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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

Nuclear import of Vpr is a signal-mediated process. Indirect immunofluorescence of HeLa cells transfected with an expression plasmid encoding either βgal (A) or Vpr–βgal fusion  protein (B). Import of FITC-labeled βgal (C), IBB–βgal (D), and  Vpr–βgal (E) in digitonin-permeabilized cells. Reactions shown  here and in all of the following figures contain rabbit reticulocyte  lysate with an energy-regenerating system unless otherwise indicated.
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Figure 1: Nuclear import of Vpr is a signal-mediated process. Indirect immunofluorescence of HeLa cells transfected with an expression plasmid encoding either βgal (A) or Vpr–βgal fusion protein (B). Import of FITC-labeled βgal (C), IBB–βgal (D), and Vpr–βgal (E) in digitonin-permeabilized cells. Reactions shown here and in all of the following figures contain rabbit reticulocyte lysate with an energy-regenerating system unless otherwise indicated.

Mentions: Vpr has been previously shown to localize to the nucleus, both in transiently transfected cells and in cells infected with HIV (Lu et al., 1993; Zhao et al., 1994; Yao et al., 1995). Since the molecular mass of Vpr (14 kD) is substantially smaller than the 40–60-kD size limit for passive diffusion of proteins through the NPC, the observed nuclear location of Vpr could result from at least two possible mechanisms: (a) Vpr is imported via a nuclear targeting signal, or (b) Vpr enters the nucleus by passive diffusion and is retained in that cellular compartment by binding to nuclear proteins. To distinguish between these two possibilities, we constructed a chimeric protein consisting of Vpr fused at its COOH terminus to βgal, and analyzed its subcellular localization both in vivo after transfection in HeLa cells and in vitro using digitonin-permeabilized HeLa cells and fluorescently labeled recombinant Vpr–βgal fusion proteins (Fig. 1). The size of the resulting fusion protein (∼130 kD for the monomer and ∼520 kD for the tetramer) is sufficiently large to preclude passive diffusion into the nucleus.


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

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

Nuclear import of Vpr is a signal-mediated process. Indirect immunofluorescence of HeLa cells transfected with an expression plasmid encoding either βgal (A) or Vpr–βgal fusion  protein (B). Import of FITC-labeled βgal (C), IBB–βgal (D), and  Vpr–βgal (E) in digitonin-permeabilized cells. Reactions shown  here and in all of the following figures contain rabbit reticulocyte  lysate with an energy-regenerating system unless otherwise indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Nuclear import of Vpr is a signal-mediated process. Indirect immunofluorescence of HeLa cells transfected with an expression plasmid encoding either βgal (A) or Vpr–βgal fusion protein (B). Import of FITC-labeled βgal (C), IBB–βgal (D), and Vpr–βgal (E) in digitonin-permeabilized cells. Reactions shown here and in all of the following figures contain rabbit reticulocyte lysate with an energy-regenerating system unless otherwise indicated.
Mentions: Vpr has been previously shown to localize to the nucleus, both in transiently transfected cells and in cells infected with HIV (Lu et al., 1993; Zhao et al., 1994; Yao et al., 1995). Since the molecular mass of Vpr (14 kD) is substantially smaller than the 40–60-kD size limit for passive diffusion of proteins through the NPC, the observed nuclear location of Vpr could result from at least two possible mechanisms: (a) Vpr is imported via a nuclear targeting signal, or (b) Vpr enters the nucleus by passive diffusion and is retained in that cellular compartment by binding to nuclear proteins. To distinguish between these two possibilities, we constructed a chimeric protein consisting of Vpr fused at its COOH terminus to βgal, and analyzed its subcellular localization both in vivo after transfection in HeLa cells and in vitro using digitonin-permeabilized HeLa cells and fluorescently labeled recombinant Vpr–βgal fusion proteins (Fig. 1). The size of the resulting fusion protein (∼130 kD for the monomer and ∼520 kD for the tetramer) is sufficiently large to preclude passive diffusion into the nucleus.

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