The molecular mechanisms that allow HIV to integrate into particular sites of the host genome are poorly understood. Here we tested if the nuclear pore complex (NPC) facilitates the targeting of HIV integration by acting on chromatin topology. We show that the integrity of the nuclear side of the NPC, which is mainly composed of Tpr, is not required for HIV nuclear import, but that Nup153 is essential. Depletion of Tpr markedly reduces HIV infectivity, but not the level of integration. HIV integration sites in Tpr-depleted cells are less associated with marks of active genes, consistent with the state of chromatin proximal to the NPC, as analysed by super-resolution microscopy. LEDGF/p75, which promotes viral integration into active genes, stabilizes Tpr at the nuclear periphery and vice versa. Our data support a model in which HIV nuclear import and integration are concerted steps, and where Tpr maintains a chromatin environment favourable for HIV replication.

One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.

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.

How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of ∼100 nm, largely exceeding that of the NPC channel (∼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

HIV integration occurs in chromatin sites that favor the release of high levels of viral progeny; alternatively, the virus is also able to discreetly coexist with the host. The viral infection perturbs the cellular environment inducing the remodelling of the nuclear landscape. Indeed, HIV-1 triggers the nuclear clustering of the host factor CPSF6, but the underlying mechanism is poorly understood. Our data indicate that HIV usurps a recently discovered biological phenomenon, called liquid–liquid phase separation, to hijack the host cell. We observed CPSF6 clusters as part of HIV-induced membraneless organelles (HIV-1 MLOs) in macrophages, one of the main HIV target cell types. We describe that HIV-1 MLOs follow phase-separation rules and represent functional biomolecular condensates. We highlight HIV-1 MLOs as hubs of nuclear reverse transcription, while the double-stranded viral DNA, once formed, rapidly migrates outside these structures. Transcription-competent proviruses localize outside but near HIV-1 MLOs in LEDGF-abundant regions, known to be active chromatin sites. Therefore, HIV-1 MLOs orchestrate viral events prior to the integration step and create a favorable environment for the viral replication. This study uncovers single functional host–viral complexes in their nuclear landscape, which is markedly restructured by HIV-1.