Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5

Amanda R Panfil,Mamuka Kvaratskhelia,Jacob Al-Saleem,Cory M Howard,Patrick L Green,Nikoloz Shkriabai

doi:10.3389/fmicb.2018.00080

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) encodes a protein derived from the antisense strand of the proviral genome designated HBZ (HTLV-1 basic leucine zipper factor). HBZ is the only viral gene consistently expressed in infected patients and adult T-cell leukemia/lymphoma (ATL) tumor cell lines. It functions to antagonize many activities of the Tax viral transcriptional activator, suppresses apoptosis, and supports proliferation of ATL cells. Factors that regulate the stability of HBZ are thus important to the pathophysiology of ATL development. Using affinity-tagged protein and shotgun proteomics, we identified UBR5 as a novel HBZ-binding partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Herein, we investigated the role of UBR5 in HTLV-1-mediated T-cell transformation and leukemia/lymphoma development. The UBR5/HBZ interaction was verified in vivo using over-expression constructs, as well as endogenously in T-cells. shRNA-mediated knockdown of UBR5 enhanced HBZ steady-state levels by stabilizing the HBZ protein. Interestingly, the related HTLV-2 antisense-derived protein, APH-2, also interacted with UBR5 in vivo. However, knockdown of UBR5 did not affect APH-2 protein stability. Co-immunoprecipitation assays identified ubiquitination of HBZ and knockdown of UBR5 resulted in a decrease in HBZ ubiquitination. MS/MS analysis identified seven ubiquitinated lysines in HBZ. Interestingly, UBR5 expression was upregulated in established T lymphocytic leukemia/lymphoma cell lines and the later stage of T-cell transformation in vitro. Finally, we demonstrated loss of UBR5 decreased cellular proliferation in transformed T-cell lines. Overall, our study provides evidence for UBR5 as a host cell E3 ubiquitin-protein ligase responsible for regulating HBZ protein stability. Additionally, our data suggests UBR5 plays an important role in maintaining the proliferative phenotype of transformed T-cell lines.

Highlights

Categorized as a tumorigenic virus, human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that infects and transforms CD4+ T-cells
PBL-ACH were maintained in RPMI 1640 supplemented with 20% fetal bovine serum (FBS), 20 U/mL recombinant human interleukin2, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL)
HBZ has been linked to HTLV-1-mediated oncogenic transformation and plays a role in the pathogenic process (Ma et al, 2016)

Summary

Introduction

Categorized as a tumorigenic virus, human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that infects and transforms CD4+ T-cells. HTLV-1 is responsible for a highly aggressive and chemotherapy-resistant peripheral T-cell malignancy called adult T-cell leukemia/lymphoma (ATL) (Uchiyama et al, 1977; Poiesz et al, 1980; Yoshida et al, 1982) and a chronic progressive neurodegenerative disease termed HTLV-1associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al, 1985; Osame et al, 1986). Not all HTLV-1 carriers will go on to develop disease. Disease penetrance is roughly 5% for the lifetime of an infected individual (Ishitsuka and Tamura, 2014). The discrepancy between individuals with disease development and lifelong healthy carriers is still not well understood

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