Clustering and reverse transcription of HIV‐1 genomes in nuclear niches of macrophages

In order to replicate, human immunodeficiency virus (HIV‐1) reverse‐transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non‐dividing target cells that constitute reservoirs of HIV‐1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription‐competent viral DNA. These findings change our understanding of the early HIV‐1 replication cycle and may have implications for addressing HIV‐1 persistence. Synopsis To replicate, HIV reverse transcribes its RNA genome into DNA, a process that is believed to occur exclusively in the cytoplasm. We use imaging methods to revisit the early replication cycle in macrophages, and find that reverse transcription can occur in the nucleus. Multiple HIV‐1 RNA and DNA genomes cluster in large nuclear foci. HIV‐1 genomes cluster in nuclear speckles and are enriched in CPSF6. Reverse transcription can occur in the nuclear HIV‐1 RNA clusters. Nuclear reverse transcription can lead to transcription competent viral DNA. Graphical Abstract Imaging of the early viral replication cycle reveals that conversion of incoming viral DNA into DNA can occur inside target cell nuclei, challenging the prevalent notion that HIV‐1 genome reverse transcription and host chromosomal integration are spatiotemporally separate.