Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a computer model.

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.

Single-cell analysis of population context advances RNAi screening at multiple levels.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.

An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections.

BACKGROUND: Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatitis A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA. RESULTS: Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections. CONCLUSIONS: We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes without the risk of assay specific artifacts.

Interaction and functional interference of glucocorticoid receptor and SOCS1.

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNgamma and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNgamma treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling.

Insulin-like growth factor binding protein-3 (IGFBP-3) in the prostate and in prostate cancer: local production, distribution and secretion pattern indicate a role in stromal-epithelial interaction.

BACKGROUND: Insulin-like growth factor binding protein 3 (IGFBP-3) exerts inhibitory and proapoptotic effects on prostate cancer cells. Serum levels of IGFBP-3 were found to be associated with the risk of prostate cancer, but the data are still inconclusive. We present a detailed analysis of the expression and localization of IGFBP-3 in the prostate and a comparison with its expression pattern in tumors. METHODS: Expression and localization of IGFBP-3 were analyzed in cellular models and tissue by real-time RT-PCR, ELISA, immunohistochemistry, and immunofluorescence. RESULTS: All cell types of a panel of benign epithelial, stromal and tumor prostate cells expressed IGFBP-3. Significantly higher expression levels were registered in stromal cells. TGF-beta stimulation boosted IGFBP-3 levels 60-fold in stromal cells. The pattern of expression was confirmed in microdissected tissue samples. Protein levels measured by ELISA paralleled the mRNA levels and more than 80% of IGFBP-3 was secreted. On tissue immunostaining, IGFBP-3 was found to be mainly located in the epithelium. The pattern suggested secretion of IGFBP-3, which was confirmed in prostate tissue cultured ex vivo and the ejaculate of vasectomized men. IGFBP-3 levels were increased in primary tumors but did not differ from benign epithelium in metastases and local recurrent tumors. CONCLUSIONS: We registered a significant local production of IGFBP-3 in the prostate, which may well override the effect of protein entering from blood. The stroma--particularly reactivated stroma--is the main source of IGFBP-3 in the prostate, suggesting that this peptide acts as a mediator of stromal-epithelial interactions.

Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus.

In addition to its extracellular function as a secreted protein, IGF-binding protein (IGFBP)-5 has been postulated to act as a signaling molecule in the nucleus. This study aims to assess the significance of this postulated nuclear localization. By confocal immunofluorescence microscopy, we detected IGFBP-5 in the vesicular compartment of mammary epithelial cells in culture, while no nuclear staining was observed. Immunohistochemistry performed on paraffin sections of the involuting mammary gland revealed IGFBP-5 positive staining of epithelial cells only outside the nucleus. To evaluate the contribution of reuptake of extracellular IGFBP-5, T47D cells were incubated with Alexa Fluor 647-labeled IGFBP-5. The protein was taken up into intracellular vesicles and again was neither detectable in the cytoplasm outside of vesicular structures nor in the nucleus. Quantification of the time and concentration dependence of uptake by immunoblotting revealed that the process was saturable at IGFBP-5 concentrations between 1 and 2 mum and partially reversible with 30% remaining in the cell after a 1-h chase. The observation of nuclear uptake of IGFBP-5 was restricted to artificial conditions such as expression of non-secreted forms of IGFBP-5 or selective permeabilization of the plasma membrane by digitonin.