Two Putative α-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms

Kamata, Masakazu; Aida, Yoko

doi:10.1128/jvi.74.15.7179-7186.2000

Cited by 49 publications

(59 citation statements)

References 42 publications

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“…The ␣-helical structures found in the core of the Vpr protein are essential for binding to hCG1, whereas the N-terminal region, the "leucine zipper" motif, and the C-terminal basic region are not required. These findings are in agreement with previous data indicating that deleted forms of Vpr lacking the N-or C-terminal regions of the protein still displayed a nuclear rim localization (47). Our results support a correlation between Vpr binding to hCG1 and the docking to FIG.…”

Section: Discussionsupporting

confidence: 93%

“…Finally, these experiments documented the tendency of a significant fraction of Vpr to concentrate at the NE. Such a localization of Vpr at steady state with NPC markers has been previously reported (30,47) and was related to the capacity of Vpr to associate with NPC components (26,29,30).…”

Section: Discussionsupporting

confidence: 69%

“…Vpr was initially reported as a karyophilic protein, but no identifiable classical nuclear import signal has been described within Vpr to account for this property (49,50). Recent data indicate that the ␣-helical domains and the C-terminal arginine-rich region of Vpr each contribute independently to the nuclear localization of the protein (28,47), although the role of the latter domain is still debated (47,51). Moreover, it was also reported that Vpr contains a functional CRM1-dependent NES in the distal part of the ␣-helical domains (28).…”

Section: Discussionmentioning

confidence: 99%

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Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1

Rouzic¹,

Mousnier²,

Rustum³

et al. 2002

Journal of Biological Chemistry

103

109

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The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast twohybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.In contrast to oncoretroviruses that replicate only in dividing cells and enter the nucleus upon nuclear breakdown during mitosis, HIV-1 1 and other lentiviruses have the ability to infect non-dividing cells, such as macrophages and quiescent T lymphocytes. After entry of the virus into the cell, the HIV capsid seems to uncoat rapidly. The genomic HIV-1 RNA is reverse transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex, called the preintegration complex (PIC). The viral DNA is then imported into the nucleus through the nuclear envelope (NE) via an active mechanism within 4 -6 h after infection (1).In eukaryotic cells, the NE creates distinct nuclear and cytoplasmic compartments. This structure consists of two concentric membranes, the inner and outer nuclear membranes which are continuous with the endoplasmic reticulum. The NE is stabilized by the nuclear lamina, a tight meshwork of intermediate filament proteins underlying the inner nuclear membrane (for review, see Ref.2), whereas on the outer side, cytoplasmic intermediate filaments in close contact with the nucleus serve to suspend the nucleus in the cytoplasm (3). Spanning both membranes are nuclear pore complexes (NPC) that form aqueous channels, which allow selective traffic between nucleus and cytoplasm and impose a permeability barrier to free diffusion of macromolecules or complexes. The NPC is a large supramolecular structure (4) formed of ϳ30 unique proteins in vertebrates, termed nucleoporins (Nups) giving rise to an estimated molecular mass of 60 MDa (Ref. 5; for reviews, see Refs. 6 and 7). High resolution electron microscopic images of NPCs reveal an 8-fold symmetric structure, formed by nuclear and cytoplasmic rings and a central spoke complex. Peripheral filaments emanate from the core of the complex into the nucle...

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1

Rouzic¹,

Mousnier²,

Rustum³

et al. 2002

Journal of Biological Chemistry

103

109

View full text Add to dashboard Cite

show abstract

“…Developing clones were screened for HIV-1-specific CTL activity by 51 chromium-release assay (17) against autologous B cell lines (BCLs) pulsed with the peptides recognized in the ELISPOT assays or infected with recombinant vaccinia virus (rVV) expressing either HIV-1 Vpr or Vif (provided by G. P. Mazzara, Therion Biologics, Cambridge, MA). HIV-1-specific clones were maintained by stimulation every 14 -21 days with an anti-CD3 mAb and irradiated allogeneic PBMC.…”

Section: Generation Of Ctl Clonesmentioning

confidence: 99%

Vpr Is Preferentially Targeted by CTL During HIV-1 Infection

Altfeld

Addo

Eldridge

et al. 2001

The Journal of Immunology

101

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The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-γ production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T cell lines. CD8+ T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8+ T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8+ T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.

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“…Flagtagged Vpr encoding lentiviral vector (Vpr+/CCR-X) or control vector (Vpr-/CCR-X) were produced by cotransfection of pRRL-cPPT-CMV-PRE-SIN [23], which carries Flag-tagged Vpr sequence [24] with or without stop codon right after Flag tag sequence, pCMVΔR8.2ΔVpr, and pCMV VSV-G, respectively. VLPs and the lentiviral vectors were harvested, concentrated as described previously [25], and titratedto ensure that approximately 50 % (for Vpr+/VLP) and 90% (for Vpr+/CCR-X) of HeLa cells were arrested inG 2 phase at 24 h postinfection.…”

Section: Cell Virus Like Particles (Vlp) and Lentiviral Vectorsmentioning

confidence: 99%

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

Kamata

et al. 2006

Biochemical and Biophysical Research Communications

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Viral protein R (Vpr), one of the human immunodeficiency virus type 1 (HIV-1) accessory proteins, contributes to multiple cytopathic effects, G 2 cell cycle arrest and apoptosis. The mechanisms of Vpr have been intensely studied because it is believed that they underlie HIV-1 pathogenesis. We here report a cell-based small molecule screen on Vpr induced cell death in the context of HIV-1 infection. From the screen of 504 bioactive compounds, we identified Damnacanthal (Dam), a component of noni fruit, as an inhibitor of Vpr induced cell death. Our studies illustrate a novel efficient platform for drug discovery and development in anti-HIV therapy which should also be applicable to other viruses.

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Two Putative α-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms

Cited by 49 publications

References 42 publications

Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1

Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1

Vpr Is Preferentially Targeted by CTL During HIV-1 Infection

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

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