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

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.

Nucleolar stress enhances lytic reactivation of the Kaposi’s sarcoma-associated herpesvirus

“…As shown in Figure 4 , FACS analysis of mCherry-positive cells revealed that treatment with Nutlin-3 alone induced a minor increase in lytic reactivation, which was enhanced when combined with Dox treatment. This result is in agreement with a previous report [ 45 ]. Combined treatment with Nutlin-3 and Dox together with induction of nucleolar stress with Act D or CX-5461 resulted in a further additive increase in the fraction of cells expressing mCherry.…”

“…Previous studies employing latently KSHV-infected cells have shown that inhibition of MDM2 by Nutlin-3, as well as genetic knockdown of MDM2 [ 45 ], result in activation of the tumor suppressor p53 pathway and stabilization of K-Rta leading to cell cycle arrest, massive cell death and disruption of viral latency. Based on these observations, we hypothesized that nucleolar stress, described as failures in ribosome biogenesis or function, which are often coupled with inhibition of MDM2 and p53 activation, might promote lytic reactivation of KSHV.…”

Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase

“…Coimmunoprecipitation analysis was performed as described previously ( 42 ). Briefly, 293T cells were cotransfected with expression vectors encoding myc-tagged NTPase and FLAG-tagged proteins as indicated in Fig.…”

Diabetes and risk of Kaposi's sarcoma: effects of high glucose on reactivation and infection of Kaposi's sarcoma-associated herpesvirus

“…The target sequence of the c-Jun shRNA is 5′-ATTCGATCTCATTCAGTATTA. Preparation of shRNA lentiviral particles has been described previously [ 41 ].…”

“…After washing with PBS to remove unbound virions, cells were cultured in normal or high glucose for another 24 hours. Immunofluorecent staining was performed as described previously [ 41 ]. For infecting B cells, BJAB cells were co-cultivated with 293T(BAC16) at a 3:1 ratio in normal or high glucose-containing media for 4 days.…”