Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

Burdick, Ryan C.; Hu, Wei-Shau; Pathak, Vinay K.

doi:10.1073/pnas.1315996110

Cited by 65 publications

(96 citation statements)

References 56 publications

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“…Interestingly, the low percentage of EV detected in the nuclear compartment may suggest that nuclear import either occurs during mitosis or does not strictly depend on genomic signals and that reverse transcription might not be an absolute requirement for nuclear import. 29,30,31 Finally, we prove that the number of HIV-1 complexes detected in the nuclei of infected cells correlates with the titer of viral supernatants used for infection, thus further supporting the intact virological nature of the HIV-IN-EGFP system.…”

Section: Discussionsupporting

confidence: 63%

Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

Francis

Primio

Quercioli

et al. 2014

AIDS Research and Human Retroviruses

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The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.

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Section: Discussionsupporting

confidence: 63%

Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

Francis

Primio

Quercioli

et al. 2014

AIDS Research and Human Retroviruses

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“…Recognition and processing of such intermediates by HLTF could impede an ordered synthesis of the cDNA copy of the HIV-1 RNA genome. Although integration competent HIV-1 preintegration complexes form in the cytoplasm, the completion of reverse transcription does not appear to be a prerequisite for HIV-1 entry to the nucleus (55), where partially reverse-transcribed viral cDNA can be accessed by DNA repair proteins. Alternatively, HLTF could modulate processing of the integrated proviral DNA, as it is known to control the choice between two pathways of postreplication repair of DNA lesions at the replication fork: error-free, by template switching, and error-prone, through recruitment of mutagenic translesion DNA polymerases (45,56).…”

Section: Discussionmentioning

confidence: 99%

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

Hrecka

Hao

Shun

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

103

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HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4DCAF1 E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.

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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.…”

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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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Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

Cited by 65 publications

References 56 publications

Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 Complexes

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

Imaging HIV-1 Genomic DNA from Entry through Productive Infection

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