DYRK1A Controls HIV-1 Replication at a Transcriptional Level in an NFAT Dependent Manner

Abstract

Background: Transcription of the HIV-1 provirus is regulated by both viral and host proteins and is very important in the context of viral latency. In latently infected cells, viral gene expression is inhibited as a result of the sequestration of host transcription factors and epigenetic modifications.

Results: In our present study we analyzed the effect of host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) on HIV-1 replication. We show that DYRK1A controls HIV-1 replication by regulating provirus transcription. Downregulation or inhibition of DYRK1A increased LTR-driven transcription and viral replication in cell lines and primary PBMC. Furthermore, inhibition of DYRK1A resulted in reactivation of latent HIV-1 provirus to a similar extent as two commonly used broad-spectrum HDAC inhibitors. We observed that DYRK1A regulates HIV-1 transcription via the Nuclear Factor of Activated T-cells (NFAT) by promoting its translocation from the nucleus to the cytoplasm. Therefore, inhibition of DYRK1A results in increased nuclear levels of NFAT and increased NFAT binding to the viral LTR and thus increasing viral transcription.

Conclusions: Our data indicate that host factor DYRK1A plays a role in the regulation of viral transcription and latency. Therefore, DYRK1A might be an attractive candidate for therapeutic strategies targeting the viral reservoir.

Fig 1. The effect of DYRK1A knockdown or inhibition on HIV-1 replication.

(A) To test the shRNA targeting against DYRK1A, HEK293T cells were transfected with either 6.25 ng, 12.5 ng or 25 ng of shDYRK1A or shControl and 48-hours after transfection DYRK1A protein expression was determined by Western blot. The results shown are representative for 3 independent experiments. The ratio is calculated by dividing the intensity of the DYRK1A bands by the intensity of the β-actin band as determined with ImageJ. (B) The effect of DYRK1A downregulation on HIV-1 replication was tested by transfecting HEK293T cells in 96-wells plates with either 6.25 ng, 12.5 ng or 25 ng of shDYRK1A or corresponding concentrations of the shControl. Forty-eight hours after DYRK1A downregulation cells were inoculated at a MOI of 0.01 with a VSV-G-pseudotyped single-round luciferase virus. Luciferase activity was determined 48 hours post infection as a measure for viral replication and expressed relative to the corresponding shControl. Data is shown as mean and SD of three independent experiments. (C) The effect of DYRK1A inhibition on HIV-1 replication in HEK293T was analyzed by infection with a VSV-G-pseudotyped single-round luciferase virus. Twenty-four hours post infection cells were treated with 24 μM, 48 μM or 120 μM of either INDY or DMSO control and after an additional 24-hours luciferase activity was determined as a measure for viral replication and expressed relative to DMSO control (No Drug). Data is shown as mean and SD of three independent experiments. (D) The effect of DYRK1A inhibition on HIV-1 replication in PBMC was analyzed by infecting PHA-stimulated PBMC from four healthy blood donors with a VSV-G-pseudotyped single-round luciferase virus at a MOI of 0.1. Twenty-four hours post infection, cells were treated with either 24 μM or 48 μM of INDY or DMSO control and after another 24-hours luciferase activity was determined as a measure for viral replication and expressed relative to the DMSO control (No drug). Data is shown as mean and SD of 4 independent donors. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01, ***p<0.001.

Fig 2. The effect of DYRK1A knockdown or inhibition on LTR driven transcription.

(A) The effect of DYRK1A downregulation on LTR driven transcription was analyzed by co-transfection of HEK293T cells in 96-wells plates with 5 ng of LTR-luciferase reporter construct and 12.5 ng, 25 ng or 50 ng of shDYRK1A or the shControl vector. Luciferase activity was analyzed 48-hours post-transfection as a measure for LTR activity and expressed relative to the shControl. (B) The effect of DYRK1A inhibition on LTR driven transcription was analyzed by transfecting HEK293T cells in 96-wells plates with 5 ng of LTR-luciferase reporter construct. Twenty-four hours post transfection cells were treated with 24 μM, 48 μM or 120 μM of either INDY or the DMSO control and after an additional 24-hours luciferase activity was determined and expressed relative to DMSO control (No Drug). (C) Comparison of basal transcriptional levels of the LTR-luciferase reporter construct and LTR-luciferase reporter construct lacking the NFAT and NF-kB binding sites was performed by transfecting HEK293T cells in 96-wells plates with 5 ng of either construct. Luciferase activity was analyzed 48-hours post-transfection as a measure for LTR activity. The effect of DYRK1A downregulation (D) or inhibition (E) on LTR driven transcription was analyzed by co-transfection of HEK293T cells in 96-wells plates with 5 ng of LTR-luciferase reporter construct lacking the NFAT and NF-kB binding sites and 12.5 ng, 25 ng or 50 ng of shDYRK1A or the shControl vector or treatment with 24 μM, 48 μM or 120 μM INDY. Luciferase activity was analyzed 48-hours post-transfection as a measure for LTR activity and expressed relative to the shControl. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01. Data is shown as mean and SD of three independent experiments.

Fig 3. The effect of DYRK1A inhibition on reactivation of HIV-1 LTRs.

(A) The effect of DYRK1A inhibition on reactivation of silent HIV-1 provirus was studied in TZM-bl cells. TZM-bl were treated with either 48 μM, 120 μM or 240 μM of INDY or the DMSO control and 24-hours later LTR-driven luciferase activity was determined as a measure for viral reactivation. Results are expressed relative to the DMSO control. Data is shown as mean and SD of three independent experiments. (B-E) J-Lat cells were treated for twenty four hours with 24, μM, 48 μM, 120 μM or 240 μM of INDY or 12.5 ng/ml TNFα as a positive control. Subsequently the percentage of GFP expressing cells and the mean fluorescent intensity was determined by FACS. Results are expressed relative to the control). Data is shown as mean and SD of two independent experiments. (F) The effect of DYRK1A inhibition on the reactivation of silent HIV-1 provirus was compared to reactivation by two HDAC inhibitors sodium butyrate and TSA. TZM-bl were treated with either 120 μM or 240 μM of INDY, 5 mM or 10 mM of Sodium butyrate, or 9 nM or 18 nM of TSA or the appropriate vehicle control. Twenty-four hours later, LTR-driven luciferase activity was determined and expressed relative to vehicle control. Data is shown as mean and SD of three independent experiments. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01, ***p<0.001.

Fig 4. The effect of DYRK1A inhibition and downregulation is mediated via NFAT.

(A) To analyze the effect of DYRK1A inhibition on the amount of NFAT or NF-kB bound to the viral LTR, a ChIP-qPCR analysis was performed in TZM-bl cells treated with either 240 μM of INDY, the DMSO control, or 12.5 ng/ml TNFα. Sheared DNA was immunoprecipitated with either control IgG, anti-NFAT, or anti-NF-κB antibodies and levels of bound LTR DNA were analyzed by qPCR. (B) The effect of DYRK1A downregulation on LTR driven transcription in the presence of 10μM NF-kB inhibitor BAY or 300 ng/ml NFAT inhibitor FK506 (C) was analyzed by co-transfection of HEK293T cells in 96-wells plates with 5 ng of LTR-luciferase reporter construct and 12.5 ng, 25 ng or 50 ng of shDYRK1A or the shControl vector. Luciferase activity was analyzed 48-hours post-transfection as a measure for LTR activity and expressed relative to the shControl. Data is shown as mean and SD of three independent experiments. (D) Nuclear localization of NFAT was studied in TZM-bl cells cultured for 24 hours in the absence or presence of 240 μM INDY. Subsequently, cells were stained with Hoechst and anti-NFAT and analysed by confocal fluorescent microscopy. Results are representative of at least two independent experiments. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01, ***p<0.001.