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.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) evolution has been marked by the rapid accumulation of mutations, among which cytosine-to-uracil (C-to-U) transitions represent a major proportion of observed genomic changes. These mutations have been proposed to result from the activity of host APOBEC3 cytidine deaminases, innate immune enzymes capable of editing viral RNA. However, the molecular mechanisms underlying APOBEC3 involvement in SARS-CoV-2 biology remain poorly understood. Here, we systematically investigated physical interactions between APOBEC family members and the SARS-CoV-2 proteins using a Gaussia princeps protein complementation assay. Screening of APOBEC family proteins against the viral proteome identified specific interactions between APOBEC3G (A3G) and APOBEC3H (A3H) with the viral nucleocapsid (N) protein. These interactions were validated by co-immunoprecipitation and were found to be conserved across nucleocapsid proteins from all seven human coronaviruses, suggesting conserved structural determinants. Mechanistic analyses revealed that the RNA-binding and oligomerization capacities of A3G and A3H are key for their interaction with the SARS-CoV-2 nucleocapsid. Mapping experiments further showed that the C-terminal domain of N constitutes the minimal interacting region, with stronger binding observed in larger constructs encompassing adjacent regions, indicating cooperative stabilization. Further work will be needed to determine whether A3G and/or A3H can restrict viral replication and whether their interaction with the nucleocapsid allows them to access and mutate the viral genome.

Human adenoviruses (HAdVs) are a large family of viruses comprising more than 100 genotypes distributed across seven species (A to G). Depending on the genotype, they cause respiratory, ocular, urinary tract, or gastrointestinal infections. Despite the considerable diversity within the human adenovirus genus, most of our current knowledge comes from studies on HAdV-C5. The availability of experimental tools, including antibodies and reverse genetics systems, for the study of adenoviruses other than the C5 strain remains very limited. Here, we describe a reverse genetics tool to engineer HAdV-A12, named AdamiForge. HAdV-A12 is phylogenetically distant from HAdV-C5 and differs markedly in its clinical manifestations, warranting the development of dedicated experimental tools. In principle, AdamiForge is similar to the widely used reverse genetics tool for HAdV-C5 known as AdenoBuilder. Briefly, the viral genome is divided into several blocks and cloned into high-copy plasmids. These genomic fragments are released using a restriction enzyme and recombined in vitro by Gibson assembly. The reconstituted genome is then delivered into permissive cells to generate new virions. The main advantage of this approach lies in the fragmentation of the viral genome into small plasmids, which can be easily modified. In addition to the reconstitution of the wild-type virus, we engineered two recombinant viruses expressing modified versions of the viral DNA-binding protein fused either to eGFP or to a FLAG-tag. These modifications compensated for the lack of specific antibodies and enabled successful detection of the HAdV-A12 DBP by both Western blotting and immunofluorescence. IMPORTANCE: Adenoviruses belong to a large family of viruses comprising seven species (A–G) and more than 100 genotypes. Beyond their role as human pathogens, adenoviruses have been extensively studied as vectors in gene therapy, vaccine development, and oncolytic virotherapy. To date, research has largely focused on a limited number of strains. However, strain-specific characteristics can substantially influence both pathogenic potential and vector performance. It is therefore essential to expand research to other genotypes and to develop new tools for the study of less-characterized strains such as human adenovirus A12.