNL4-3, ChIP (chrmatin immunoprecipitation) assays were performed to analyze effect of RBBP4 on the binding of HDAC1/2 and NR2F1 to HIV LTR, and the changes of RBBP4, HDAC1, HDAC2 and NR2F1 binding on LTR in HIV latent and activated CEM-Bru cells.Results1. We found that the expression of RBBP4 in HIV infected patients with no treatment was significantly higher than in HIV infected patients with effective treatment and health controls (P<0.01, P<0.01); RBBP4 mRNA and protein expression in HIV infected CEM-ss and TZM-bl cells were significantly higher than in uninfected control cells; interference of RBBP4 expression led to a marked increase in HIV virus particles in supernatant in HIV infected CEM-ss and TZM-bl cells.2. Over expression of RBBP4 reduced the supernatant HIV virus particles、viral load、single spliced transcripts、multiple spliced transcripts and unspliced transcripts; RBBP4 repressed HIV LTR basal transcription and NFκB or tat activated transcription; and RBBP4 respectively bound to NF-κB (-115～-75) and NR2F1 (-345～-319) binding sites on HIV LTR (-454 to+181).3. The change of RBBP4 on HIV LTR affected the binding of HDAC1/2 and NR2F1 to LTR, that is when the binding of RBBP4 on HIV LTR was increased, the binding of HD AC 1/2 and NR2F1 to LTR was also increased, which was accompanied by reduced level of histone acetylation, and finally HIV P24 and virus particles in supernatant were reduced; in HIV latent CEM-Bru cells, the binding of RBBP4、 HDAC1/2 and NR2F1 was higher than in activated cells; Interfering with RBBP4 expression promoted HIV transcription initiation in HIV latent cells.Conclusions1. We shown for the first time that RBBP4 expression was increased in HIV infected patients with no treatment. And RBBP4 expression was significantly increased after TZM-bl and CEM-ss cells were infected by HIV. It indicates that HIV infection can induce the increase of RBBP4 expression.2. RBBP4 could inhibit HIV LTR basal transcription; RBBP4 could repress LTR transcription activated by NF-κB and Tat; and RBBP4 could bind to the NF-κB (-115～-75) and NR2F1 (-345 ～-319) binding site on HIV LTR.3. RBBP4 inhibited HIV LTR transcription by affecting the binding of NR2F1 and HDAC1/2 to the HIV LTR promoter; RBBP4、NR2F1 and HDAC1/2 collectively maintained HIV latent state.