Inhibition of human immunodeficiency virus type 1 (HIV-1) nuclear import via Vpr–Importin α interactions as a novel HIV-1 therapy

2009 ◽  
Vol 380 (4) ◽  
pp. 838-843 ◽  
Author(s):  
Tatsunori Suzuki ◽  
Norio Yamamoto ◽  
Mizuho Nonaka ◽  
Yoshie Hashimoto ◽  
Go Matsuda ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Importin Α ◽  
Hiv 1

scholarly journals Novel Nuclear Import of Vpr Promoted by Importin α Is Crucial for Human Immunodeficiency Virus Type 1 Replication in Macrophages

2007 ◽  
Vol 81 (10) ◽  
pp. 5284-5293 ◽  
Author(s):  
Yuko Nitahara-Kasahara ◽  
Masakazu Kamata ◽  
Takuya Yamamoto ◽  
Xianfeng Zhang ◽  
Yoichi Miyamoto ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Accessory Gene ◽  
Cytoplasmic Extract ◽  
Immunodeficiency Virus ◽  
Importin Α ◽  
Hiv 1

ABSTRACT Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin α alone, in an importin β-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin α from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin α at low levels, whereas the expression of three major importin α isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin α is required for nuclear import of Vpr. Furthermore, interaction between importin α and the N-terminal α-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin α, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

scholarly journals A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions

2000 ◽  
Vol 74 (24) ◽  
pp. 11811-11824 ◽  
Author(s):  
Kalpana Gupta ◽  
David Ott ◽  
Thomas J. Hope ◽  
Robert F. Siliciano ◽  
Jef D. Boeke
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Productive Infection ◽  
Genomic Rna ◽  
Virion Production ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55Gag and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4+ T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55Gag and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

scholarly journals Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

2002 ◽  
Vol 76 (23) ◽  
pp. 12087-12096 ◽  
Author(s):  
Jeffrey D. Dvorin ◽  
Peter Bell ◽  
Gerd G. Maul ◽  
Masahiro Yamashita ◽  
Michael Emerman ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Situ Hybridization ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

scholarly journals The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

1999 ◽  
Vol 19 (2) ◽  
pp. 1210-1217 ◽  
Author(s):  
Ray Truant ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

scholarly journals The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase

2009 ◽  
Vol 84 (1) ◽  
pp. 397-406 ◽  
Author(s):  
Lavanya Krishnan ◽  
Kenneth A. Matreyek ◽  
Ilker Oztop ◽  
Kyeongeun Lee ◽  
Christopher H. Tipper ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Critical Role ◽  
Bovine Immunodeficiency Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (HIV-1) infection and preintegration complex (PIC) nuclear import. Moreover, HIV-1 integrase interacted with recombinant transportin 3 protein under conditions whereby Moloney murine leukemia virus (MLV) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral PIC nuclear import. Here we correlate infectivity defects in transportin 3 knockdown cells with in vitro protein binding affinities for an expanded set of retroviruses that include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Rous sarcoma virus (RSV) to critically address the role of integrase-transportin 3 interactions in viral infection. Lentiviruses, with the exception of FIV, display a requirement for transportin 3 in comparison to MLV and RSV, yielding an infection-based dependency ranking of SIV > HIV-1 > BIV and EIAV > MLV, RSV, and FIV. In vitro pulldown and surface plasmon resonance assays, in contrast, define a notably different integrase-transportin 3 binding hierarchy: FIV, HIV-1, and BIV > SIV and MLV > EIAV. Our results therefore fail to support a critical role for integrase binding in dictating transportin 3 dependency during retrovirus infection. In addition to integrase, capsid has been highlighted as a retroviral nuclear import determinant. Accordingly, MLV/HIV-1 chimera viruses pinpoint the genetic determinant of sensitization to transportin 3 knockdown to the HIV-1 capsid protein. We therefore conclude that capsid, not integrase, is the dominant viral factor that dictates transportin 3 dependency during HIV-1 infection.

scholarly journals Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type

2006 ◽  
Vol 80 (20) ◽  
pp. 10262-10269 ◽  
Author(s):  
Nathalie Arhel ◽  
Sandie Munier ◽  
Philippe Souque ◽  
Karine Mollier ◽  
Pierre Charneau
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cell ◽  
Nuclear Import ◽  
Target Cells ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.

scholarly journals Nuclear Export of Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type 1 in Tissue Macrophages

2003 ◽  
Vol 77 (13) ◽  
pp. 7582-7589 ◽  
Author(s):  
Michael P. Sherman ◽  
Carlos M. C. de Noronha ◽  
Lauren A. Eckstein ◽  
Jason Hataye ◽  
Pamela Mundt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nucleocytoplasmic Shuttling ◽  
Viral Spread ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

scholarly journals Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection

2004 ◽  
Vol 78 (21) ◽  
pp. 11563-11573 ◽  
Author(s):  
Tamako Ikeda ◽  
Hironori Nishitsuji ◽  
Xin Zhou ◽  
Nobuo Nara ◽  
Takashi Ohashi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Acute Infection ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Viral Cdna ◽  
Hiv 1

ABSTRACT Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP>KL, and KKK>AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP>KL, and KKK>AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP>KL, and KKK>AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.

scholarly journals Isolation of Human Immunodeficiency Virus Type 1 Cores: Retention of Vpr in the Absence of p6gag

2000 ◽  
Vol 74 (13) ◽  
pp. 6198-6202 ◽  
Author(s):  
Molly A. Accola ◽  
Åsa Öhagen ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Viral Genome ◽  
Structural Protein ◽  
Immunodeficiency Virus ◽  
Primate Lentiviruses ◽  
Hiv 1

ABSTRACT Mature human immunodeficiency virus type 1 (HIV-1) virions contain a typically cone-shaped core that encases the viral genome. In this study, we established conditions which allowed the efficient isolation of morphologically intact HIV-1 cores from virions. The isolated cores consisted mostly of cones which appeared uniformly capped at both ends but were heterogeneous with respect to the shape of the broad cap as well as the dimensions and angle of the cone. Vpr, a nonstructural virion component implicated in the nuclear import of the viral genome, was recovered in core preparations of HIV-1 and simian immunodeficiency viruses from African green monkeys. Unexpectedly, p6 gag , a structural protein required for the incorporation of Vpr, was absent from HIV-1 core preparations. Taken together, our results indicate that the incorporation of Vpr into the virion core is a conserved feature of primate lentiviruses and that the interactions required for the uptake of Vpr into assembling particles differ from those which confine Vpr within the core.

scholarly journals Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr

2001 ◽  
Vol 75 (3) ◽  
pp. 1522-1532 ◽  
Author(s):  
Michael P. Sherman ◽  
Carlos M. C. de Noronha ◽  
Marina I. Heusch ◽  
Spencer Greene ◽  
Warner C. Greene
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Fusion Proteins ◽  
Nucleocytoplasmic Shuttling ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin α/β-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1–71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK–Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich α-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

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