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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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Comparison of  NLS-mediated (A–D) and  Vpr-mediated (E–H) nuclear  import in vitro. Import of  IBB–βgal as a probe of the  NLS pathway or Vpr–βgal  was performed in the presence of lysate alone (A and  E), lysate supplemented  with WGA (50 μg/ml) (B  and F), or lysate containing 8  μM importin β (71–876) (C  and G). Import reactions  containing IBB–βgal (D) or  Vpr–βgal (H) fusion proteins  were also performed at 0°C  instead of 25°C.
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Figure 2: Comparison of NLS-mediated (A–D) and Vpr-mediated (E–H) nuclear import in vitro. Import of IBB–βgal as a probe of the NLS pathway or Vpr–βgal was performed in the presence of lysate alone (A and E), lysate supplemented with WGA (50 μg/ml) (B and F), or lysate containing 8 μM importin β (71–876) (C and G). Import reactions containing IBB–βgal (D) or Vpr–βgal (H) fusion proteins were also performed at 0°C instead of 25°C.

Mentions: To investigate whether Vpr–βgal nuclear uptake reflected active transport across the nuclear pore, the effects of wheat germ agglutinin (WGA) were examined. This lectin binds to N-acetyl-d-glucosamine residues present on many of the nucleoporins and blocks NLS-mediated import without restricting passive diffusion of small molecules (Finlay et al., 1987). Addition of WGA markedly inhibited nuclear uptake of both IBB–βgal (Fig. 2 B) and Vpr–βgal (Fig. 2 F). Vpr-mediated nuclear import was also examined in the presence of a dominant-negative importin β deletion mutant, importin β (71–876). This mutant lacks a RanGTP binding domain and has been shown to inhibit multiple pathways of nuclear import and export across the NPC (Izaurralde et al., 1997a; Kutay et al., 1997b). Addition of importin β (71–876) effectively blocked nuclear localization of both IBB–βgal (Fig. 2 C) and Vpr–βgal (Fig. 2 G). Finally, we examined the temperature dependence of the Vpr-mediated import reaction. When the import reaction was performed on ice (0°C) instead of at room temperature (25°C), nuclear localization of both IBB–βgal (Fig. 2 D) and Vpr–βgal was inhibited (Fig. 2 H). Taken together, these results indicate that nuclear import of Vpr is a temperature-dependent, signal-mediated process that proceeds through the NPC.


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

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

Comparison of  NLS-mediated (A–D) and  Vpr-mediated (E–H) nuclear  import in vitro. Import of  IBB–βgal as a probe of the  NLS pathway or Vpr–βgal  was performed in the presence of lysate alone (A and  E), lysate supplemented  with WGA (50 μg/ml) (B  and F), or lysate containing 8  μM importin β (71–876) (C  and G). Import reactions  containing IBB–βgal (D) or  Vpr–βgal (H) fusion proteins  were also performed at 0°C  instead of 25°C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132945&req=5

Figure 2: Comparison of NLS-mediated (A–D) and Vpr-mediated (E–H) nuclear import in vitro. Import of IBB–βgal as a probe of the NLS pathway or Vpr–βgal was performed in the presence of lysate alone (A and E), lysate supplemented with WGA (50 μg/ml) (B and F), or lysate containing 8 μM importin β (71–876) (C and G). Import reactions containing IBB–βgal (D) or Vpr–βgal (H) fusion proteins were also performed at 0°C instead of 25°C.
Mentions: To investigate whether Vpr–βgal nuclear uptake reflected active transport across the nuclear pore, the effects of wheat germ agglutinin (WGA) were examined. This lectin binds to N-acetyl-d-glucosamine residues present on many of the nucleoporins and blocks NLS-mediated import without restricting passive diffusion of small molecules (Finlay et al., 1987). Addition of WGA markedly inhibited nuclear uptake of both IBB–βgal (Fig. 2 B) and Vpr–βgal (Fig. 2 F). Vpr-mediated nuclear import was also examined in the presence of a dominant-negative importin β deletion mutant, importin β (71–876). This mutant lacks a RanGTP binding domain and has been shown to inhibit multiple pathways of nuclear import and export across the NPC (Izaurralde et al., 1997a; Kutay et al., 1997b). Addition of importin β (71–876) effectively blocked nuclear localization of both IBB–βgal (Fig. 2 C) and Vpr–βgal (Fig. 2 G). Finally, we examined the temperature dependence of the Vpr-mediated import reaction. When the import reaction was performed on ice (0°C) instead of at room temperature (25°C), nuclear localization of both IBB–βgal (Fig. 2 D) and Vpr–βgal was inhibited (Fig. 2 H). Taken together, these results indicate that nuclear import of Vpr is a temperature-dependent, signal-mediated process that proceeds through the NPC.

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