HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains

AbstractThe early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

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HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

10.1101/2021.03.18.436028 ◽

2021 ◽

Author(s):

Anabel Guedán ◽

Callum D Donaldson ◽

Ophélie Cosnefroy ◽

Ian A Taylor ◽

Kate N. Bishop

Keyword(s):

Reverse Transcription ◽

Integration Site ◽

Productive Infection ◽

Nuclear Pore ◽

Virus Infectivity ◽

Nuclear Speckles ◽

Nuclear Entry ◽

The Core ◽

Fold Reduction ◽

Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

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HIV-1 uncoats in the nucleus near sites of integration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920631117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5486-5493 ◽

Cited By ~ 38

Author(s):

Ryan C. Burdick ◽

Chenglei Li ◽

MohamedHusen Munshi ◽

Jonathan M. O. Rawson ◽

Kunio Nagashima ◽

...

Keyword(s):

Reverse Transcription ◽

Nuclear Import ◽

Host Protein ◽

Productive Infection ◽

Protein Cleavage ◽

Integration Sites ◽

Specificity Factor ◽

Infected Cells ◽

Viral Dna Integration ◽

Hiv 1

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 μm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.

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HIV Capsid and Integration Targeting

Viruses ◽

10.3390/v13010125 ◽

2021 ◽

Vol 13 (1) ◽

pp. 125

Author(s):

Alan N. Engelman

Keyword(s):

Nuclear Import ◽

Integration Site ◽

Nuclear Periphery ◽

Viral Gene Expression ◽

Viral Gene ◽

Nuclear Speckles ◽

Host Interactions ◽

Cleavage And Polyadenylation ◽

Site Targeting ◽

Transcription Complexes

Integration of retroviral reverse transcripts into the chromosomes of the cells that they infect is required for efficient viral gene expression and the inheritance of viral genomes to daughter cells. Before integration can occur, retroviral reverse transcription complexes (RTCs) must access the nuclear environment where the chromosomes reside. Retroviral integration is non-random, with different types of virus-host interactions impacting where in the host chromatin integration takes place. Lentiviruses such as HIV efficiently infect interphase cells because their RTCs have evolved to usurp cellular nuclear import transport mechanisms, and research over the past decade has revealed specific interactions between the HIV capsid protein and nucleoporin (Nup) proteins such as Nup358 and Nup153. The interaction of HIV capsid with cleavage and polyadenylation specificity factor 6 (CPSF6), which is a component of the cellular cleavage and polyadenylation complex, helps to dictate nuclear import as well as post-nuclear RTC invasion. In the absence of the capsid-CPSF6 interaction, RTCs are precluded from reaching nuclear speckles and gene-rich regions of chromatin known as speckle-associated domains, and instead mis-target lamina-associated domains out at the nuclear periphery. Highlighting this area of research, small molecules that inhibit capsid-host interactions important for integration site targeting are highly potent antiviral compounds.

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TRIM5α restriction of HIV-1-N74D viruses in lymphocytes is caused by a loss of cyclophilin A protection

10.1101/2020.09.29.318121 ◽

2020 ◽

Author(s):

Anastasia Selyutina ◽

Lacy M. Simons ◽

Angel Bulnes-Ramos ◽

Judd F. Hultquist ◽

Felipe Diaz-Griffero

Keyword(s):

T Cells ◽

Integration Site ◽

Cyclophilin A ◽

Site Preference ◽

Primary Cells ◽

Protein Cleavage ◽

Wild Type ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Hiv 1

ABSTRACTThe core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein cleavage and polyadenylation specificity factor subunit 6 (CPSF6). In addition, HIV-1-N74D viruses have altered patterns of integration site preference in human cell lines. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit infectivity defects when compared to wild type. The reason for this loss of infectivity in primary cells is unknown. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5αhu) restriction in CD4+ T cells, we tested whether TRIM5αhu was involved in the decreased infectivity observed for HIV-1-N74D. Depletion of TRIM5αhu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5αhu. Accordingly, TRIM5αhu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5αhu restriction activity.

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HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

PLoS Pathogens ◽

10.1371/journal.ppat.1009484 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009484

Author(s):

Anabel Guedán ◽

Callum D. Donaldson ◽

Eve R. Caroe ◽

Ophélie Cosnefroy ◽

Ian A. Taylor ◽

...

Keyword(s):

Reverse Transcription ◽

Integration Site ◽

Productive Infection ◽

Nuclear Pore ◽

Virus Infectivity ◽

Nuclear Speckles ◽

Nuclear Entry ◽

The Core ◽

Fold Reduction ◽

Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

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HIV-1 cores retain their integrity until minutes before uncoating in the nucleus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019467118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2019467118

Author(s):

Chenglei Li ◽

Ryan C. Burdick ◽

Kunio Nagashima ◽

Wei-Shau Hu ◽

Vinay K. Pathak

Keyword(s):

Fluorescent Protein ◽

Integration Site ◽

Fluid Phase ◽

Proteolytic Processing ◽

Preintegration Complex ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Capsid Integrity ◽

Green Fluorescent ◽

Hiv 1

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

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HumanImmunodeficiency Virus Type 1 DNA Nuclear Import and Integration AreMitosis Independent in CyclingCells

Journal of Virology ◽

10.1128/jvi.77.24.13412-13417.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13412-13417 ◽

Cited By ~ 41

Author(s):

Richard A. Katz ◽

James G. Greger ◽

Pamela Boimel ◽

Anna Marie Skalka

Keyword(s):

Virus Type ◽

Nuclear Import ◽

Replication Fork ◽

Integration Site ◽

Nuclear Entry ◽

Preintegration Complex ◽

Daughter Cells ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT An essential step in human immunodeficiency virus type 1 (HIV-1) replication is the movement of the viral preintegration complex from the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA nuclear entry in cycling cells have not been established. Here, we show that if cycling cells are infected before S phase, viral DNA can be integrated prior to passage of the host DNA replication fork through the integration site, as indicated by stable inheritance in both daughter cells. We conclude that efficient nuclear entry can occur independently of mitotic nuclear disassembly in cycling cells.

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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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Factors that mold the nuclear landscape of HIV-1 integration

Nucleic Acids Research ◽

10.1093/nar/gkaa1207 ◽

2020 ◽

Author(s):

Gregory J Bedwell ◽

Alan N Engelman

Keyword(s):

Nuclear Import ◽

Integration Site ◽

Preintegration Complex ◽

Retroviral Integration ◽

Host Interactions ◽

Aids Pandemic ◽

Nuclear Environment ◽

Site Targeting ◽

Viral Dna Integration ◽

Hiv 1

Abstract The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.

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The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010534 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5081-5094

Author(s):

Evan Chaudhuri ◽

Sabyasachi Dash ◽

Muthukumar Balasubramaniam ◽

Adrian Padron ◽

Joseph Holland ◽

...

Keyword(s):

Molecular Mechanisms ◽

Mammalian Species ◽

Cellular Protein ◽

Luciferase Reporter ◽

Down Regulation ◽

Host Interaction ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Viral Dna Integration ◽

Hiv 1

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3′UTR of CPSF6 contains a miR-125b–binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3′UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3′UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3′UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3′UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

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