Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

Francis, Ashwanth C.; Primio, Cristina Di; Quercioli, Valentina; Valentini, Paola; Boll, Annegret; Girelli, Gabriele; Demichelis, Francesca; Arosio, Daniele; Cereseto, Anna

doi:10.1089/aid.2013.0277

Cited by 28 publications

(43 citation statements)

References 32 publications

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“…S1). In contrast to the viral particles used in previous studies495354, we now retained the wild-type (WT) IN sequence (and not the catalysis defective D64E mutant5355) in the packaging construct, resulting in viral particles that maintain 100% of the WT single-round infectivity (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…By using an HIV packaging construct that encodes WT IN instead of the catalytically defective IN D64E mutant20535455, fluorescently labeled viral particles were generated with WT infectivity (Fig. 1B).…”

Section: Discussionmentioning

confidence: 99%

“…S2) offers an alternative for methods which count a limited number of HIV complexes by manually selecting a region of interest (ROI) and sum or average pixel intensities65570. In our routine ROIs are automatically determined using an intensity threshold, which results in a higher precision and the unbiased processing of thousands of complexes within a few hours.…”

Section: Discussionmentioning

confidence: 99%

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Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Borrenberghs

Dirix

Wit

et al. 2016

Sci Rep

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Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Borrenberghs

Dirix

Wit

et al. 2016

Sci Rep

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“…Importantly, antibody-based imaging of HIV-1 and fluorescence in situ hybridization (FISH) strategies to visualize vRNA and vDNA (Pezzella et al, 1987; Singer et al, 1989), as well as the use of chimeric viral proteins (Campbell and Hope, 2008; Francis et al, 2014), have also improved our knowledge of HIV-1 infection at a single cell level. Some of the acknowledged limitations of these approaches are a lack of sensitivity and disruptive preparative conditions (i.e.…”

Section: Introductionmentioning

confidence: 99%

Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration

et al. 2015

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Summary Direct visualization of HIV-1 replication would improve our understanding of the viral lifecycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’ integration into gene dense regions.

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“…Numerous refinements of imaging approaches have allowed the detection and localization of nuclear HIV-1 genomic structures, including the use of fluorescently tagged proteins associated with viral preintegration complexes (PICs) (22)(23)(24)(25), DNA fluorescence in situ hybridization (FISH) (13,26), staining of surrogate markers of DNA damage following the cleavage of a specific restriction site within the integrated provirus (27), and the incorporation of the thymidine nucleoside analog 5-ethynyl-2=-deoxyuridine (EdU) and subsequent fluorescent labeling (15). These approaches have provided valuable insights into intranuclear transport and integration site selection in infected cell nuclei.…”

mentioning

confidence: 99%

Imaging HIV-1 Genomic DNA from Entry through Productive Infection

Stultz

Cenker

McDonald

2017

J Virol

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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, EdUlabeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNApositive 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. IMPORTANCEThe 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.KEYWORDS fluorescent-image analysis, human immunodeficiency virus, macrophages H uman immunodeficiency virus type 1 (HIV-1) infection can be viewed as occurring in early (entry) stages, including reverse transcription (RT), nuclear import, and the integration of the viral genome, and late (productive) stages, including viral transcr...

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Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

Cited by 28 publications

References 32 publications

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration

Imaging HIV-1 Genomic DNA from Entry through Productive Infection

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