PLoS One. 2014 Oct 27;9(10):e111256. doi: 10.1371/journal.pone.0111256.

The HBx oncoprotein of hepatitis B virus deregulates the cell cycle by promoting the intracellular accumulation and re-compartmentalization of the cellular deubiquitinase USP37.

Saxena N, Kumar V.

Virology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

 

Abstract

The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonists in driving hepatocarcinogenesis. HBx exerts its influence over the cell cycle progression by potentiating the activity of cyclin A/E-CDK2 complex, the Cyclin A partner of which is a well-known target of cellular deubiquitinase USP37. In the present study, we observed the intracellular accumulation of cyclin A and USP37 proteins under the HBx microenvironment. Flow cytometry analysis of the HBx-expressing cells showed deregulation of cell cycle apparently due to the enhanced gene expression and stabilization of USP37 protein and deubiquitination of Cyclin A by USP37. Our co-immunoprecipitation and confocal microscopic studies suggested a direct interaction between USP37 and HBx. This interaction promoted the translocation of USP37 outside the nucleus and prevented its association and ubiquitination by E3 ubiquitin ligases – APC/CDH1 and SCF/β-TrCP. Thus, HBx seems to control the cell cycle progression via the cyclin A-CDK2 complex by regulating the intracellular distribution and stability of deubiquitinase USP37.

PMID: 25347529

 

Summary

Viral oncoprotein HBx engages cellular deubiquitinase USP37 to deregulate cell cycle

Hepatitis B Virus (HBV) through its HBx oncoprotein reins various physiological processes to gain leverage inside the host cell. Like any oncovirus, HBV manipulates the cell cycle to augur unchecked host cell proliferation. HBx deregulates the cell cycle by exacerbating the activity of Cyclin E/A-CDK2 complex and maintaining Cyclin A protein levels all through the cell cycle [1] as a prominent deviation from the normal waxing and waning of Cyclin A levels [2]. Recently, deubiquitinating enzyme USP37 was shown to stabilize Cyclin A by reversing the activity of Cyclin A- E3 ubiquitin ligase- CDH1 at the G1/S boundary of cell cycle [3]. However, the role of USP37 in oncogenesis is still unclear. The overexpression of USP37 is associated with poor prognosis of non-small cell lung cancer. Further, it plays a pivotal role in the stabilization of tumor suppressor p27 in medulloblastoma cells and promyelocytic leukemia zinc finger and retinoic acid receptor alpha (PLZF-RARA) oncogenic fusion protein in Acute promyelocytic leukemia [4-6]. With this background, we investigated the role of USP37 in the enhancement of Cyclin A stability in the HBx microenvironment. We found that while the normal USP37 conferred stability to Cyclin A, the deubiquitinase (DUB) dead mutant of USP37 (USP37-DD) failed to stabilize Cyclin A in the presence of HBx. Using BrdU incorporation and FACS assays, we report that USP37 along with HBx hastened the cell entry into the S phase of cell cycle while this was reversed in the presence of USP37-DD mutant [7].

After elucidating the role of USP37 in HBx-mediated cell cycle deregulation, we next probed a possible regulatory interaction between HBx and USP37. We found that both mRNA as well as protein levels of USP37 were elevated in the presence of HBx. The increased USP37 mRNA levels was attributed to the increased E2F1 activity. The stability of USP37, as determined by half-life and ubiquitination assays, was also enhanced in presence of HBx. The Cyclin /CDK2 complex is known to enhance the stability of USP37 by mediating phosphorylation at Ser-628 in USP37 [3]. Akin to the known enhancement in the Cyclin A/CDK2 activity in the HBx microenvironment, we observed a marked reduction in the levels of USP37 protein following inhibition of CDK2 activity in the HBx expressing cells. This was substantiated by a drastic reduction in the phosphorylated USP37 levels. Thus, Cylin A/CDK2-mediated phosphorylation seems to confer stability to USP37 in presence of HBx.

To explore other operating mechanisms contributing towards USP37 stability, we performed co-immunoprecipitation studies, ubiquitination assays and confocal experiments with some interesting results [7]. We uncovered an intricate mechanism wherein USP37, primarily a nuclear protein [8], was chaperoned out of the nucleus by HBx. Interestingly, this transport was compromised in the presence of a nuclear export signal mutant of HBx (HBx-NESM) [9]. Previous studies have indicated the presence of USP37-E3 ligases β-TrCP and CDH1 inside the nucleus [10,11]. We also confirmed a nuclear localization of β-TrCP and CDH1 in our confocal studies.  We observed that the interaction between USP37 and its E3 ligases was disrupted in the presence of HBx but not by its HBx-NESM mutant. Further, in agreement with earlier observations, we found that the ubiquitination of USP37 was also attenuated in the presence of HBx but not by its HBx-NESM mutant. Thus, we hypothesize that USP37 protein levels are stabilized due to physical segregation of USP37 from its E3 ligases in the HBx microenvironment (Figure 1).

 

Figure  - Saxena and KumarFigure 1. Schematic representation of molecular events leading to the stabilization and intracellular redistribution of USP37 in the presence of viral HBx. Events that occur in a normal cell are highlighted in gray and the events that occur in the presence of HBx oncoprotein have been highlighted in red.

 

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