Biology subjects

Di Nunzio, F.

Publications and source records attributed to Di Nunzio, F..

7 recordsLinked to original sources

bioRxiv2025

Tracking HIV-1 DNA fate from Cell Culture to Humanized mice Tissues.

Unambiguous identification of HIV reservoirs is essential for their characterization and for developing curative strategies. A major obstacle to curing HIV is the persistence of silent viral genomes in host cells that evade immune detection and resist therapy. Here, we present a fluorescence microscopy-based method for direct in vivo visualization of full-length double-stranded HIV-1 DNA, an achievement not previously realized. To achieve this, we developed an advanced imaging platform adapted for replication-competent HIV-1. This system leverages a bacterial-derived two-component tagging strategy, where a fluorescent protein (OR-GFP), efficiently expressed in xenografted human immune cells, specifically binds to an engineered ANCH3 tag sequence integrated into the viral genome. The tagged virus infects CD4+ T cells both in vitro and in vivo, producing bright nuclear puncta corresponding to individual viral genomes. Multiple nucleation sites within ANCH3 enable a stable, shortened tag form for persistent and long-term tracking. Transcriptional profiling during acute infection revealed both transcriptionally active and silent genomes in spleen, lymph nodes, and bone marrow, with silent forms enriched in lymph nodes and marrow, supporting early reservoir establishment. This live-cell imaging strategy enables high-specificity detection of latent HIV, offering a transformative tool for studying reservoir dynamics and guiding future cure strategies.

microbiology↗

bioRxiv2024

Decoding the biogenesis of HIV-induced CPSF6 puncta and their fusion with the nuclear speckle

Viruses rely on host cellular machinery for replication. After entering the nucleus, the HIV genome accumulates in nuclear niches where it undergoes reverse transcription and integrates into neighboring chromatin, promoting high transcription rates and new virus progeny. Despite antiretroviral treatment, viral genomes can persist in these nuclear niches and reactivate upon treatment interruption, raising the possibility that they could play a role in the establishment of viral reservoirs. The post-nuclear entry dynamics of HIV remain unclear, and understanding these steps is critical for revealing how viral reservoirs are established. In this study, we elucidate the formation of HIV-induced CPSF6 puncta and the domains of CPSF6 essential for this process. We also explore the roles of nuclear speckle scaffold factors, SON and SRRM2, in the biogenesis of these puncta. Through genetic manipulation and depletion experiments, we demonstrate the key role of the intrinsically disordered region of SRRM2 in enlarging nuclear speckles in the presence of the HIV capsid. We identify the FG domain of CPSF6 as essential for both puncta formation and binding to the viral core, which serves as the scaffold for CPSF6 puncta. While the low-complexity regions (LCRs) modulate CPSF6 binding to the viral capsid, they do not contribute to puncta formation, nor do the disordered mixed charge domains (MCDs) of CPSF6. Interestingly, the FG peptide facilitates viral replication. These results demonstrate how HIV evolved to hijack host nuclear factors, enabling its persistence in the host. Of note, this study provides new insights into the underlying interactions between host factors and viral components, advancing our understanding of HIV nuclear dynamics and offering potential therapeutic targets for preventing viral persistence. HighlightsO_LIThe formation of HIV-induced CPSF6 puncta is critical for restoring HIV-1 nuclear reverse transcription. C_LIO_LICPSF6 protein lacking the FG peptide cannot bind to the viral core, thereby failing to form HIV-induced CPSF6 puncta. C_LIO_LIThe FG peptide, rather than low-complexity regions (LCRs) or the mixed charge domains (MCDs) of the CPSF6 protein, drives the formation of HIV-induced CPSF6 puncta. C_LIO_LIThe presence of the FG peptide plays a pivotal role in facilitating viral replication within macrophage-like cells. C_LIO_LIHIV-induced CPSF6 puncta form individually and later fuse with nuclear speckles (NS) via the intrinsically disordered region (IDR) of SRRM2. C_LI

microbiology↗

bioRxiv2024

A versatile reporter system to study cell-to-cell and cell-free bovine leukemia virus infection

Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus of the genus Deltaretrovirus that infects dairy cattle worldwide and is the causative agent of enzootic bovine leukosis. BLV demonstrates remarkably low efficiency in infecting cells via free viral particles derived from infected B cells, as virions are rarely detected in the bloodstream of infected cattle. However, transmission efficacy significantly increases upon the establishment of direct cell-to-cell interactions. Syncytium formation assays are the main tool in the study of BLV infectivity. Although this traditional method is highly robust, the complexity of visually counting syncytia poses a significant technical challenge. Using lentiviral vectors, we generated a stable reporter cell line, in which the GFP reporter gene is under the control of the full-length BLV-LTR. We have demonstrated that the BLV-Tax protein and histone deacetylases inhibitors, such as VPA and TSA, can transactivate the BLV LTR. Upon co-culturing the reporter cell line with BLV-infected cells, fluorescent syncytia can be visualized. By implementing automated scanning and image acquisition using a confocal microscope, together with the development of an analysis software, we can detect and measure single GFP cells and fluorescent multinucleated cells. In summary, our reporter cell line, combined with the development of analysis software, is a useful tool for understanding the role of cell fusion and cell-free mechanism of transmission in BLV infection.

microbiology↗

bioRxiv2020

Direct tracking of single proviruses reveals HIV-1/LEDGF complexes excluded from virus-induced membraneless organelles

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 remodeling 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 (LLPS), to hijack the host cell. We observed CPSF6 clusters as part of HIV-induced membraneless organelles (HIV-1 MLOs) in macrophages, which are one of the main HIV target cells. 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.

microbiology↗

bioRxiv2020

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

In order to replicate, the 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 replication cycle in macrophages, 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 and viral RNA, in which multiple genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6 and localize to nuclear speckles. Strikingly, we show that viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where RT is pharmacologically suppressed and in untreated cells. We demonstrate that, after temporary inhibition, RT can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear RT can result in transcription competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle, and may have implications for understanding HIV-1 persistence.

microbiology↗

bioRxiv2020

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes.

Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. To enter the nucleus, the viral DNA must be led through the nuclear pore complex (NPC). During HIV-1 cytoplasmic journey, the viral core acts like a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for the reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core reshapes into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated to the pre-integration complex (PIC) during and after nuclear translocation, in particular, in human lymphocytes, the main target cells of HIV-1, is still enigmatic. In this study, we analysed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immuno-gold labelling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby uncovering the architecture of the potential HIV-1 PIC. Thus, we revealed DNA- and CA-positive complexes by correlated light- and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodelled in a "pearl necklace" shape. Thus, we observed how CA proteins reshape around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed by the integrase and the retrotranscribed DNA leads HIV-1 genome inside the host nucleus to potentially replicate. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. ImportanceHow the reverse transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. 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 proteins organization is required to achieve effective nuclear translocation. The mechanistic of this process remains undefined due to the lack of a technology capable to visualize potential CA sub-complexes 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 intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed in the outer and inner side of the NE, and they represent 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.

microbiology↗

bioRxiv2019

Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the viral Core

Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand the mechanism, we used Jurkat cells, human PBMCs, and human CD4+ T cells. Our results showed that the inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human TRIM5 (TRIM5hu), suggesting that TRIM5hu restricts HIV-1. Accordingly, we found that TRIM5hu binds to the HIV-1 core. Disruption of CypA-capsid interactions failed to affect HIV-1-A92E infection, correlating with the loss of TRIM5hu binding to HIV-1-A92E cores. Disruption of CypA-capsid interactions in PBMCs and CD4+ T cells had a greater inhibitory effect on HIV-1 when compared to Jurkat cells. HIV-1-A92E infection of PBMCs and CD4+ T cells was unaffected by disruption of CypA-capsid interactions. Consistent with TRIM5 restriction, disruption of CypA-capsid interactions in CD4+ T cells inhibited reverse transcription. Overall, our results showed that CypA binding to the core protects HIV-1 from TRIM5hu restriction.

microbiology↗