A quantitative analysis of the interplay of environment, neighborhood, and cell state in 3D spheroids.

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell-intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems-level studies of single-cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell-intrinsic and cell-extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.

The HSV-1 Transcription Factor ICP4 Confers Liquid-Like Properties to Viral Replication Compartments.

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid-liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid-liquid phase-separated condensates.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-ß-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.

High-content, arrayed compound screens with rhinovirus, influenza A virus and herpes simplex virus infections.

Viruses are genetically and structurally diverse, and outnumber cells by orders of magnitude. They can cause acute and chronic infections, suppress, or exacerbate immunity, or dysregulate survival and growth of cells. To identify chemical agents with pro- or antiviral effects we conducted arrayed high-content image-based multi-cycle infection screens of 1,280 mainly FDA-approved compounds with three human viruses, rhinovirus (RV), influenza A virus (IAV), and herpes simplex virus (HSV) differing in genome organization, composition, presence of an envelope, and tropism. Based on Z'-factors assessing screening quality and Z-scores ranking individual compounds, we identified potent inhibitors and enhancers of infection: the RNA mutagen 5-Azacytidine against RV-A16; the broad-spectrum antimycotic drug Clotrimazole inhibiting IAV-WSN; the chemotherapeutic agent Raltitrexed blocking HSV-1; and Clobetasol enhancing HSV-1. Remarkably, the topical antiseptic compound Aminacrine, which is clinically used against bacterial and fungal agents, inhibited all three viruses. Our data underscore the versatility and potency of image-based, full cycle virus propagation assays in cell-based screenings for antiviral agents.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

Microscopy deep learning predicts virus infections and reveals mechanics of lytic-infected cells.

Imaging across scales reveals disease mechanisms in organisms, tissues, and cells. Yet, particular infection phenotypes, such as virus-induced cell lysis, have remained difficult to study. Here, we developed imaging modalities and deep learning procedures to identify herpesvirus and adenovirus (AdV) infected cells without virus-specific stainings. Fluorescence microscopy of vital DNA-dyes and live-cell imaging revealed learnable virus-specific nuclear patterns transferable to related viruses of the same family. Deep learning predicted two major AdV infection outcomes, non-lytic (nonspreading) and lytic (spreading) infections, up to about 20 hr prior to cell lysis. Using these predictive algorithms, lytic and non-lytic nuclei had the same levels of green fluorescent protein (GFP)-tagged virion proteins but lytic nuclei enriched the virion proteins faster, and collapsed more extensively upon laser-rupture than non-lytic nuclei, revealing impaired mechanical properties of lytic nuclei. Our algorithms may be used to infer infection phenotypes of emerging viruses, enhance single cell biology, and facilitate differential diagnosis of non-lytic and lytic infections.

The Adenovirus Death Protein - a small membrane protein controls cell lysis and disease.

Human adenoviruses (HAdVs) cause widespread acute and persistent infections. Infections are usually mild and controlled by humoral and cell-based immunity. Reactivation of persistently infected immune cells can lead to a life-threatening disease in immunocompromised individuals, especially children and transplant recipients. To date, no effective therapy or vaccine against HAdV disease is available to the public. HAdV-C2 and C5 are the best-studied of more than 100 HAdV types. They persist in infected cells and release their progeny by host cell lysis to neighbouring cells and fluids, a process facilitated by the adenovirus death protein (ADP). ADP consists of about 100 amino acids and harbours a single membrane-spanning domain. It undergoes post-translational processing in endoplasmic reticulum and Golgi compartments, before localizing to the inner nuclear membrane. Here, we discuss the current knowledge on how ADP induces membrane rupture. Membrane rupture is essential for both progression of disease and efficacy of therapeutic viruses in clinical applications, in particular oncolytic therapy.

A high-content image-based drug screen of clinical compounds against cell transmission of adenovirus.

Human adenoviruses (HAdVs) are fatal to immuno-suppressed individuals, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HTS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny and second-round infections. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and performed robust image analyses and hit filtering. We present the entire set of the screening data including all images, image analyses and data processing pipelines. The data are made available at the Image Data Resource (IDR, idr0081). Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation.

BACKGROUND: RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. RESULTS: We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. CONCLUSIONS: SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Concepts in Light Microscopy of Viruses.

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells.

Cytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference.IMPORTANCE This study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions. It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM.

Immunotherapy With the PreS-based Grass Pollen Allergy Vaccine BM32 Induces Antibody Responses Protecting Against Hepatitis B Infection.

BACKGROUND: We have constructed and clinically evaluated a hypoallergenic vaccine for grass pollen allergy, BM32, which is based on fusion proteins consisting of peptides from the IgE binding sites of the major grass pollen allergens fused to preS (preS1+preS2), a domain of the hepatitis B virus (HBV) large envelope protein which mediates the viral attachment and entry. Aim of this study was the characterization of the HBV-specific immune response induced by vaccination of allergic patients with BM32 and the investigation of the vaccines' potential to protect against infection with HBV. METHODS: Hepatitis B-specific antibody and T cell responses of patients vaccinated with BM32 were studied using recombinant preS and synthetic overlapping peptides spanning the preS sequence. The specificities of the antibody responses were compared with those of patients with chronic HBV infection. Furthermore, the capacity of BM32-induced antibodies, to inhibit HBV infection was investigated using HepG2-hNTCP cell-based in vitro virus neutralization assays. FINDINGS: IgG antibodies from BM32-vaccinated but not of HBV-infected individuals recognized the sequence motif implicated in NTCP (sodium-taurocholate co-transporting polypeptide)-receptor interaction of the hepatitis B virus and inhibited HBV infection. INTERPRETATION: Our study demonstrates that the recombinant hypoallergenic grass pollen allergy vaccine BM32 induces hepatitis B-specific immune responses which protect against hepatitis B virus infection in vitro.

Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange.

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.