Journal:
Article Title: Two Putative ?-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms
doi:
Figure Lengend Snippet: Nuclear import by predicted α-helical domains αH1 and αH2. (A) Construction of plasmids derived from pEGFP-N1 that encode wild-type Vpr, αH1, αH2, or a substitution mutant form of Vpr fused at the carboxyl terminus to GFP and at the amino terminus to the Flag tag. The helical regions of Vpr encoded by each plasmid are in gray; the positions of the substituted amino acids are numbered, and amino acids are indicated in the single-letter code. (B) Amino acid sequences of αH1, αH2, and the NLS of the large T antigen of SV40. (C) Analysis by radiolabeling and immunoprecipitation of transfected HeLa cells that express the indicated fusion proteins. One day after transfection with the pEGFP-N1 plasmids indicated above the lanes, HeLa cells were incubated with Redivue Pro-Mix (a mixture of [35S]methionine and [35S]cysteine [1,000 Ci/mmol]; Amersham Pharmacia Biotech) at a concentration of 200 μCi/ml for 2 h. The cells were lysed at 4°C in lysis buffer {0.15 M NaCl, 0.05 M Tris-HCl [pH 8.5], 5 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid [CHAPS; Sigma]} with a cocktail of protease inhibitors (Boehringer Mannheim Biochemicals) and centrifuged for 20 min at 17,000 × g and 4°C to remove cell debris. Supernatants were subjected to immunoprecipitation with Flag-specific MAb M2 and protein A-Sepharose 4FF (50% [vol/vol]; Amersham Pharmacia Biotech.). The immunoprecipitates were dissolved in 0.05 M Tris-HCl (pH 6.8)–0.1 M dithiothreitol–2% sodium dodecyl sulfate–10% glycerol–0.001% bromphenol blue, heated at 100°C for 5 min, and analyzed by sodium dodecyl sulfate–5 to 20% polyacrylamide gel electrophoresis with subsequent fluorography. Arrowheads indicate the positions of chimeric proteins. (D) Photomicrographs of transfected HeLa cells that express the indicated fusion constructs. HeLa cells were transfected with derivatives of pEGFP-N1 that encode Flag-Vpr-GFP (e and f), Flag-αH1-GFP (g and h), Flag-αLA/αH1-GFP (i and j), Flag-αH2-GFP (k and l), Flag-I60P/αH2-GFP (m and n), Flag-L67P/αH2-GFP (o and p), Flag-SV40 NLS-GFP (c and d), or the control protein Flag-GFP (a and b). At 24 h after transfection, cells were treated (+) with digitonin at 40 μg/ml and then with 0.025% Triton X-100 (b, d, f, h, j, l, n, and p) or were not treated (−) with these reagents (a, c, e, g, i, k, m, and o) and then they were fixed in 1% formalin. The fusion proteins were detected by indirect immunofluorescence staining with Flag-specific MAb M2 and Cy3-conjugated antibodies against mouse IgG. Then they were examined by confocal laser scanning microscopy at a focal plane near the center of each nucleus. Bar, 20 μm.
Article Snippet: The fragment obtained by PCR was subcloned in the GFP expression vector pEGFP-N1 (Clontech Laboratories) to yield pGFP-Fvpr.
Techniques: Derivative Assay, Mutagenesis, FLAG-tag, Plasmid Preparation, Radioactivity, Immunoprecipitation, Transfection, Incubation, Concentration Assay, Lysis, Polyacrylamide Gel Electrophoresis, Construct, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy
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![Nuclear import by predicted α-helical domains αH1 and αH2. (A) Construction of plasmids derived from <t>pEGFP-N1</t> that encode wild-type Vpr, αH1, αH2, or a substitution mutant form of Vpr fused at the carboxyl terminus to <t>GFP</t> and at the amino terminus to the Flag tag. The helical regions of Vpr encoded by each plasmid are in gray; the positions of the substituted amino acids are numbered, and amino acids are indicated in the single-letter code. (B) Amino acid sequences of αH1, αH2, and the NLS of the large T antigen of SV40. (C) Analysis by radiolabeling and immunoprecipitation of transfected HeLa cells that express the indicated fusion proteins. One day after transfection with the pEGFP-N1 plasmids indicated above the lanes, HeLa cells were incubated with Redivue Pro-Mix (a mixture of [35S]methionine and [35S]cysteine [1,000 Ci/mmol]; Amersham Pharmacia Biotech) at a concentration of 200 μCi/ml for 2 h. The cells were lysed at 4°C in lysis buffer {0.15 M NaCl, 0.05 M Tris-HCl [pH 8.5], 5 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid [CHAPS; Sigma]} with a cocktail of protease inhibitors (Boehringer Mannheim Biochemicals) and centrifuged for 20 min at 17,000 × g and 4°C to remove cell debris. Supernatants were subjected to immunoprecipitation with Flag-specific MAb M2 and protein A-Sepharose 4FF (50% [vol/vol]; Amersham Pharmacia Biotech.). The immunoprecipitates were dissolved in 0.05 M Tris-HCl (pH 6.8)–0.1 M dithiothreitol–2% sodium dodecyl sulfate–10% glycerol–0.001% bromphenol blue, heated at 100°C for 5 min, and analyzed by sodium dodecyl sulfate–5 to 20% polyacrylamide gel electrophoresis with subsequent fluorography. Arrowheads indicate the positions of chimeric proteins. (D) Photomicrographs of transfected HeLa cells that express the indicated fusion constructs. HeLa cells were transfected with derivatives of pEGFP-N1 that encode Flag-Vpr-GFP (e and f), Flag-αH1-GFP (g and h), Flag-αLA/αH1-GFP (i and j), Flag-αH2-GFP (k and l), Flag-I60P/αH2-GFP (m and n), Flag-L67P/αH2-GFP (o and p), Flag-SV40 NLS-GFP (c and d), or the control protein Flag-GFP (a and b). At 24 h after transfection, cells were treated (+) with digitonin at 40 μg/ml and then with 0.025% Triton X-100 (b, d, f, h, j, l, n, and p) or were not treated (−) with these reagents (a, c, e, g, i, k, m, and o) and then they were fixed in 1% formalin. The fusion proteins were detected by indirect immunofluorescence staining with Flag-specific MAb M2 and Cy3-conjugated antibodies against mouse IgG. Then they were examined by confocal laser scanning microscopy at a focal plane near the center of each nucleus. Bar, 20 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2238/pmc00112238/pmc00112238__jv1500346003.jpg)