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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 contains two independent nuclear targeting signals.  (A and B) Subcellular localization was determined for Vpr  (1–71)–βgal and Vpr(73–96)–βgal in transiently transfected  HeLa cells. Fusion proteins were detected by indirect immunofluorescence using antibodies specific for βgal. (C and D) Import  of Vpr(1–71)–βgal and Vpr(73–96)–βgal fusion proteins was also  analyzed in vitro in the digitonin-permeabilized HeLa cell system.
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Figure 3: Vpr contains two independent nuclear targeting signals. (A and B) Subcellular localization was determined for Vpr (1–71)–βgal and Vpr(73–96)–βgal in transiently transfected HeLa cells. Fusion proteins were detected by indirect immunofluorescence using antibodies specific for βgal. (C and D) Import of Vpr(1–71)–βgal and Vpr(73–96)–βgal fusion proteins was also analyzed in vitro in the digitonin-permeabilized HeLa cell system.

Mentions: We next investigated what sequences within Vpr are both necessary and sufficient for the observed nuclear uptake of Vpr–βgal. As a first step, two Vpr fragments containing amino acids 1–71 and 73–96 were individually fused to βgal and tested. Unexpectedly, both of these Vpr fragments effectively targeted the βgal protein to the nucleus in vivo (Fig. 3, A and B) and in vitro (Fig. 3, C and D). These findings suggest that Vpr contains at least two functional 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 contains two independent nuclear targeting signals.  (A and B) Subcellular localization was determined for Vpr  (1–71)–βgal and Vpr(73–96)–βgal in transiently transfected  HeLa cells. Fusion proteins were detected by indirect immunofluorescence using antibodies specific for βgal. (C and D) Import  of Vpr(1–71)–βgal and Vpr(73–96)–βgal fusion proteins was also  analyzed in vitro in the digitonin-permeabilized HeLa cell system.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Vpr contains two independent nuclear targeting signals. (A and B) Subcellular localization was determined for Vpr (1–71)–βgal and Vpr(73–96)–βgal in transiently transfected HeLa cells. Fusion proteins were detected by indirect immunofluorescence using antibodies specific for βgal. (C and D) Import of Vpr(1–71)–βgal and Vpr(73–96)–βgal fusion proteins was also analyzed in vitro in the digitonin-permeabilized HeLa cell system.
Mentions: We next investigated what sequences within Vpr are both necessary and sufficient for the observed nuclear uptake of Vpr–βgal. As a first step, two Vpr fragments containing amino acids 1–71 and 73–96 were individually fused to βgal and tested. Unexpectedly, both of these Vpr fragments effectively targeted the βgal protein to the nucleus in vivo (Fig. 3, A and B) and in vitro (Fig. 3, C and D). These findings suggest that Vpr contains at least two functional 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