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

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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A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

10.1101/2020.08.10.245522 ◽

2020 ◽

Author(s):

Qi Shen ◽

Chaoyi Xu ◽

Sooin Jang ◽

Qiancheng Xiong ◽

Swapnil C. Devarkar ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Dna Origami ◽

Nuclear Pore ◽

Nuclear Entry ◽

Cellular Factors ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Human Immunodeficiency Virus 1

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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Selection for Loss of Ref1 Activity in Human Cells Releases Human Immunodeficiency Virus Type 1 from Cyclophilin A Dependence during Infection

Journal of Virology ◽

10.1128/jvi.78.21.12066-12070.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 12066-12070 ◽

Cited By ~ 34

Author(s):

David M. Sayah ◽

Jeremy Luban

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Mammalian Cells ◽

Leukemia Virus ◽

Cyclophilin A ◽

Cellular Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Capsid (CA)-specific restrictions are determinants of retroviral tropism in mammalian cells. One such restriction, human Ref1, targets strains of murine leukemia virus bearing an arginine at CA residue 110 (N-MLV), resulting in decreased accumulation of viral cDNA. The cellular factors accounting for Ref1 activity are unknown. As2O3 increases N-MLV titer in Ref1-positive cells, possibly by counteracting Ref1. Restriction factor saturation experiments suggest that Ref1 may also target human immunodeficiency virus type 1 (HIV-1), but only if its CA is not bound to the cellular protein cyclophilin A (CypA). As a step towards understanding the genetic determinants of Ref1, we subjected Ref1-positive TE671 cells to three sequential rounds of selection with N-MLV reporter viruses. We isolated a subclone, 17H1, that was permissive for N-MLV infection and therefore deficient in Ref1 activity. Stimulation of N-MLV replication by As2O3 was attenuated in 17H1, confirming that the drug acts by overcoming Ref1 activity. HIV-1 infection of 17H1 cells was resistant to disruption of the CA-CypA interaction, demonstrating that Ref1 restricts CypA-free HIV-1. Our results suggest that interaction with CypA evolved to protect HIV-1 from this human antiviral activity.

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MxB sensitivity of HIV-1 is determined by a highly variable and dynamic capsid surface

eLife ◽

10.7554/elife.56910 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Richard J Miles ◽

Claire Kerridge ◽

Laura Hilditch ◽

Christopher Monit ◽

David A Jacques ◽

...

Keyword(s):

Nuclear Import ◽

Cytotoxic T Lymphocyte ◽

Cyclophilin A ◽

Conformational Flexibility ◽

Restriction Factors ◽

Nuclear Entry ◽

Linear Forms ◽

Cofactor Binding ◽

Cofactor Recruitment ◽

Hiv 1

The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.

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Disruption of Human TRIM5α Antiviral Activity by Nonhuman Primate Orthologues

Journal of Virology ◽

10.1128/jvi.79.12.7883-7888.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7883-7888 ◽

Cited By ~ 45

Author(s):

Lionel Berthoux ◽

Sarah Sebastian ◽

David M. Sayah ◽

Jeremy Luban

Keyword(s):

Leukemia Virus ◽

Coiled Coil ◽

Cyclophilin A ◽

Susceptibility To Infection ◽

Coiled Coil Domain ◽

Immunodeficiency Virus ◽

Species Specific ◽

Hiv 1 ◽

Modest Effect

ABSTRACT TRIM5 is a determinant of species-specific differences in susceptibility to infection by retroviruses bearing particular capsids. Human immunodeficiency virus type 1 (HIV-1) infection is blocked by the alpha isoform of macaque TRIM5 (TRIM5αrh) or by the product of the owl monkey TRIM5-cyclophilin A gene fusion (TRIMCyp). Human TRIM5α potently restricts specific strains of murine leukemia virus (N-MLV) but has only a modest effect on HIV-1. The amino termini of TRIM5 orthologues are highly conserved and possess a coiled-coil domain that promotes homomultimerization. Here we show that heterologous expression of TRIM5αrh or TRIMCyp in human cells interferes with the anti-N-MLV activity of endogenous human TRIM5α (TRIM5αhu). Deletion of the cyclophilin domain from TRIMCyp has no effect on heteromultimerization or colocalization with TRIM5αhu but prevents interference with anti-N-MLV activity. These data demonstrate that TRIM5 orthologues form heteromultimers and indicate that C-terminal extensions alter virus recognition by multimers of these proteins.

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

10.7554/elife.41800.030 ◽

2018 ◽

Author(s):

Alan Engelman ◽

Gregory J Towers

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Nuclear Pore ◽

Hiv 1

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Cyclophilin A and TRIM5α Independently Regulate Human Immunodeficiency Virus Type 1 Infectivity in Human Cells

Journal of Virology ◽

10.1128/jvi.80.6.2855-2862.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 2855-2862 ◽

Cited By ~ 81

Author(s):

Elena Sokolskaja ◽

Lionel Berthoux ◽

Jeremy Luban

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Leukemia Virus ◽

Cyclophilin A ◽

Human Cells ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5α-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5α orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5α-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5α on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5α does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5α. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5α and the putative CypA-regulated restriction factor.

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Cone-shaped HIV-1 capsids are transported through intact nuclear pores

10.1101/2020.07.30.193524 ◽

2020 ◽

Cited By ~ 3

Author(s):

Vojtech Zila ◽

Erica Margiotta ◽

Beata Turonova ◽

Thorsten G. Müller ◽

Christian E. Zimmerli ◽

...

Keyword(s):

Nuclear Import ◽

Light And Electron Microscopy ◽

Productive Infection ◽

Nuclear Pore ◽

Major Health ◽

Replication Complex ◽

Empty Capsid ◽

Immunodeficiency Virus ◽

Subtomogram Averaging ◽

Hiv 1

AbstractHuman immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

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

10.7554/elife.41800.031 ◽

2018 ◽

Author(s):

David Alejandro Bejarano ◽

Ke Peng ◽

Vibor Laketa ◽

Kathleen Börner ◽

K Laurence Jost ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Author Response ◽

Nuclear Pore ◽

Hiv 1

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Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery

Journal of Virology ◽

10.1128/jvi.01532-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 1

Author(s):

Elina Mäntylä ◽

Vesa Aho ◽

Michael Kann ◽

Maija Vihinen-Ranta

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Nuclear Import ◽

Laser Scanning ◽

Mixed Model ◽

Laser Scanning Confocal Microscopy ◽

Nuclear Pore ◽

Cancer Therapies ◽

Nuclear Entry ◽

Scanning Confocal Microscopy

ABSTRACT Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin β-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin β-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin β-capsid complexes into the nucleus. IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.

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