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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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Related in: MedlinePlus

Vpr(1–71) and  Vpr(73–96) block nuclear import mediated through both  the classical NLS and M9  pathways. (A–D) Import of  IBB–βgal and GST-M9 in the  presence of lysate (control)  (A and C) or with lysate containing 50 μM Vpr(1–71) (B  and D). (E–H) Import of  IBB–βgal and GST-M9 with  lysate containing Vpr(96–73)  reverse peptide (control) (E  and G) or containing  Vpr(73–96) forward peptide  (F and H). Peptides were  used at a final concentration  of 500 μM.
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Figure 7: Vpr(1–71) and Vpr(73–96) block nuclear import mediated through both the classical NLS and M9 pathways. (A–D) Import of IBB–βgal and GST-M9 in the presence of lysate (control) (A and C) or with lysate containing 50 μM Vpr(1–71) (B and D). (E–H) Import of IBB–βgal and GST-M9 with lysate containing Vpr(96–73) reverse peptide (control) (E and G) or containing Vpr(73–96) forward peptide (F and H). Peptides were used at a final concentration of 500 μM.

Mentions: We next analyzed the effects of the Vpr fragments on nuclear import mediated through the NLS and M9 pathways. Although saturation of either the NLS or the M9 pathway failed to block import mediated by Vpr(1–71) or Vpr(73– 96) (Fig. 4), addition of an excess of either Vpr(1–71) or Vpr(73–96) fragments markedly inhibited import mediated by either the classical NLS (Fig. 7, A vs. B) or the M9 (Fig. 7, C vs. D) signal. Together, these experiments suggest that Vpr(1–71) associates with the NPC either at the site where the NLS and the M9 pathways converge or downstream of this junction. Vpr(73–96) also enters this pathway but even further downstream of the site of Vpr(1–71) engagement, thus accounting for the ability of Vpr(73–96) to block import mediated by the Vpr(1–71), NLS, and M9 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(1–71) and  Vpr(73–96) block nuclear import mediated through both  the classical NLS and M9  pathways. (A–D) Import of  IBB–βgal and GST-M9 in the  presence of lysate (control)  (A and C) or with lysate containing 50 μM Vpr(1–71) (B  and D). (E–H) Import of  IBB–βgal and GST-M9 with  lysate containing Vpr(96–73)  reverse peptide (control) (E  and G) or containing  Vpr(73–96) forward peptide  (F and H). Peptides were  used at a final concentration  of 500 μM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Vpr(1–71) and Vpr(73–96) block nuclear import mediated through both the classical NLS and M9 pathways. (A–D) Import of IBB–βgal and GST-M9 in the presence of lysate (control) (A and C) or with lysate containing 50 μM Vpr(1–71) (B and D). (E–H) Import of IBB–βgal and GST-M9 with lysate containing Vpr(96–73) reverse peptide (control) (E and G) or containing Vpr(73–96) forward peptide (F and H). Peptides were used at a final concentration of 500 μM.
Mentions: We next analyzed the effects of the Vpr fragments on nuclear import mediated through the NLS and M9 pathways. Although saturation of either the NLS or the M9 pathway failed to block import mediated by Vpr(1–71) or Vpr(73– 96) (Fig. 4), addition of an excess of either Vpr(1–71) or Vpr(73–96) fragments markedly inhibited import mediated by either the classical NLS (Fig. 7, A vs. B) or the M9 (Fig. 7, C vs. D) signal. Together, these experiments suggest that Vpr(1–71) associates with the NPC either at the site where the NLS and the M9 pathways converge or downstream of this junction. Vpr(73–96) also enters this pathway but even further downstream of the site of Vpr(1–71) engagement, thus accounting for the ability of Vpr(73–96) to block import mediated by the Vpr(1–71), NLS, and M9 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