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The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins - PubMed

The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins

Andrea Gomez Corredor et al. J Virol. 2012 May.

Abstract

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.

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Figures

Fig 1
Fig 1

Nuclear import of the BIV Rev protein in HeLa cells is an active transport mechanism dependent on cytosolic factors, the nucleopore, energy, and Ran protein. The nuclear import of BIV EGFP Rev-His and rhodamine-conjugated BSA-NLSSV40 was examined by using an in vitro nuclear import assay. (A) Digitonin-permeabilized HeLa cells were incubated at 4 or 37°C with 2 μM BIV EGFP Rev-His or BSA-NLSSV40 in 50 μl of transport buffer alone or complete transport buffer containing an ATP regeneration system and rabbit reticulocyte lysate (identified as cytosol in the figure). For WGA treatment, permeabilized cells were preincubated with 0.8 mg/ml of WGA for 30 min at room temperature and incubated for 30 min at 37°C with complete transport buffer. The cells were washed with transport buffer and then fixed and stained with DAPI. The cells were visualized by fluorescence microscopy. (B) The nuclear import assay was performed as indicated above for panel A in the presence or absence of ATP regeneration system. For apyrase experiment, cells were preincubated with buffer containing 25 U/ml of apyrase for 15 min at 37°C and then incubated with complete transport buffer. (C) Digitonin-permeabilized HeLa cells were incubated with 2 μM BIV EGFP Rev-His or BSA-NLSSV40 in 50 μl of complete transport buffer in the presence of a 2 μM concentration of either Ran or RanQ69L. The cells were washed with transport buffer and then fixed and stained with DAPI. The cells were visualized by fluorescence microscopy.

Fig 2
Fig 2

The BIV Rev protein is not imported into the nucleus by importin β alone. Digitonin-permeabilized HeLa cells were incubated with a 2 μM concentration of either GST-FITC, BIV EGFP Rev-His, BSA-NLSSV40, or HIV-1 EGFP Rev-His proteins in 50 μl of transport buffer alone or in 50 μl of transport buffer containing ATP regeneration system, 2 μM Ran WT, and 2 μM GST-importin β. The cells were washed with transport buffer, fixed, and stained with DAPI. The cells were visualized by fluorescence microscopy.

Fig 3
Fig 3

The BIV Rev protein is transported into the nucleus by the classical nuclear import pathway involving importins α3 and α5. (A) Nuclear import assay using importin α1. (Left) Digitonin-permeabilized HeLa cells were pretreated with 50 μl of transport buffer containing 2 μg/μl of importin β-specific antibody, washed with transport buffer, and then incubated with 2 μM either BIV EGFP Rev-His or BSA-NLSSV40 in 50 μl of transport buffer alone or 50 μl of transport buffer containing ATP regeneration system, 2 μM Ran WT, 2 μM GST-importin α alone or used in combination with 2 μM GST-importin β. (Right) Import efficiencies of BIV EGFP Rev-His or BSA-NLSSV40 in the presence of buffer alone, GST-importin α alone or GST-importin α used in combination with importin β were quantified by measuring the fluorescence intensity in the nucleus and the cytoplasm. The results were expressed as the mean nucleus/cytoplasm (N/C) fluorescence ratio plus standard error of the mean (error bars). The values that were significantly different (P < 0.0001) in the importin α and importin α/β conditions using a two-tailed t test are indicated by four asterisks. Imp, importin. (B) Nuclear import assay using importin α3. (C) Nuclear import assay using importin α5. (D) Nuclear import in the presence of importin α ΔIBB. The nuclear import assay was conducted as described above for panel A, but only in the presence of importin α ΔIBB and not importin β. (E) GST pulldown assay. GST, GST-importin α1, GST-importin α3, GST-importin α5, and GST-importin β were immobilized on beads and incubated with BIV Rev-His. As a positive control in the GST pulldown assay, the HIV Rev-His protein was incubated with GST and GST-importin β. Bound proteins were analyzed by 12% SDS-PAGE followed by Western blotting (WB) using an anti-His6 tag antibody. (Bottom) Coomassie brilliant blue-stained gel to illustrate the purified GST and GST-importin input.

Fig 4
Fig 4

The nuclear export of the BIV Rev protein is CRM1 dependent. HEK 293T cells were cotransfected with either pEGFPRevWT or pEGFPRevΔ3 and pRed-C1Nucleolin plasmids. After 24 h of transfection, cells were incubated in the presence of 5 nM LMB or left untreated for 4 h. The cells were fixed, and the nuclei were counterstained with DAPI for cellular localization of the proteins. Expression of the proteins was detected via the fluorescence of EGFP (in green) or DsRed (in red). The merge image represents the superposition of either the BIV EGFP-Rev or BIV EGFP-Rev Δ3 image and the DsRed-Nucleolin image. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells.

Fig 5
Fig 5

The region encompassing amino acids (aa) 111 and 130 of BIV Rev is associated with the nuclear export function of the protein. (A) Cellular localization of BIV Rev WT and BIV Rev M6 mutant both fused to EGFP. HEK 293T cells were cotransfected with pEGFPRevWT or pEGFPRevM6 in combination with the pRed-C1Nucleolin plasmid. At 24 h after transfection, the cells were fixed, and the nuclei were counterstained with DAPI. The expression of the proteins was detected via the fluorescence of EGFP (in green) or DsRed (in red). The merged image represents the superposition of both EGFP and DsRed images. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells. (B) The nuclear export activity of EGFP-RevM6 (harboring the deletion of aa 111 to 130 within the BIV Rev WT sequence) fusion protein was determined using a CAT gene reporter assay performed with 50 μg of cell lysate (HEK 293T). The CAT levels were normalized to the β-galactosidase activity. The results represent mean values of triplicate samples of three separate experiments. Rev activity is expressed as the mean ratio of BIV EGFP-Rev (Rev WT) or mutant CAT expression versus basal expression of pRRE-BIV alone. The error bars indicate the standard errors of the means. Values that were significantly different (P < 0.0001) from the value for BIV EGFP-Rev using a two-tailed t test are indicated by four asterisks. (C) Bioinformatics analysis of the region deleted in Rev M6 showed that a sequence of 10 aa can harbor the NES. The putative NES peptide (112L-E-D-L-V-R-H-M-S-L121) was fused to the C-terminal end of EGFP. (D) HEK 293T cells were transfected with pEGFP or pEGFP-NES. At 24 h after transfection, the cells were incubated with LMB as described in the legend to Fig. 4. The cells were fixed and counterstained with DAPI (in blue). The merge image represents the superposition of EGFP and DAPI images. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells. (E) Alanine substitutions were introduced into pcDNA3.1Myc/His RevWT targeting the putative NES (112L-E-D-L-V-R-H-M-S-L121) sequence to generate pcDNA3.1Myc/HisRev NES mutant constructs. (F) The nuclear export activity of Rev-Myc/His BIV and Rev-Myc/His BIV NES mutants with the mutations in the sequence shown in panel E was determined using a CAT reporter assay as described above for panel B. Also shown is the expression of BIV Rev-Myc/His and BIV Rev-Myc/His mutants as determined by Western blotting of HEK 293T cells transfected with the appropriate plasmid constructs. Total cell proteins (50 μg) were separated on 12% SDS-polyacrylamide gels, electroblotted onto nitrocellulose membranes, and probed with Myc-specific antibody. α-Tubulin immunostaining was used as a loading control.

Fig 6
Fig 6

BIV Rev protein contains a strong nuclear export signal. (A) Schematic representation of the HIV-1 Rev(1.4)-EGFP nuclear export system. Rev(1.4) contains a nuclear localization signal (NLS) and disrupted nuclear export signal (NES), and the NES BIV sequence is inserted between Rev(1.4) and EGFP to recovery the NES function of HIV-1 Rev. The NLS-mediated import of Rev is blocked by treatment of cells with actinomycin D (ActD) (29), whereas NES-mediated export is prevented by treatment with leptomycin B (LMB). (B) The pRev(1.4)EGFP vectors were transfected into HeLa cells; 24 h after transfection, the cells were left untreated (Unt) or exposed to ActD or ActD/LMB and fixed. The subcellular localization of EGFP was determined as nuclear (N), nuclear cytoplasmic (NC), or cytoplasmic (C). The Rev(1.4)EGFP construct was used as a negative control. Rev(1.4)-NES3-EGFP was used as a positive control. The activities of Rev(1.4), Rev(1.4)-NES3, BIV Rev NES, and the different NES mutants were analyzed and scored by the method of Henderson and Eleftheriou (29). A very strong export function for BIV Rev NES sequence with a NES score of 9 was found. Values are the mean percentages of positive cells plus standard errors of the means (error bars) from three independent experiments.

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References

    1. Adam SA, Marr RS, Gerace L. 1990. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111:807–816 - PMC - PubMed
    1. Andersen JS, et al. 2005. Nucleolar proteome dynamics. Nature 433:77–83 - PubMed
    1. Arnold M, Nath A, Hauber J, Kehlenbach RH. 2006. Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1. J. Biol. Chem. 281:20883–20890 - PubMed
    1. Bian XL, Rosas-Acosta G, Wu YC, Wilson VG. 2007. Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal. J. Virol. 81:2899–2908 - PMC - PubMed
    1. Bogerd HP, Echarri A, Ross TM, Cullen BR. 1998. Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J. Virol. 72:8627–8635 - PMC - PubMed

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