Genomic profiling of HIV-1 integration in microglia cells links viral integration to the topologically associated domains.

HIV-1 encounters the hierarchically organized host chromatin to stably integrate and persist in anatomically distinct latent reservoirs. The contribution of genome organization in HIV-1 infection has been largely understudied across different HIV-1 targets. Here, we determine HIV-1 integration sites (ISs), associate them with chromatin and expression signatures at different genomic scales in a microglia cell model, and profile them together with the primary T cell reservoir. HIV-1 insertions into introns of actively transcribed genes with IS hotspots in genic and super-enhancers, characteristic of blood cells, are maintained in the microglia cell model. Genome organization analysis reveals dynamic CCCTC-binding factor (CTCF) clusters in cells with active and repressed HIV-1 transcription, whereas CTCF removal impairs viral integration. We identify CTCF-enriched topologically associated domain (TAD) boundaries with signatures of transcriptionally active chromatin as HIV-1 integration determinants in microglia and CD4+ T cells, highlighting the importance of host genome organization in HIV-1 infection.

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters.

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+ /NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a "shock and kill effect" decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells.

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.

When viruses infect human cells, they hijack the cell's machinery to produce the proteins they need to replicate. Retroviruses like HIV-1 do this by entering the nucleus and inserting their genetic information into the genome of the infected cell. This requires HIV-1 to convert its genetic material into DNA, which is then released from the protective shell surrounding it (known as the capsid) via a process called uncoating. The nucleus is enclosed within an envelope containing pores that molecules up to a certain size can pass through. Until recently these pores were thought to be smaller than the viral capsid, which led scientists to believe that the HIV-1 genome must shed this coat before penetrating the nucleus. However, recent studies have found evidence for HIV-1 capsid proteins and capsid structures inside the nucleus of some infected cells. This suggests that the capsid may not be removed before nuclear entry or that it may even play a role in helping the virus get inside the nucleus. To investigate this further, Müller et al. attached fluorescent labels to the newly made DNA of HIV-1 and some viral and cellular proteins. Powerful microscopy tools were then used to monitor the uncoating process in various cells that had been infected with the virus. Müller et al. found large amounts of capsid protein inside the nuclei of all the infected cells studied. During the earlier stages of infection, the capsid proteins were mostly associated with viral DNA and the capsid structure appeared largely intact. At later time points, the capsid structure had been broken down and the viral DNA molecules were gradually separating themselves from these remnants. These findings suggest that the HIV-1 capsid helps the virus get inside the nucleus and may protect its genetic material during conversion into DNA until right before integration into the cell's genome. Further experiments studying this process could lead to new therapeutic approaches that target the capsid as a way to prevent or treat HIV-1.

Viruses in the Nucleus.

Viral infection is intrinsically linked to the capacity of the virus to generate progeny. Many DNA and some RNA viruses need to access the nuclear machinery and therefore transverse the nuclear envelope barrier through the nuclear pore complex. Viral genomes then become chromatinized either in their episomal form or upon integration into the host genome. Interactions with host DNA, transcription factors or nuclear bodies mediate their replication. Often interfering with nuclear functions, viruses use nuclear architecture to ensure persistent infections. Discovering these multiple modes of replication and persistence served in unraveling many important nuclear processes, such as nuclear trafficking, transcription, and splicing. Here, by using examples of DNA and RNA viral families, we portray the nucleus with the virus inside.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4+ T Cells.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4+ T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.

Spatial-Temporal Variations in Atmospheric Factors Contribute to SARS-CoV-2 Outbreak.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has reached over five million confirmed cases worldwide, and numbers are still growing at a fast rate. Despite the wide outbreak of the infection, a remarkable asymmetry is observed in the number of cases and in the distribution of the severity of the COVID-19 symptoms in patients with respect to the countries/regions. In the early stages of a new pathogen outbreak, it is critical to understand the dynamics of the infection transmission, in order to follow contagion over time and project the epidemiological situation in the near future. While it is possible to reason that observed variation in the number and severity of cases stems from the initial number of infected individuals, the difference in the testing policies and social aspects of community transmissions, the factors that could explain high discrepancy in areas with a similar level of healthcare still remain unknown. Here, we introduce a binary classifier based on an artificial neural network that can help in explaining those differences and that can be used to support the design of containment policies. We found that SARS-CoV-2 infection frequency positively correlates with particulate air pollutants, and specifically with particulate matter 2.5 (PM2.5), while ozone gas is oppositely related with the number of infected individuals. We propose that atmospheric air pollutants could thus serve as surrogate markers to complement the infection outbreak anticipation.

Alterations of redox and iron metabolism accompany the development of HIV latency.

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.

NHC-gold compounds mediate immune suppression through induction of AHR-TGFß1 signalling in vitro and in scurfy mice.

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar N-heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies showed that the lead compound (MC3) activates TGFß1 signaling and suppresses CD4+ T-cell activation in vitro, in human and mouse T cells. Conversely, genetic knockdown or chemical inhibition of AHR activity or of TGFß1-SMAD-mediated signaling offsets the MC3-mediated immunosuppression. In scurfy mice, a mouse model of human immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, MC3 treatment reduced autoimmune phenotypes and extended lifespan from 24 to 58 days. Our findings suggest that the immunosuppressive activity of gold compounds can be improved by introducing planar NHC ligands to activate the AHR-associated immunosuppressive pathway, thus expanding their potential clinical application for autoimmune diseases.

Multifunctional Roles of the N-Terminal Region of HIV-1SF2Nef Are Mediated by Three Independent Protein Interaction Sites.

HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1SF2Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread.IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1SF2Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

Navigating through the nucleus with a virus.

In each cell, the hierarchical organisation of the ∼2m DNA fibre ensures different nuclear functions, particularly proper gene expression. Chromosomes are non-randomly positioned occupying specific chromosome territories in the 3D nuclear space and circumventing several nuclear landmarks the Nuclear Envelope with embedded Nuclear Pore Complexes, Splicing Speckles, PML bodies and many others. At a higher level of organisation, similarly regulated chromatin regions cluster together in so called Topologically Associated Domains, TADs, while on a smaller scale, DNA sequences wrapped around histones dictate binding of transcription factors or inhibitors that determine the level of chromatin compaction. As intracellular pathogens, viruses explore different cellular structures and functions to either promote their lytic infection or control the latent state of their replication cycles. Here we highlight the most recent discoveries on how different levels of nuclear architecture and genome are exploited by various human viruses.

Potential impact of the antirheumatic agent auranofin on proviral HIV-1 DNA in individuals under intensified antiretroviral therapy: Results from a randomised clinical trial.

Antiretroviral therapy (ART) is typically composed of a combination of three antiretroviral drugs and is the treatment of choice for people with human immunodeficiency virus type 1/acquired immune deficiency syndrome (HIV-1/AIDS). However, it is unable to impact on viral reservoirs, which harbour latent HIV-1 genomes that are able to reignite the infection upon treatment suspension. The aim of this study was to provide an estimate of the safety of the disease-modifying antirheumatic agent auranofin and its impact on the HIV-1 reservoir in humans under intensified ART. For this purpose, an interim analysis was conducted of three of the six arms of the NCT02961829 clinical trial (five patients each) with: no intervention, i.e. continuation of first-line ART; intensified ART (ART + dolutegravir and maraviroc); and intensified ART plus auranofin. Auranofin treatment was found to be well tolerated. No major adverse events were detected apart from a transient decrease in CD4+ T-cell counts at Weeks 8 and 12. Auranofin decreased total viral DNA in peripheral blood mononuclear cells compared with ART-only regimens at Week 20 (P = 0.036) and induced a decrease in integrated viral DNA as quantified by Alu PCR. Despite the limited number of patient-derived sequences available in this study, phylogenetic analyses of nef sequences support the idea that auranofin may impact on the viral reservoir. [ClinicalTrials.gov ID: NCT02961829].

Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression.

Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.