Chromatin remodeling controls Kaposi's sarcoma-associated herpesvirus reactivation from latency

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, the endothelial cell cancer Kaposi's sarcoma, and two B cell cancers, Primary Effusion Lymphoma and multicentric Castleman's disease. KSHV has latent and lytic phases of the viral life cycle, and while both contribute to viral pathogenesis, lytic proteins contribute to KSHV-mediated oncogenesis. Reactivation from latency is driven by the KSHV lytic gene transactivator RTA, and RTA transcription is controlled by epigenetic modifications. To identify host chromatin-modifying proteins that are involved in the latent to lytic transition, we screened a panel of inhibitors that target epigenetic regulatory proteins for their ability to stimulate KSHV reactivation. We found several novel regulators of viral reactivation: an inhibitor of Bmi1, PTC-209, two additional histone deacetylase inhibitors, Romidepsin and Panobinostat, and the bromodomain inhibitor (+)-JQ1. All of these compounds stimulate lytic gene expression, viral genome replication, and release of infectious virions. Treatment with Romidepsin, Panobinostat, and PTC-209 induces histone modifications at the RTA promoter, and results in nucleosome depletion at this locus. Finally, silencing Bmi1 induces KSHV reactivation, indicating that Bmi1, a member of the Polycomb repressive complex 1, is critical for maintaining KSHV latency.

Fig 1. Inhibitor screen for epigenetic regulatory proteins that induce KSHV reactivation from latency.

BJAB-KSHV.219 cells were incubated with 1 μM of each compound for 48 hours in (A) or 10 μM of each compound for 120 hours in (B), and the percentage of GFP positive cells expressing RFP (percent reactivation) was determined by FACS analysis. (C) BJAB-KSHV.219 cells were incubated with EZH2 inhibitors at concentrations of 50, 16.7, 5.6, and 1.9 μM for 120 hours. Percent reactivation was determined by FACS analysis. Data are representative of two independent experiments performed in duplicate; mean and standard error are shown.

Fig 2. Romidepsin, Panobinostat, (+)-JQ1, and PTC-209 induce KSHV lytic gene expression.

(A) RT-qPCR analysis for KSHV lytic transcript levels was performed on PEL cells treated with DMSO, NaB+TPA, or 1 μM Romidepsin or Panobinostat for 48 hours. Lytic transcript levels were normalized to GAPDH, and the fold difference between DMSO and compound treated cells was calculated. (B) Immunoblot analysis for vIL-6, ORF45, or actin was performed on lysates from BCBL-1 cells treated as in (A). Approximate molecular mass (kDa) marker positions are indicated to the left of each blot. (C) RT-qPCR analysis for KSHV lytic transcript levels was performed as in (A) on PEL cells treated with DMSO, NaB+TPA, or 10 μM (+)-JQ1 or PTC-209 for 120 hours. (D) Immunoblot analysis was performed on lysates from BCBL-1 cells treated as in (C). Data are representative of at least two independent experiments performed in triplicate; mean and standard error are shown.

Fig 3. Romidepsin, Panobinostat, (+)-JQ1, and PTC-209 induce global KSHV gene expression.

KSHV whole genome transcriptional profiling was performed on BCBL-1 cells treated with DMSO or 1 μM Romidepsin or Panobinostat for 48 hours (A), or with 10μM (+)-JQ1 or PTC-209 for 120 hours (B). mRNA levels of viral genes were normalized to the mRNA levels of multiple cellular housekeeping genes to yield dCt as a measure of relative expression. Higher transcript expression levels are indicated by red and lower expression levels by blue.

Fig 4. Romidepsin, Panobinostat, and PTC-209 induce KSHV genome replication and release of infectious virions.

(A) qPCR analysis for cell-associated KSHV genome copies was performed on BCBL-1 cells treated with DMSO or 1 μM Romidepsin or Panobinostat for five days. Naïve Vero cells were infected with supernatants collected from BCBL-1 cells treated as in (A), and KSHV genome copies in the infected cells were determined by qPCR (B), and KSHV lytic transcript levels were assayed by RT-qPCR (C). Lytic transcript levels were normalized to GAPDH, and the fold difference between DMSO and compound treated cells was calculated. (D) qPCR analysis for cell-associated KSHV genome copies was performed on BCBL-1 cells treated with DMSO or 10 μM PTC-209 for seven days. Naïve Vero cells were infected with supernatants collected from BCBL-1 cells treated as in (D), and KSHV genome copies in the infected cells were determined by qPCR (E), and KSHV lytic transcript levels were assayed by RT-qPCR (F). Lytic transcript levels were normalized to GAPDH, and the fold difference between DMSO and PTC-209 treated cells was calculated.

Fig 5. Romidepsin, Panobinostat, and PTC-209 impact histone modifications at the RTA promoter.

(A) BCBL-1 cells were treated with DMSO or 1 μM Romidepsin or Panobinostat for four hours, and levels of H3Ac and total H3 at the RTA promoter were determined by ChIP-qPCR. Levels of H3Ac were normalized to total levels of H3, and the fold difference between DMSO and compound treated cells was calculated. (B) BCBL-1 cells were treated with DMSO or 10 μM PTC-209 for five days, and levels of H2AK119ub and total H2A at the RTA promoter were determined by ChIP-qPCR. Levels of H2AK119ub were normalized to total levels of H2A, and the fold difference between DMSO and PTC-209 treated cells was calculated. Data are representative of three independent experiments.

Fig 6. Romidepsin, Panobinostat, and PTC-209 induce nucleosome depletion at the RTA promoter.

Nucleosome density was assayed by FAIRE-qPCR in 293-KSHV.219 cells treated with DMSO or 1 μM Romidepsin or Panobinostat (A), or DMSO or 10 μM PTC-209 (B). Data are representative of at least three independent experiments.

Fig 7. Romidepsin and Panobinostat, but not PTC-209, alter chromatin accessibility across the KSHV genome.

Nucleosome density was assayed by FAIRE-seq in 293-KSHV.219 cells treated with DMSO or 1 μM Romidepsin or Panobinostat (A), or DMSO or 10 μM PTC-209 (B). Sequencing reads were mapped to the KSHV reference genome NC_009333, and mapped reads from biological replicates were merged. Statistically significant FAIRE peaks were identified using CLC Genomics Workbench with input DNA as a control. Red bars indicate regions of FAIRE enrichment. The black rectangles highlight the RTA promoter.

Fig 8. Bmi1 is essential for maintenance of KSHV latency.

BCBL-1 cells were mock transfected or transfected with control or Bmi1 siRNAs, and cells were lysed at 0 and 96 hours post-transfection. KSHV lytic proteins were assayed by immunoblot.