Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but virus-induced expansion of inflammatory macrophages.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

COVID-19: a fatal case of acute liver failure associated with SARS-CoV-2 infection in pre-existing liver cirrhosis.

BACKGROUND: The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is challenging, particularly in post-mortem human tissues. However, there is increasing evidence for viral SARS-CoV-2 manifestation in non-respiratory tissues. In this context, it is a current matter of debate, whether SARS-CoV-2 shows hepatotropism. CASE PRESENTATION: Here, we report a case of an 88-year-old women with massive SARS-CoV-2 viremia, severe jaundice and clinical signs of an acute hepatitis, who died within a few days from an acute liver failure without showing any clinical signs of pneumonia. Autopsy revealed a severe chronic and acute liver damage with bile duct infestation by SARS-CoV-2 that was accompanied by higher expressions of angiotensin-converting enzyme-2 (ACE2), Cathepsin L and transmembrane serine protease 2 (TMPRSS2). CONCLUSION: Our findings indicate an enhanced biliary susceptibility to viral infection with SARS-CoV-2, that might have resulted from pre-existing severe liver damage. Furthermore, our findings emphasize the differential diagnosis of coronavirus disease 2019 (COVID-19)-associated liver failure in the clinical setting of an inexplicable jaundice.

Influence of RNA extraction methods and library selection schemes on RNA-seq data.

BACKGROUND: Gene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions. RESULTS: Using HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods. CONCLUSIONS: We could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.

SARS-CoV-2 tropism to intestinal but not gastric epithelial cells is defined by limited ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily affects the lung but can also cause gastrointestinal (GI) symptoms. In vitro experiments confirmed that SARS-CoV-2 robustly infects intestinal epithelium. However, data on infection of adult gastric epithelium are sparse and a side-by-side comparison of the infection in the major segments of the GI tract is lacking. We provide this direct comparison in organoid-derived monolayers and demonstrate that SARS-CoV-2 robustly infects intestinal epithelium, while gastric epithelium is resistant to infection. RNA sequencing and proteome analysis pointed to angiotensin-converting enzyme 2 (ACE2) as a critical factor, and, indeed, ectopic expression of ACE2 increased susceptibility of gastric organoid-derived monolayers to SARS-CoV-2. ACE2 expression pattern in GI biopsies of patients mirrors SARS-CoV-2 infection levels in monolayers. Thus, local ACE2 expression limits SARS-CoV-2 expression in the GI tract to the intestine, suggesting that the intestine, but not the stomach, is likely to be important in viral replication and possibly transmission.

Correction: State-of-the-art analytical methods of viral infections in human lung organoids.

[This corrects the article DOI: 10.1371/journal.pone.0276115.].

A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods.

Preserving the original RNA orientation information in RNA-Sequencing (RNA-Seq) experiment is essential to the analysis and understanding of the complexity of mammalian transcriptomes. We describe herein a simple, robust, and time-effective protocol for generating strand-specific RNA-seq libraries suited for the Illumina sequencing platform. We modified the Illumina TruSeq RNA sample preparation by implementing the strand specificity feature using the dUTP method. This protocol uses low amounts of starting material and allows a fast processing within two days. It can be easily implemented and requires only few additional reagents to the original Illumina kit.

State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

Crystallization and preliminary X-ray crystallographic analysis of the [NiFe]-hydrogenase maturation factor HypF1 from Ralstonia eutropha H16.

The hydrogenase maturation factor HypF1 is a truncated but functional version of the HypF protein. HypF is known to be involved in the supply of the CN(-) ligands of the active site of [NiFe]-hydrogenases, utilizing carbamoyl phosphate as a substrate. The first crystallization and preliminary X-ray studies of HypF1 from Ralstonia eutropha H16 are reported here. Crystals of HypF1 (394 amino acids, 40.7 kDa) were obtained by the sitting-drop vapour-diffusion technique using sodium formate as a precipitant. The crystals belonged to space group I222, with unit-cell parameters a = 79.7, b = 91.6, c = 107.2 A. Complete X-ray diffraction data sets were collected at 100 K from native crystals and from a platinum derivative to a maximum resolution of 1.65 A.

The Roborovski Dwarf Hamster Is A Highly Susceptible Model for a Rapid and Fatal Course of SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated an unprecedented and yet-unresolved health crisis worldwide. Different mammals are susceptible to SARS-CoV-2; however, few species examined so far develop robust clinical disease that mirrors severe human cases or allows testing of vaccines and drugs under conditions of severe disease. Here, we compare the susceptibilities of three dwarf hamster species (Phodopus spp.) to SARS-CoV-2 and introduce the Roborovski dwarf hamster (P. roborovskii) as a highly susceptible COVID-19 model with consistent and fulminant clinical signs. Particularly, only this species shows SARS-CoV-2-induced severe acute diffuse alveolar damage and hyaline microthrombi in the lungs, changes described in patients who succumbed to the infection but not reproduced in any experimentally infected animal. Based on our findings, we propose the Roborovski dwarf hamster as a valuable model to examine the efficacy and safety of vaccine candidates and therapeutics, particularly for use in highly susceptible individuals.