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. 2014 Jun;88(12):6873-84.
doi: 10.1128/JVI.00283-14. Epub 2014 Apr 2.

Inhibition of KAP1 enhances hypoxia-induced Kaposi's sarcoma-associated herpesvirus reactivation through RBP-Jκ

Liming Zhang  1 , Caixia Zhu  2 , Yi Guo  3 , Fang Wei  4 , Jie Lu  5 , Jing Qin  6 , Shuvomoy Banerjee  5 , Junwen Wang  6 , Hong Shang  3 , Subhash C Verma  7 , Zhenghong Yuan  2 , Erle S Robertson  8 , Qiliang Cai  9
Affiliations

Inhibition of KAP1 enhances hypoxia-induced Kaposi's sarcoma-associated herpesvirus reactivation through RBP-Jκ

Liming Zhang et al. J Virol. 2014 Jun.

Abstract

Hypoxia-inducible factor 1α (HIF-1α) has been frequently implicated in many cancers as well as viral pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. It can stabilize HIF-1α during latent infection and undergoes lytic replication in response to hypoxic stress. However, the mechanism by which KSHV controls its latent and lytic life cycle through the deregulation of HIF-1α is not fully understood. Our previous studies showed that the hypoxia-sensitive chromatin remodeler KAP1 was targeted by the KSHV-encoded latency-associated nuclear antigen (LANA) to repress expression of the major lytic replication and transcriptional activator (RTA). Here we further report that an RNA interference-based knockdown of KAP1 in KSHV-infected primary effusion lymphoma (PEL) cells disrupted viral episome stability and abrogated sub-G1/G1 arrest of the cell cycle while increasing the efficiency of KSHV lytic reactivation by hypoxia or using the chemical 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate (NaB). Moreover, KSHV genome-wide screening revealed that four hypoxia-responsive clusters have a high concurrence of both RBP-Jκ and HIF-1α binding sites (RBS+HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-Jκ with the HIF-1α complex for driving RTA expression not only in normoxia but also in hypoxia. These results suggest that both KAP1 and the concurrence of RBS+HRE within the RTA promoter are essential for KSHV latency and hypoxia-induced lytic reactivation.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV), a DNA tumor virus, is an etiological agent linked to several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). HIF-1α, a key hypoxia-inducible factor, is frequently elevated in KSHV latently infected tumor cells and contributes to KSHV lytic replication in hypoxia. The molecular mechanisms of how KSHV controls the latent and lytic life cycle through deregulating HIF-1α remain unclear. In this study, we found that inhibition of hypoxia-sensitive chromatin remodeler KAP1 in KSHV-infected PEL cells leads to a loss of viral genome and increases its sensitivity to hypoxic stress, leading to KSHV lytic reactivation. Importantly, we also found that four hypoxia-responsive clusters within the KSHV genome contain a high concurrence of RBP-Jκ (a key cellular regulator involved in Notch signaling) and HIF-1α binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-Jκ, and HIF-1α play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.

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Figures

FIG 1
FIG 1
Establishment of PEL stable cell lines with KAP1 or Sin3A knockdown. (A) Constitutive knockdown of KAP1 and Sin3A in PEL cells. PEL cell lines BC3 and BCBL1, parental and with KAP1 (shKAP1) or Sin3A (shSin3A) knockdown or scramble control (shCtrl), were individually generated by lentivirus-mediated transduction followed by selection with 2 μg/ml of selection. IB, immunoblotting. (B) Lower KSHV episome copy number in PEL cells with KAP1 or Sin3A constitutive knockdown. The genome DNA extracted from PEL cell lines BC3 and BCBL1 with shKAP1 or shSin3A or scramble control shRNA (shCtrl) from panel A were used to detect the relative intracellular viral episome copy number by quantitative PCR with TR as a target. GAPDH was used as an internal control. The results are presented as the average relative fold compared with parental cells from 3 independent knockdown clones from panel A. Double asterisks indicate a P value less than 0.05.
FIG 2
FIG 2
Loss of KAP1 or Sin3A dramatically enhances the relative efficiency of KSHV virion induction by TPA-NaB or hypoxia. (A) Equal amounts of parental BC3 cells with constitutive knockdown of KAP1 (shKAP1) or Sin3A (shSin3A) or scramble control (shCtrl) were cultured and exposed to TPA and butyrate (NaB) or 1% oxygen treatment for 0, 18, and 36 h. The supernatant of culture medium was subjected to quantitative PCR with TR or K8 as a target for virion production. The efficiency of extracellular KSHV DNA (virion) induction by TPA-NaB or hypoxia was detected and is presented as relative fold compared with mock treatment before induction. (B) A representative image of human PBMC infection with 36-h hypoxia induction virion from panel A at day 4 postinfection by LANA and DAPI nuclear staining.
FIG 3
FIG 3
Inhibition of KAP1 or Sin3A alters sensitivity of PEL cells to different extracellular stress. (A) Inhibition of KAP1 or Sin3A alters the sensitivity of PEL cells in response to lytic replication stimuli. BC3 cells with constitutively knocked down KAP1 or Sin3A were individually subjected to treatment with hypoxia (1% O2) or TPA-NaB (20 ng/ml, 1.5 mM) for 36 h. Cell lysates were subjected to immunoblotting as indicated. The relative density (RD) of RTA and LANA was quantified and is shown in the middle. (B) BC3 cells with constitutive KAP1 or Sin3A knockdown were individually subjected to treatment with or without 1% hypoxia overnight, followed by analysis of cell cycle profile. The average percentage of different phases (sub-G1, G1, S, G2/M) from three repeats is presented in a histogram.
FIG 4
FIG 4
Genome-wide screening of LANA, KAP1, Sin3A, HIF-1α, RTA, and RBP-Jκ binding sites within the KSHV genome. (A) (ChIP) assay was done by real-time PCR using a genome-wide array of 100 pair primers spaced across the KSHV genome using specific antibody as indicated. The enrichment of specific antibody bound to DNA was calculated by compared with input DNA and verified by an affinity more than 2-fold higher than for the nonspecific IgG parallel control. Graphs represent means ± standard deviations of two independent experiments. Chromatin DNA were prepared from KSHV-positive cell lines BC3 and BCBL1 with 21% (normoxia) or 1% (hypoxia) oxygen treatment for overnight before harvest. The results reveal four hypoxia-responsive clusters (HRC I, II, III, and IV) within the KSHV genome based on the HIF-1α–DNA binding region during hypoxia and indicated in light green. The main genes within these four clusters are shown in Fig. 5A. (B) Summary of KSHV whole-genome peak analysis for LANA, KAP1, Sin3A, HIF-1α, RBP-Jκ, and RTA binding chromatin from BC3 and BCBL1 cells under normoxic and hypoxic conditions. The amount of each protein binding site on KSHV genome is summarized as fold more than 2 from panel A, in parentheses.
FIG 5
FIG 5
RBP-Jκ is involved in hypoxia-induced transcription of KSHV lytic genes. (A) Quantitative PCR of transcriptional levels of HRE-containing viral genes from BC3 cells induction by hypoxia or TPA-NaB at different time points. The viral genes within the four hypoxia-responsive clusters (HRC) are presented at the top. The gene promoters containing HRE with an RTA (RtaBS) or RBP-Jκ (RBS) binding site are individually indicated by squares and circles. The relative ratio of each gene stimulated by hypoxia (H) and TPA-NaB (TB) was calculated as average value of induction at three time points. R/L, ratio of RTA with LANA. (B) ChIP assay of the HRE or HRE+RBS (RBP-Jκ binding site)-containing promoter. Chromatin DNA prepared from KSHV-positive cell line BC3 with 21% (−) or 1% oxygen (O2) treatment overnight before harvest was subjected to ChIP assays by antibodies as indicated. The DNA binding level of each protein was detected by qPCR, and the results are presented as percentages compared with input. Asterisks indicate P values as follows: *, P > 0.05, and **, P < 0.01.
FIG 6
FIG 6
The transcription activities of gene promoters containing HREs in hypoxia are impaired by both KAP1 and RBP-Jκ. (A) Inhibition of KAP1 enhances the association of RBP-Jκ with HIF complex in hypoxia. In vitro DNA-binding assays of RBP-Jκ were performed under normoxic and hypoxic conditions. Nuclear cell extracts from BC3/shCtrl or BC3/shKAP1 with 21% or 1% oxygen treatment overnight were individually incubated with wild-type (Wt) or mutant (Mut) HIF-1α-binding DNA oligonucleotide followed by three washes, and the precipitations were Western blotted with RBP-Jκ antibodies. The relative density (RD) of RBP-Jκ binding affinity is presented. (B) Inhibition of KAP1 dramatically enhances the transcriptional level of the RTA promoter with a RBP-Jκ binding site in hypoxia. 293 cells were transfected with the luciferase reporter of the RTA promoter (pRta-Luc) with RBS and HRE or HRE alone in the presence or absence of KAP1 knockdown (shKAP1). At 36 h posttransfection, cells were subjected to treatment with or without 1% oxygen for 12 h before harvest. The results are shown as the RLU (relative luciferase unit) fold induction of pRta-Luc compared with pGL-3 vector alone. Data are presented as means ± standard deviations of three independent experiments. (C) Percentages of HREs and RBP-Jκ binding sites (RBS) within the same gene promoter of total HREs in host cellular and KSHV genomes. The data are summarized from Table 2. The sequence logo of conserved RBS used for analysis is shown at the bottom.
FIG 7
FIG 7
Schematic illustrating the role of both KAP1 and RBP-Jκ in hypoxia-induced KSHV reactivation. There are at least four hypoxia-responsive clusters (HRC I, II, III, and IV) within KSHV chromatin. During latent infection, to efficiently silence HRE-responsive gene expression and maintain the viral episome, LANASIM-mediated chromatin silencer KAP1 targets the gene promoter of each HRC region with a high concurrence of RBP-Jκ binding site and HRE to inhibit viral gene expression (i.e., RTA) in normoxia. In contrast, each HRC region of KSHV chromatin in hypoxia (1% O2) undergoes three steps to reactivate lytic replication. In step 1, loss of SUMO-modified KAP1 induced by hypoxia (confirmed by shRNA-mediated inhibition of KAP1) remodels the chromatin structure of RBP-Jκ and HIF-1α binding sites; in step 2, the formation of RBP-Jκ–LANA–HIF-1α active complex in hypoxia greatly induces RTA expression; and in step 3, the expressed RTA will feed back to further globally activate KSHV lytic replication to produce virion progeny.
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