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

Chin, Christopher R.; Perreira, Jill M.; Savidis, George; Portmann, Jocelyn M.; Aker, Aaron M.; Feeley, Eric M.; Smith, Miles; Brass, Abraham L.

doi:10.1016/j.celrep.2015.10.036

Cited by 139 publications

(241 citation statements)

References 37 publications

(65 reference statements)

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“…The decreased colocalization of EdU and CA that we observed in MDM cultured with SIV-VLP may be a consequence of accelerated reverse transcription due to increased dNTP pools and thus a faster uncoating of the RTCs. These data demonstrating the association of HIV-1 DNA and CA in the nucleus of HIV-1-infected MDM are consistent with results from previous studies (13,15) and further validate the model that capsid fragments assist the HIV RTC in entering the nucleus during primary cell infection.…”

Section: Resultssupporting

confidence: 80%

“…Interestingly, in addition to modulating uncoating and RT, the interaction of CA with host proteins can dictate the nuclear import pathway and integration site selection, suggesting that CA may play a role in the nucleus of infected cells (9)(10)(11)(12). Support for this finding has been observed in several recent imaging studies in which CA was shown to be associated with HIV-1 in the nuclei of infected cells (13)(14)(15).…”

supporting

confidence: 64%

“…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%

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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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Section: Resultssupporting

confidence: 80%

supporting

confidence: 64%

mentioning

confidence: 99%

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Imaging HIV-1 Genomic DNA from Entry through Productive Infection

Stultz

Cenker

McDonald

2017

J Virol

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“…It also will be of interest to more precisely locate this block to infection, which might occur during uncoating, docking at the nuclear envelope, or nuclear entry itself. Future experiments to address this issue could include measures of viral uncoating, such as CsA-washout (90), fate-of-capsid (91), or imaging assays (reviewed in reference 19) or visualization of the infectious RTC/PIC (30,92). In contrast to overexpression, we found that silencing SUN2 in cell lines did not change the percentage of HIV-infected cells, although experiments in other cell types, including primary cells, could provide clarification.…”

Section: Discussionmentioning

confidence: 47%

“…CPSF6 and CypA binding to CA may also limit innate immune recognition of HIV (29). Nuclear (fulllength) CPSF6 also mediates the depth of nuclear entry of the preintegration complex (PIC) (30). The importance of nuclear topology for integration into active transcriptional units was underscored by recent findings that nucleoporin-mediated entry of the PIC into the nucleus, and provirus integration into the nuclear periphery, are intimately linked (31,32).…”

mentioning

confidence: 92%

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

Donahue

Amraoui

Nunzio

et al. 2016

J Virol

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In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCECells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection.

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