Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

On a path toward a broad-spectrum anti-viral: inhibition of HIV-1 and coronavirus replication by SR kinase inhibitor harmine.

IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.

An Effective Method for Quantifying RNA Expression of IbsC-SibC, a Type I Toxin-Antitoxin System in Escherichia coli.

Toxin and antitoxin (TA) systems are small genetic modules consisting of a toxin protein and an RNA or protein antitoxin. It is difficult to study their functions in a large part due to the lack of effective methods to study toxin RNAs, which usually exist at exceptionally low levels. Herein, we describe a sensitive reverse transcription quantitative PCR (RT-qPCR) method that is able to quantitate such RNA species. The method was directed at detection of the toxin mRNA of the ibsC-sibC TA pair, and its high specificity was validated by sequencing. The approach was used to determine relative expression of the IbsC and SibC RNAs at different cell-growth phases; this revealed an expression pattern that cannot be explained by the prevailing notion of growth stasis by the toxin and rescue by the antitoxin. The usefulness of the method was further showcased by the determination of average cellular copy numbers of the IbsC-SibC RNAs in wild-type E. coli cells and RNA abundance in E. coli cells engineered with extra copies of the ibsC-sibC genes. With a robust method to quantitate cellular small RNAs at very low concentrations, we are now equipped to study the expression of TA systems under different conditions to gain useful insights about their functions.

RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing.

The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named "RiboFACSeq". Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.

A dual reporter system for detecting RNA interactions in bacterial cells.

Detecting RNA-partner interactions in cells is often difficult due to a lack of suitable tools. Here we describe a dual reporter system capable of detecting intracellular interactions in which one of the partners is an RNA. The system utilizes two fluorescent proteins with similar maturation rates but distinct spectral properties, specifically cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). By placing the CFP gene upstream and the YFP gene downstream of an RNA gene of interest, the production of YFP becomes sensitive to RNA-partner interaction, whereas the synthesis of CFP is not disturbed. Therefore, the RNA-partner interaction can be simply measured by the change in the ratio of fluorescence of YFP over CFP. The utility of our approach is demonstrated through verification of three known RNA-partner interactions in the model bacterium Escherichia coli. Our two-reporter strategy should be broadly useful to the study of RNA-targeted interactions in bacteria.