scholarly journals HIV-1 uncoats in the nucleus near sites of integration

2020 ◽  
Vol 117 (10) ◽  
pp. 5486-5493 ◽  
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.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ashwanth C. Francis ◽  
Mariana Marin ◽  
Parmit K. Singh ◽  
Vasudevan Achuthan ◽  
Mathew J. Prellberg ◽  
...  
Keyword(s):  
Viral Replication ◽  
Nuclear Import ◽  
Integration Site ◽  
Nuclear Speckles ◽  
Nuclear Entry ◽  
Primary Monocyte ◽  
Integration Sites ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Hiv 1

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.

scholarly journals Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

2020 ◽  
Author(s):  
Anastasia Selyutina ◽  
Mirjana Persaud ◽  
Kyeongeun Lee ◽  
Vineet KewalRamani ◽  
Felipe Diaz-Griffero
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Viral Genome ◽  
Cellular Compartment ◽  
Nuclear Compartment ◽  
Nuclear Compartments ◽  
The Core ◽  
Biochemical Assays ◽  
Infected Cells ◽  
Hiv 1

SUMMARYHIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

scholarly journals HIV-1 Vpu promotes phagocytosis of infected CD4+ T cells by macrophages through downregulation of CD47

2021 ◽  
Author(s):  
Lijun Cong ◽  
Scott M. Sugden ◽  
Pascal Leclair ◽  
Chinten James Lim ◽  
Tram NQ. Pham ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Host Protein ◽  
Productive Infection ◽  
Free Form ◽  
Accessory Protein ◽  
Inhibitory Receptor ◽  
Macrophage Infection ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus (HIV) remodels the cell surface of infected cells to facilitate viral dissemination and promote immune evasion. The membrane-associated Vpu accessory protein encoded by HIV-1 plays a key role in this process by altering cell surface levels of multiple host proteins. Using an unbiased quantitative plasma membrane profiling approach, we previously identified CD47 as a putative host target downregulated by Vpu. CD47 is a ubiquitously-expressed cell surface protein that interacts with the myeloid cell inhibitory receptor SIRPα to deliver a “don’t-eat-me” signal, thus protecting cells from phagocytosis. In this study, we investigate whether CD47 modulation by HIV-1 Vpu might promote the susceptibility of macrophages to viral infection via phagocytosis of infected CD4+ T cells. Indeed, we find that Vpu downregulates CD47 expression on infected CD4+ T cells leading to an enhanced capture and phagocytosis by macrophages. Interestingly, it is through this process that a CCR5-tropic transmitted/founder (T/F) virus, which otherwise poorly infects macrophages in its cell-free form, becomes infectious in macrophages. Importantly, we show that HIV-1-infected cells expressing a Vpu-resistant CD47 mutant are less prone to infect macrophages through phagocytosis. Mechanistically, Vpu forms a physical complex with CD47 through its transmembrane domain and targets the latter for lysosomal degradation. These results reveal a novel role of Vpu in modulating macrophage infection, which has important implications for HIV-1 transmission in early stages of infection and the establishment of viral reservoir.IMPORTANCEMacrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus. Selective capture and engulfment of HIV-1 infected T cells was shown to drive efficient macrophage infection suggesting that this mechanism represents an important mode of infection notably for weakly macrophage-tropic T/F viruses. In this study, we provide insight into the signals that regulate this process. We show that the HIV-1 accessory protein Vpu downregulates cell surface levels of CD47, a host protein that interacts with the inhibitory receptor SIRPα to deliver a “don’t-eat-me” signal to macrophages. This allows for enhanced capture and phagocytosis of infected T cells by macrophages, ultimately leading to their productive infection even with T/F virus. These findings provide new insights into the mechanisms governing the intercellular transmission of HIV-1 to macrophages with implications for the establishment of the macrophage reservoir and early HIV-1 dissemination in vivo.

scholarly journals HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David Alejandro Bejarano ◽  
Ke Peng ◽  
Vibor Laketa ◽  
Kathleen Börner ◽  
K Laurence Jost ◽  
...  
Keyword(s):  
Stimulated Emission ◽  
Host Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Cleavage ◽  
Nuclear Entry ◽  
Successful Infection ◽  
Specificity Factor ◽  
Pore Complexes ◽  
Hiv 1

Nuclear entry of HIV-1 replication complexes through intact nuclear pore complexes is critical for successful infection. The host protein cleavage-and-polyadenylation-specificity-factor-6 (CPSF6) has been implicated in different stages of early HIV-1 replication. Applying quantitative microscopy of HIV-1 reverse-transcription and pre-integration-complexes (RTC/PIC), we show that CPSF6 is strongly recruited to nuclear replication complexes but absent from cytoplasmic RTC/PIC in primary human macrophages. Depletion of CPSF6 or lack of CPSF6 binding led to accumulation of HIV-1 subviral complexes at the nuclear envelope of macrophages and reduced infectivity. Two-color stimulated-emission-depletion microscopy indicated that under these circumstances HIV-1 complexes are retained inside the nuclear pore and undergo CA-multimer dependent CPSF6 clustering adjacent to the nuclear basket. We propose that nuclear entry of HIV-1 subviral complexes in macrophages is mediated by consecutive binding of Nup153 and CPSF6 to the hexameric CA lattice.

scholarly journals Imaging HIV-1 Genomic DNA from Entry through Productive Infection

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Ryan D. Stultz ◽  
Jennifer J. Cenker ◽  
David McDonald
Keyword(s):  
Reverse Transcription ◽  
Genomic Dna ◽  
Modified Nucleosides ◽  
Productive Infection ◽  
Nuclear Accumulation ◽  
Progeny Virus ◽  
Pcr Analysis ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdU-labeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNA-positive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis. IMPORTANCE The fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used this technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and nonproductively infected macrophages. We visualized transcriptionally active HIV-1 DNA, revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, this approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.

scholarly journals HIV-1 Uncoating and Nuclear Import Precede the Completion of Reverse Transcription in Cell Lines and in Primary Macrophages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1234
Author(s):  
Ashwanth C. Francis ◽  
Mariana Marin ◽  
Mathew J. Prellberg ◽  
Kristina Palermino-Rowland ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Nuclear Import ◽  
Cyclophilin A ◽  
Target Cells ◽  
Nuclear Pore ◽  
Virus Escape ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1

An assembly of capsid proteins (CA) form the mature viral core enclosing the HIV-1 ribonucleoprotein complex. Discrepant findings have been reported regarding the cellular sites and the extent of core disassembly (uncoating) in infected cells. Here, we combined single-virus imaging and time-of-drug-addition assays to elucidate the kinetic relationship between uncoating, reverse transcription, and nuclear import of HIV-1 complexes in cell lines and monocyte-derived macrophages (MDMs). By using cyclophilin A-DsRed (CDR) as a marker for CA, we show that, in contrast to TZM-bl cells, early cytoplasmic uncoating (loss of CDR) is limited in MDMs and is correlated with the efficiency of reverse transcription. However, we find that reverse transcription is dispensable for HIV-1 nuclear import, which progressed through an uncoating step at the nuclear pore. Comparison of the kinetics of nuclear import and the virus escape from inhibitors targeting distinct steps of infection, as well as direct quantification of viral DNA synthesis, revealed that reverse transcription is completed after nuclear import of HIV-1 complexes. Collectively, these results suggest that reverse transcription is dispensable for the uncoating step at the nuclear pore and that vDNA synthesis is completed in the nucleus of unrelated target cells.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Highly Productive Infection with Pseudotyped Human Immunodeficiency Virus Type 1 (HIV-1) Indicates No Intracellular Restrictions to HIV-1 Replication in Primary Human Astrocytes

2001 ◽  
Vol 75 (17) ◽  
pp. 7925-7933 ◽  
Author(s):  
Mario Canki ◽  
Janice Ngee Foong Thai ◽  
Wei Chao ◽  
Anuja Ghorpade ◽  
Mary Jane Potash ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Productive Infection ◽  
Human Astrocytes ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Virus Expression ◽  
Hiv 1

ABSTRACT Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 μg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

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 Factors that mold the nuclear landscape of HIV-1 integration

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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