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


OBJECTIVE
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.
MOE& MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China Department of Clinical Laboratory, Central Hospital of Taizhou City, Taizhou, People's Republic of China.
Liming
L
Zhang
MOE& MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.
Caixia
C
Zhu
Department of Gynecology, Key Laboratory of AIDS Immunology of Ministry of Health, First Affiliated Hospital of China Medical University, Shenyang, People's Republic of China.
Yi
Y
Guo
ShengYushou Center of Cell Biology and Immunology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
Fang
F
Wei
Department of Microbiology and Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Jie
J
Lu
Department of Biochemistry and Center for Genome Science, The University of Hong Kong, Hong Kong, People's Republic of China.
Jing
J
Qin
Department of Microbiology and Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Shuvomoy
S
Banerjee
Department of Biochemistry and Center for Genome Science, The University of Hong Kong, Hong Kong, People's Republic of China.
Junwen
J
Wang
Department of Gynecology, Key Laboratory of AIDS Immunology of Ministry of Health, First Affiliated Hospital of China Medical University, Shenyang, People's Republic of China.
Hong
H
Shang
Department of Microbiology and Immunology, School of Medicine, University of Nevada, Reno, Nevada, USA.
Subhash C
SC
Verma
MOE& MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.
Zhenghong
Z
Yuan
Department of Microbiology and Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA qiliang@fudan.edu.cn erle@upenn.edu.
Erle S
ES
Robertson
MOE& MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China qiliang@fudan.edu.cn erle@upenn.edu.
Qiliang
Q
Cai
D002453:Cell Cycle; D045744:Cell Line, Tumor; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology / Q000821:virology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012514:Sarcoma, Kaposi / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology / Q000821:virology; D000076183:Tripartite Motif-Containing Protein 28; D014775:Virus Activation; D017735:Virus Latency
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C497570:RBPJ protein, human; D012097:Repressor Proteins; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; D010100:Oxygen