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Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

Jenkins Y, McEntee M, Weis K, Greene WC - J. Cell Biol. (1998)

Bottom Line: Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy.Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites.Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, California 94141-9100, USA.

ABSTRACT
While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

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Vpr does not use  the classical NLS or the M9  pathway for entry into the nucleus. (A–H) Import of IBB– βgal, Vpr–βgal, Vpr(1–71)– βgal, and Vpr(73–96)–βgal  with lysate (control) (A, C, E,  and G) or with lysate containing 30 μM unlabeled IBB as a  competitor (B, D, F, and H).  (I–P) Import of GST-M9  (FITC-labeled), Vpr–βgal,  Vpr(1–71)–βgal, and Vpr(73– 96)–βgal with lysate (control)  (I, K, M, and O) or with lysate  containing 15 μM unlabeled  GST-M9 (J, L, N, and P). Inhibition of the nuclear import  reaction may be manifested  by a cytoplasmic pattern of  staining (B) or a general decrease in the fluorescence intensity reflecting exit from the  permeabilized cell (J).
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Figure 4: Vpr does not use the classical NLS or the M9 pathway for entry into the nucleus. (A–H) Import of IBB– βgal, Vpr–βgal, Vpr(1–71)– βgal, and Vpr(73–96)–βgal with lysate (control) (A, C, E, and G) or with lysate containing 30 μM unlabeled IBB as a competitor (B, D, F, and H). (I–P) Import of GST-M9 (FITC-labeled), Vpr–βgal, Vpr(1–71)–βgal, and Vpr(73– 96)–βgal with lysate (control) (I, K, M, and O) or with lysate containing 15 μM unlabeled GST-M9 (J, L, N, and P). Inhibition of the nuclear import reaction may be manifested by a cytoplasmic pattern of staining (B) or a general decrease in the fluorescence intensity reflecting exit from the permeabilized cell (J).

Mentions: A prior study had indicated that saturation of the classical NLS pathway using a peptide corresponding to the SV-40 large T antigen sequence had no inhibitory effect on Vpr import (Gallay et al., 1996). This result suggests that Vpr does not bind to importin α at the NLS cargo site but does not exclude direct binding to importin β. In this regard, importin β–dependent and importin α–independent nuclear entry has been reported for translocation of U snRNPs (Palacios et al., 1997). To further confirm the lack of importin α involvement and to probe the use of importin β by Vpr, we performed nuclear import assays in the presence of excess IBB as an unlabeled competitor. Inclusion of the IBB domain potently inhibited NLS-mediated import in vitro (Fig. 4, A vs. B) by saturating the importin α binding site on importin β (Görlich et al., 1996; Weis et al., 1996). In contrast, the addition of the IBB competitor did not impair nuclear uptake of Vpr–βgal (Fig. 4, C vs. D). However, since one but not both of the Vpr signals might involve the NLS pathway, each signal was individually tested. The addition of the IBB competitor did not inhibit either Vpr signal (Vpr[1–71], Fig. 4, E vs. F, or Vpr[73–96], Fig. 4, G vs. H). Together, these findings indicate that Vpr does not use the classical NLS pathway for nuclear entry via either of its nuclear targeting signals.


Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

Jenkins Y, McEntee M, Weis K, Greene WC - J. Cell Biol. (1998)

Vpr does not use  the classical NLS or the M9  pathway for entry into the nucleus. (A–H) Import of IBB– βgal, Vpr–βgal, Vpr(1–71)– βgal, and Vpr(73–96)–βgal  with lysate (control) (A, C, E,  and G) or with lysate containing 30 μM unlabeled IBB as a  competitor (B, D, F, and H).  (I–P) Import of GST-M9  (FITC-labeled), Vpr–βgal,  Vpr(1–71)–βgal, and Vpr(73– 96)–βgal with lysate (control)  (I, K, M, and O) or with lysate  containing 15 μM unlabeled  GST-M9 (J, L, N, and P). Inhibition of the nuclear import  reaction may be manifested  by a cytoplasmic pattern of  staining (B) or a general decrease in the fluorescence intensity reflecting exit from the  permeabilized cell (J).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132945&req=5

Figure 4: Vpr does not use the classical NLS or the M9 pathway for entry into the nucleus. (A–H) Import of IBB– βgal, Vpr–βgal, Vpr(1–71)– βgal, and Vpr(73–96)–βgal with lysate (control) (A, C, E, and G) or with lysate containing 30 μM unlabeled IBB as a competitor (B, D, F, and H). (I–P) Import of GST-M9 (FITC-labeled), Vpr–βgal, Vpr(1–71)–βgal, and Vpr(73– 96)–βgal with lysate (control) (I, K, M, and O) or with lysate containing 15 μM unlabeled GST-M9 (J, L, N, and P). Inhibition of the nuclear import reaction may be manifested by a cytoplasmic pattern of staining (B) or a general decrease in the fluorescence intensity reflecting exit from the permeabilized cell (J).
Mentions: A prior study had indicated that saturation of the classical NLS pathway using a peptide corresponding to the SV-40 large T antigen sequence had no inhibitory effect on Vpr import (Gallay et al., 1996). This result suggests that Vpr does not bind to importin α at the NLS cargo site but does not exclude direct binding to importin β. In this regard, importin β–dependent and importin α–independent nuclear entry has been reported for translocation of U snRNPs (Palacios et al., 1997). To further confirm the lack of importin α involvement and to probe the use of importin β by Vpr, we performed nuclear import assays in the presence of excess IBB as an unlabeled competitor. Inclusion of the IBB domain potently inhibited NLS-mediated import in vitro (Fig. 4, A vs. B) by saturating the importin α binding site on importin β (Görlich et al., 1996; Weis et al., 1996). In contrast, the addition of the IBB competitor did not impair nuclear uptake of Vpr–βgal (Fig. 4, C vs. D). However, since one but not both of the Vpr signals might involve the NLS pathway, each signal was individually tested. The addition of the IBB competitor did not inhibit either Vpr signal (Vpr[1–71], Fig. 4, E vs. F, or Vpr[73–96], Fig. 4, G vs. H). Together, these findings indicate that Vpr does not use the classical NLS pathway for nuclear entry via either of its nuclear targeting signals.

Bottom Line: Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy.Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites.Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, California 94141-9100, USA.

ABSTRACT
While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

Show MeSH
Related in: MedlinePlus