Imaging HIV-1 Genomic DNA from Entry through Productive Infection
| Autor: | Jennifer J. Cenker, Ryan D. Stultz, David McDonald |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Virus Integration Immunology Biology Virus Replication Microbiology Genome Cell Line 03 medical and health sciences chemistry.chemical_compound Virology medicine Humans In Situ Hybridization Fluorescence Cell Nucleus Staining and Labeling medicine.diagnostic_test Macrophages RNA Reverse Transcription Virus Internalization Cyclin-Dependent Kinase 9 Deoxyuridine Molecular biology Reverse transcriptase Virus-Cell Interactions genomic DNA HEK293 Cells 030104 developmental biology chemistry Insect Science DNA Viral HIV-1 Capsid Proteins Thymidine DNA SAMHD1 Fluorescence in situ hybridization |
| Zdroj: | Journal of Virology. 91 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.00034-17 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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