Presence of a functional but dispensable nuclear export signal in the HTLV-2 Tax protein

Abstract

Background: Human T-cell leukemia virus type 1 and type 2 are related human retroviruses. HTLV-1 is the etiological agent of the Adult T-cell Leukemia/Lymphoma and of the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy, whereas, HTLV-2 infection has not been formally associated with any T-cell malignancy. HTLV-1 and 2 genomes encode, respectively, the Tax1 and Tax2 proteins whose role is to transactivate the viral promoter. HTLV-1 and HTLV-2 Tax sequences display 28% divergence at the amino acid level. Tax1 is a shuttling protein that possesses both a non canonical nuclear import (NLS) and a nuclear export (NES) signal. We have recently demonstrated that Tax1 and Tax2 display different subcellular localization and that residues 90-100 are critical for this process. We investigate in the present report, whether Tax2 also possesses a functional NES.

Results: We first used a NES prediction method to determine whether the Tax2 protein might contain a NES and the results do suggest the presence of a NES sequence in Tax2. Using Green Fluorescent Protein-NES (GFP-NES) fusion proteins, we demonstrate that the Tax2 sequence encompasses a functional NES (NES2). As shown by microscope imaging, NES2 is able to mediate translocation of GFP from the nucleus, without the context of a full length Tax protein. Furthermore, point mutations or leptomycin B treatment abrogate NES2 function. However, within the context of full length Tax2, similar point mutations in the NES2 leucine rich stretch do not modify Tax2 localization. Finally, we also show that Tax1 NES function is dependent upon the positioning of the nuclear export signal "vis-à-vis" GFP.

Conclusion: HTLV-2 Tax NES is functional but dispensable for the protein localization in vitro.

Figure 1

Output after submission of the complete Tax2 amino acid sequence to the NetNES website [23]. Output consists of two parts; one is a listing of all residues with the individual scores noted (see column heading), the other is a graphical plot of the values given in the table. The prediction server calculates the NES score from the HMM and Artificial Neural Network (ANN) scores but all three values are given for each residue. If the calculated 'NES score' exceeds the threshold, then that particular residue is expected to participate in a nuclear export signal. This is denoted with a 'Yes' in the column "Predicted".

Figure 2

Without the context of the full length protein, NES2 can redirect GFP to the cytoplasm, while NES1 function depends on its positioning vis-à-vis GFP. (A): Sequence alignment of a consensus NES sequence with Tax1 NES and Tax2 putative NES. (B): HeLa cells were transiently transfected with NES-EGFP and GFP-NES plasmids. Twenty-four hours after transfection, the cells were washed with PBS, fixed with 4% paraformaldehyde, mounted with DAPI-containing mounting medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Axiocam HRc (color) camera and the Zeiss Apotome software. Images of cells that are representative of the entire population are shown. (C): Western-blot analysis of cytoplasmic and nuclear cell fractions. 293T cell fractions were subjected to electrophoresis on a 10 % TG gel and probed with a GFP antibody. The western-blot results are representative of four independent experiments.

Figure 3

Nucleocytoplasmic distribution of GFP-NES2 is altered after incubation with leptomycin B or by single point mutations. (A): Sequence alignment of wild-type Tax2 and Tax2 GFP-NES mutants. (B and D): HeLa cells were transiently transfected with the different GFP-NES plasmids. Eighteen hours post transfection, transfected cells were treated with leptomycin B (40 nM) or methanol for 3 hours. Cells were then washed, fixed, mounted with DAPI-containing medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Axiocam HRc (color) camera and the Zeiss Apotome software. Images of cells that are representative of the entire population are shown. (C and E): Western-blot analysis of GFP and GFP-NES proteins. 293T cell lysates (70 μg) were subjected to electrophoresis on a 10 % TG gel and probed with GFP or β-tubulin antibodies.

Figure 4

The presence of leucine 188 restores NES1 function within the context of a NES1-EGFP protein. (A): Western-blot analysis of the different EGFP and NES-EGFP proteins. 293T cell lysates (70 μg) were subjected to electrophoresis on a 10 % TG gel and probed with GFP or β-tubulin antibodies. (B): HeLa cells were transiently transfected with the different NES-EGFP plasmids. Eighteen hours post transfection, transfected cells were washed, fixed, mounted with DAPI-containing medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Axiocam HRc (color) camera and the Zeiss Apotome software. Images of cells that are representative of the entire population are shown. (C): 293T nuclear and cytoplasmic cell fractions were subjected to electrophoresis on a 10 % TG gel and probed with a GFP antibody. The western-blot results are representative of four independent experiments.

Figure 5

Within the context of a full length Tax2 protein, the presence of a functional NES2 domain is dispensable for the protein localization. (A): HeLa cells were transiently transfected with the GFP-Tax1, GFP-Tax2 and the different GFP-Tax2 mutants plasmids. Eighteen hours post transfection, the cells were washed, fixed, mounted with DAPI-containing medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Axiocam HRc (color) camera and the Zeiss Apotome software. Images of cells that are representative of the entire population are shown. (B): Western-blot analysis of GFP and GFP-NES proteins. 293T cell lysates (70 μg) were subjected to electrophoresis on a 10 % TG gel and probed with GFP or β-tubulin antibodies.