Regulation of the Abundance of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2.

Tzu-Hsuan Chang,Pey-Jium Chang,Shie-Shan Wang,Li-Kwan Chang,Ying-Ju Shih,Lee-Wen Chen,Shih-Tung Liu,Shou-Jiang Gao

doi:10.1371/journal.ppat.1005918

Abstract

The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.

Highlights

Kaposi’s sarcoma-associated herpesvirus (KSHV), referred to as human herpesvirus-8 (HHV-8), is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphomas (PELs) and multicentric Castleman’s disease [1,2,3]
The switch of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is an essential process for controlling viral dissemination and the pathogenesis of KSHV-associated diseases
We previously reported that a two-component protein abundance regulatory signal (PARS) region, which contains PARS-I and PARS-II, in open reading frame 50 (ORF50) influences its protein abundance

Summary

Introduction

Kaposi’s sarcoma-associated herpesvirus (KSHV), referred to as human herpesvirus-8 (HHV-8), is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphomas (PELs) and multicentric Castleman’s disease [1,2,3]. Like a typical transcription activator, ORF50 has both a DNA-binding domain (aa 1–390) and an activation domain (aa 486–691), which are located in the N-terminal and C-terminal regions, respectively [10, 11]. Earlier studies showed that nuclear translocation of ORF50 protein is mediated through a nuclear localization signal, KKRK, located between aa 527 and 530 [12]. Studies from Yu et al [9] showed that a RING-like ubiquitin E3 ligase domain is located near the N-terminal region from aa 118 to 207 (Fig 1). Izymiya et al [16] reported that ORF50 has multiple small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) distributed in the region between aa 239 and 503 (Fig 1). Due to the presence of the intrinsic ubiquitin E3 ligase activity and the capacity to bind SUMOs with high affinity, ORF50 is suggested to be a SUMO-targeting ubiquitin ligase doi:10.1371/journal.ppat.1005918.g001

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Discussion

Conclusion