Author response: HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

SummaryThe capsid of human immunodeficiency virus 1 (HIV-1) plays a pivotal role in viral nuclear import, but the mechanism by which the viral core passages the nuclear pore complex (NPC) is poorly understood. Here, we use DNA-origami mimics of the NPC, termed NuPODs (NucleoPorins Organized by DNA), to reveal the mechanistic underpinnings of HIV-1 capsid nuclear entry. We found that trimeric interface formed via three capsid protein hexamers is targeted by a triple-arginine (RRR) motif but not the canonical phenylalanine-glycine (FG) motif of NUP153. As NUP153 is located on the nuclear face of the NPC, this result implies that the assembled capsid must cross the NPC in vivo. This hypothesis is corroborated by our observations of tubular capsid assemblies penetrating through NUP153 NuPODs. NUP153 prefers to bind highly curved capsid assemblies including those found at the tips of viral cores, thereby facilitating capsid insertion into the NPC. Furthermore, a balance of capsid stabilization by NUP153 and deformation by CPSF6, along with other cellular factors, may allow for the intact capsid to pass NPCs of various sizes. The NuPOD system serves as a unique tool for unraveling the previously elusive mechanisms of nuclear import of HIV-1 and other viruses.

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Nup153 Unlocks the Nuclear Pore Complex for HIV-1 Nuclear Import in Non-Dividing Cells

SSRN Electronic Journal ◽

10.2139/ssrn.3155897 ◽

2018 ◽

Author(s):

Silvana Opp ◽

Alicia Martinez-Lopez ◽

Thomas Fricke ◽

Cindy Buffone ◽

Marco Severgnini ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Nuclear Pore ◽

Dividing Cells ◽

Hiv 1

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SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A

Journal of Virology ◽

10.1128/jvi.00229-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00229-18 ◽

Cited By ~ 5

Author(s):

Xinlong Luo ◽

Wei Yang ◽

Guangxia Gao

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Leukemia Virus ◽

Cyclophilin A ◽

Cellular Protein ◽

Wild Type Virus ◽

Nuclear Pore ◽

Nuclear Entry ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA.IMPORTANCEHIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process.

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Faculty Opinions recommendation of Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1006050.144107 ◽

2002 ◽

Author(s):

Pamela Silver

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Nuclear Pore ◽

Complex Composition

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SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016650117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28344-28354 ◽

Cited By ~ 6

Author(s):

Lisa Miorin ◽

Thomas Kehrer ◽

Maria Teresa Sanchez-Aparicio ◽

Ke Zhang ◽

Phillip Cohen ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Nuclear Translocation ◽

Nuclear Pore ◽

Interferon Signaling ◽

Antiviral Signaling ◽

Pathogenic Viruses ◽

Transcriptional Induction ◽

Global Health Problem ◽

Viral Accessory Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

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The nuclear pore complex, nuclear transport, and apoptosisThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y05-100 ◽

2006 ◽

Vol 84 (3-4) ◽

pp. 279-286 ◽

Cited By ~ 22

Author(s):

Birthe Fahrenkrog

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Morphological Changes ◽

Nuclear Transport ◽

Nuclear Pore ◽

Simple Fact ◽

Cell Death Program ◽

A Cell ◽

Cellular Compartments ◽

Selection Of

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.

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HIV-1 remodels the nuclear pore complex

Journal of Experimental Medicine ◽

10.1084/jem2086oia16 ◽

2011 ◽

Vol 208 (6) ◽

pp. i16-i16

Author(s):

Anne Monette ◽

Nelly Panté ◽

Andrew J. Mouland

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Pore ◽

Hiv 1

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Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200608141 ◽

2006 ◽

Vol 175 (4) ◽

pp. 579-593 ◽

Cited By ~ 100

Author(s):

Benjamin L. Timney ◽

Jaclyn Tetenbaum-Novatt ◽

Diana S. Agate ◽

Rosemary Williams ◽

Wenzhu Zhang ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Pore Complex ◽

Nuclear Import ◽

Yeast Cells ◽

Limiting Factor ◽

Nuclear Pore ◽

Nuclear Localization Signals ◽

The Poor ◽

Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

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Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.703 ◽

2000 ◽

Vol 11 (2) ◽

pp. 703-719 ◽

Cited By ~ 26

Author(s):

Susanne M. Steggerda ◽

Ben E. Black ◽

Bryce M. Paschal

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Translocation ◽

Living Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Permeabilized Cells ◽

Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

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