Genome Of HIV-1 Innovations
In order to trace the fate of HIV-1 particles from early entry events through productive infection, we developed a way to see HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent classify 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 are doing not divide and thus don't incorporate EdU into chromosomal DNA, which might 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 reduction of the check factor SAMHD1 resulted during 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 place 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 way to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this technique allows stable labeling and monitoring of HIV genomic DNA within infected cells
during cytoplasmic transit, nuclear import, and mRNA synthesis.
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