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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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The two nuclear  targeting signals of Vpr target the protein to distinct  import pathways. (A–F)  Import of Vpr(1–71)–βgal,  Vpr(73–96)–βgal, and full-length Vpr–βgal with lysate  (control) (A, C, and E) or  with lysate containing 50 μM  unlabeled Vpr(1–71) as a  competitor (B, D, and F).  (G–L) Import of Vpr(1–71)– βgal, Vpr(73–96)–βgal, and  Vpr–βgal with lysate containing unlabeled Vpr(96–73) reverse peptide as a control (G,  I, and K) or Vpr(73–96) forward peptide (H, J, and L).  Peptides were used at a final  concentration of 500 μM.
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Figure 6: The two nuclear targeting signals of Vpr target the protein to distinct import pathways. (A–F) Import of Vpr(1–71)–βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal with lysate (control) (A, C, and E) or with lysate containing 50 μM unlabeled Vpr(1–71) as a competitor (B, D, and F). (G–L) Import of Vpr(1–71)– βgal, Vpr(73–96)–βgal, and Vpr–βgal with lysate containing unlabeled Vpr(96–73) reverse peptide as a control (G, I, and K) or Vpr(73–96) forward peptide (H, J, and L). Peptides were used at a final concentration of 500 μM.

Mentions: To further examine receptor specificity in the two Vpr pathways, cross-competition experiments using the Vpr fragments were performed. Addition of a 300-fold molar excess of an unlabeled Vpr(1–71) protein fragment inhibited Vpr(1–71)–βgal nuclear import (Fig. 6, A vs. B). However, addition of an equimolar amount of Vpr(1–71) did not prevent nuclear entry mediated by Vpr(73–96) (Fig. 6, C vs. D) or full-length Vpr (Fig. 6, E vs. F). These findings demonstrate that nuclear import occurring via the Vpr (1–71) fragment is a saturable process mediated through a receptor distinct from that involved in Vpr(73–96) import. Additionally, the ability of Vpr–βgal to enter the nucleus in the presence of an excess of the Vpr(1–71) protein fragment indicates that the COOH-terminal signal is functional in the context of the full-length protein.


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

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

The two nuclear  targeting signals of Vpr target the protein to distinct  import pathways. (A–F)  Import of Vpr(1–71)–βgal,  Vpr(73–96)–βgal, and full-length Vpr–βgal with lysate  (control) (A, C, and E) or  with lysate containing 50 μM  unlabeled Vpr(1–71) as a  competitor (B, D, and F).  (G–L) Import of Vpr(1–71)– βgal, Vpr(73–96)–βgal, and  Vpr–βgal with lysate containing unlabeled Vpr(96–73) reverse peptide as a control (G,  I, and K) or Vpr(73–96) forward peptide (H, J, and L).  Peptides were used at a final  concentration of 500 μM.
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Related In: Results  -  Collection

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Figure 6: The two nuclear targeting signals of Vpr target the protein to distinct import pathways. (A–F) Import of Vpr(1–71)–βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal with lysate (control) (A, C, and E) or with lysate containing 50 μM unlabeled Vpr(1–71) as a competitor (B, D, and F). (G–L) Import of Vpr(1–71)– βgal, Vpr(73–96)–βgal, and Vpr–βgal with lysate containing unlabeled Vpr(96–73) reverse peptide as a control (G, I, and K) or Vpr(73–96) forward peptide (H, J, and L). Peptides were used at a final concentration of 500 μM.
Mentions: To further examine receptor specificity in the two Vpr pathways, cross-competition experiments using the Vpr fragments were performed. Addition of a 300-fold molar excess of an unlabeled Vpr(1–71) protein fragment inhibited Vpr(1–71)–βgal nuclear import (Fig. 6, A vs. B). However, addition of an equimolar amount of Vpr(1–71) did not prevent nuclear entry mediated by Vpr(73–96) (Fig. 6, C vs. D) or full-length Vpr (Fig. 6, E vs. F). These findings demonstrate that nuclear import occurring via the Vpr (1–71) fragment is a saturable process mediated through a receptor distinct from that involved in Vpr(73–96) import. Additionally, the ability of Vpr–βgal to enter the nucleus in the presence of an excess of the Vpr(1–71) protein fragment indicates that the COOH-terminal signal is functional in the context of the full-length protein.

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