First published online 16 September 2003
doi: 10.1242/jcs.00747
Intracellular transport of human immunodeficiency virus type 1 integrase
Eric Devroe1,2,
Alan Engelman3,4 and
Pamela A. Silver1,2,*
1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
2 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
3 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA
View larger version (76K):
[in a new window]
|
Fig. 1. IN colocalizes with DNA. (A,C,E) IN localization monitored by indirect fluorescence microscopy. (B,D,F) DNA visualized with DAPI staining. (E,F) Enlarged views of the dividing cells in (C,D).
|
|
View larger version (79K):
[in a new window]
|
Fig. 2. Lack of evidence for a transferable NLS within IN. (A) HeLa cells were transiently transfected with pGFP-PK (a), pGFP-PK-Vpr73-96 (b) or pGFP-PK-IN (c-f) and monitored by fluorescence microscopy. As expected (Sherman et al., 2001 ), GFP-PK was exclusively localized to the cytoplasm (a) and the NLS within Vpr actively transported GFP-PK-Vpr73-96 to the cell nucleus (b). By contrast, GFP-PK-IN localized exclusively to the cytoplasm in most cells (c-e). In rare instances, some degree of nuclear staining was also observed (e,f, arrows). (B) Western-blot analysis of cells transiently transfected with the plasmids described in (A) or pEGFP-C1 (Clontech Laboratories). Fusion proteins were detected with an anti-GFP antibody. Molecular mass markers in kDa are indicated to the left of the blot.
|
|
View larger version (39K):
[in a new window]
|
Fig. 3. Localization of IN fused to a strong NES. (A) Schematic of IN intracellular transport and colocalization with DNA. (B) Predicted intracellular dynamics of IN-NES. Following nuclear entry, IN-NES should be exported back into the cytoplasm. (C) HeLa cells were transiently transfected or stably infected with viruses expressing IN or IN-NES. In contrast to IN (a,b), IN-NES was localized to the cytoplasm of most transiently transfected cells (d,e). In rare instances, IN-NES was also observed within the nucleus of transfected cells (e, arrow). However, IN-NES was observed almost exclusively in the nucleus of stably expressing cells (f), a pattern indistinguishable from IN (c). (D) Western-blot analysis of untransfected cells (Unt) or cells transiently transfected or stably expressing IN or IN-NES. 10 µg of total protein was resolved by SDS-PAGE. IN and IN-NES were detected by probing blots with 12CA5 anti-HA antibody. The migration positions of molecular mass markers in kDa are indicated to the right of the gel. Notice the ten-amino-acid NES insertion, which resulted in a slight shift in migration compared with wild-type IN. *, cross-reacting band.
|
|
View larger version (48K):
[in a new window]
|
Fig. 4. Cytoplasmic IN is subject to proteasome-dependent degradation. (A) HeLa cells (Mock) or cells stably expressing IN (IN) were treated with DMSO (–) or 5 µM MG-132 (+) for 5 hours. Following this treatment, nuclear and cytoplasmic extracts were prepared and an equal fraction of each was analysed by western blotting with the 12CA5 antibody. The stabilized IN predominantly localized to the cytosolic fraction, although a slight increase in nuclear staining was also observed. We note that the apparent cytoplasmic abundance of IN by fractionation was due to incomplete extraction of IN from nuclear lysates (data not shown) (Cherepanov et al., 2003). (B) HeLa cells were mock transfected (–) or transiently transfected with IN (IN). 18 hours later, cells were pulsed with 5 µM MG-132 for 5 hours. Subsequently, whole cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-ubiquitin (Ub) antibodies. High-molecular-weight ubiquitin conjugates were detected in IN immunoprecipitates by anti-Ub western blotting (left). Western blotting with the anti-FLAG antibody confirms the expression and immunoprecipitation of IN (right). Following immunoprecipitation of whole cell extracts with anti-Ub antibodies, IN was detected by anti-FLAG western blotting (right). Abbreviations: IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain. Owing to the high levels of antibody used for the rabbit anti-Ub immunoprecipitation, the FLAG antibody cross-reacted with the rabbit heavy chain.
|
|
View larger version (62K):
[in a new window]
|
Fig. 5. Altered localization of an IN DNA-binding mutant. (A) Wild-type IN (a-c) or the DNA-binding defective mutant K156E/K159E (d-f) was localized in transiently transfected HeLa cells following incubation in DMSO (a,d) or 5 µM MG-132 for 5 hours (b,c,e,f). Notice that the DMSO-treated cells expressing K156E/K159E IN exhibited a considerable degree of cytoplasmic staining (d), a pattern that mirrored wild-type IN stabilized with MG-132 (b,c). Following MG-132 treatment, cells expressing K156E/K159E IN exhibited essentially diffuse nuclear and cytosolic staining (e,f), although some residual nuclear accumulation was also observed (e,f, arrows). (B) Quantitation of nuclear and cytoplasmic distribution of IN and K156E/K159E IN. Indirect immunofluorescence microscopy was performed and ten fields of view were captured for each transfection condition (total of  200 transfected cells). The intensity of nuclear IN (based on colocalization with DAPI) versus cytoplasmic IN (signal outside of DAPI) was quantified for each field of view using MetaMorph software. For each condition, the mean percentage of cytoplasmic IN signal across ten fields of view is presented. Error bars represent standard deviation between the ten fields of view. (C) HeLa cells stably expressing IN or K156E/K159E were examined by indirect immunofluorescence microscopy. K156E/K159E-IN-expressing cells frequently exhibited more cytoplasmic staining than WT IN cells, although significant nuclear accumulation was also observed.
|
|
© The Company of Biologists Ltd 2003
