HTLV-I Basic Leucine Zipper Factor (sHBZ) Actively Associates with Nucleophosmin (B23) in the Nucleolus

Abstract

Human T cell leukemia virus type 1 (HTLV 1) is an oncogenic retrovirus responsible for the development of adult T cell leukemia (ATL). The minus strand of HTLV-1 provirus encodes an oncoprotein named HTLV-1 bZIP factor (HBZ), which plays a pivotal role in viral replication and T cell proliferation. Of particular interest is the spliced HBZ isoform (sHBZ), which is predominantly expressed in ATL cells and localizes within the nucleolus, conferring immortalizing properties to T cells. Our previous study has shown that sHBZ colocalizes and associates with Nucleophosmin/B23, a nucleolar phosphoprotein with multiple functions. In this study, through an optimized nucleolar isolation method, we first confirmed sHBZ's nucleolar localization via Western blotting in transfected HEK293T cells, chronically HTLV-1-infected T cell lines, and freshly infected HeLa cells. We further demonstrated that the sHBZ/B23 association predominantly occurs in the nucleolus by co-immunoprecipitation of cell fractions. Our study highlights the nucleolar localization of sHBZ and its possibly essential interaction with this nucleolar-residing protein, leading to cell immortalization.

Keywords: ATL; HTLV-1; T cell proliferation; nucleolus; nucleophosmin/B23; sHBZ.

Figure 1

Efficiency of the nucleolar extraction method. Nucleoli were isolated from HeLa (H) and HEK293T (T) cells (A) and C8166-45 T cells (B). Proteins from subcellular fractions were separated by 12% SDS-PAGE and probed with anti-α-tubulin (cytoplasmic), anti-NUP62 (nucleoplasmic), and anti-fibrillarin (nucleolar) antibodies. Each lane was loaded with a fraction sample derived from the same number of cells. These results are representative of three independent experiments. Wc, whole cell; Cp, cytoplasm; Nu, nuclei; Np, nucleoplasm; No, nucleoli.

Figure 2

Subcellular distribution of sHBZ. (A) HeLa cells were transfected with expression vectors for myc-tagged sHBZ (vs. pcDNA3.1) and GFP-fused sHBZ (vs. GFP), usHBZ, and APH2. Fixed cells were analyzed by confocal microscopy using anti-B23 and/or anti-myc antibodies following staining with DAPI. (B) HEK293T cells were transfected with expression vectors for myc-tagged sHBZ and GFP-fused sHBZ, usHBZ, and APH2. Proteins from the different subcellular fractions were analyzed by Western blot with anti-HBZ or anti-GFP, antibodies. Each lane was loaded with a fraction sample derived from the same number of cells. These results are representative of three independent experiments. Wc, whole cell; Cp, cytoplasm; Nu, nuclei; Np, nucleoplasm; No, nucleoli.

Figure 3

sHBZ-B23 association occurs predominantly in the nucleoli. HEK293T cells were transfected with expression vectors for myc-tagged sHBZ and/or empty vectors as a negative control (-) (A). Transfected HEK293T (A), C8166-45 (B), and non-infected HeLa (-) and infected HeLa cells (C) were subjected to nucleolar isolation. Cellular fractions were co-immunoprecipitated with anti-myc or anti-HBZ antibodies. Immunoprecipitates or cellular fractions were analyzed by Western blotting using anti-B23 or anti-HBZ antibodies. The intensity of B23 signals was quantified by densitometric analyses using ImageJ 1.54f. The negative control (-) in C represents HeLa cell cultures in the absence of MT2 cells. Wc, whole cell; Cp, cytoplasm; Nu, nuclei; Np, nucleoplasm; No, nucleoli.

Figure 3

sHBZ-B23 association occurs predominantly in the nucleoli. HEK293T cells were transfected with expression vectors for myc-tagged sHBZ and/or empty vectors as a negative control (-) (A). Transfected HEK293T (A), C8166-45 (B), and non-infected HeLa (-) and infected HeLa cells (C) were subjected to nucleolar isolation. Cellular fractions were co-immunoprecipitated with anti-myc or anti-HBZ antibodies. Immunoprecipitates or cellular fractions were analyzed by Western blotting using anti-B23 or anti-HBZ antibodies. The intensity of B23 signals was quantified by densitometric analyses using ImageJ 1.54f. The negative control (-) in C represents HeLa cell cultures in the absence of MT2 cells. Wc, whole cell; Cp, cytoplasm; Nu, nuclei; Np, nucleoplasm; No, nucleoli.