Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection ameliorates secondary influenza A virus disease.

Pathogen encounter can result in epigenetic remodeling that shapes disease caused by heterologous pathogens. Here, we examined innate immune memory in the context of commonly circulating respiratory viruses. Single-cell analyses of airway-resident immune cells in a disease-relevant murine model of SARS-CoV-2 recovery revealed epigenetic reprogramming in alveolar macrophages following infection. Post-COVID-19 human monocytes exhibited similar epigenetic signatures. In airway-resident macrophages, past SARS-CoV-2 infection increased activity of type I interferon (IFN-I)-related transcription factors and epigenetic poising of antiviral genes. Viral pattern recognition and canonical IFN-I signaling were required for the establishment of this innate immune memory and augmented secondary antiviral responses. Antiviral innate immune memory mounted by airway-resident macrophages post-SARS-CoV-2 was necessary and sufficient to ameliorate secondary disease caused by influenza A virus and curtailed hyperinflammatory dysregulation and mortality. Our findings provide insights into antiviral innate immune memory in the airway that may facilitate the development of broadly effective therapeutic strategies.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor.

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

Immunological landscape of human lymphoid explants during measles virus infection.

In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected 5%-15% of cells across donors. Using single-cell RNA-Seq and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon-stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5') libraries sequenced with standard read configurations, 10X 5' libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed "10X 5' with extended R1") increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5' with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.

Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection.

Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing.

Single cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNA-Seq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended read 1 (R1) sequencing strategy maximizes the detection of sgmRNAs by increasing the number of unambiguous reads spanning leader-sgmRNA junction sites. Using this method, we show that viral gene expression is highly correlated across cells suggesting a relatively consistent proportion of viral sgmRNA production throughout infection. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air-liquid interface (HAE-ALI) Basic Protocol 2: Viral inoculation of HAE-ALI Basic Protocol 3: Immunofluorescence (IF)-based detection of infected cells in HAE-ALI.