New insights on the role of paired membrane structures in coronavirus replication

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication

Image_3_Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.tif

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells – namely, the human respiratory epithelium.</p

Table_3_Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.xlsx

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells – namely, the human respiratory epithelium.</p

Image_1_Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.tif

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells – namely, the human respiratory epithelium.</p

Image_4_Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.tif

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells – namely, the human respiratory epithelium.</p

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2

EndoU-deficient MHV induces activation of the OAS-RNase L pathway, resulting in early breakdown of ribosomal RNA.

<p>(<b>a</b>) Analysis of rRNA integrity in bone marrow-derived macrophages derived from wild type C57BL/6, Mda5^-/-, RNase L^-/-, and IFNAR^-/- mice following infection with MHV-A59 and MHV_H277A (MOI = 1). Total RNA was isolated at indicated time points and degradation of ribosomal RNA as marker for RNase L activation was assessed with a Fragment Analyzer. One representative picture and migration of 18S and 28S ribosomal RNA is displayed. The RNA Quality Number (RQN) is indicated. (<b>b</b>) The integrity of rRNA from MHV-A59 and MHV_H277A infected (MOI = 1) L929 cells, with or without IFN-I pre-treatment (12.5 U of IFN-I 16h prior to infection). Analysis was performed as in panel (<b>a</b>) and one representative image out of five is displayed.</p

Coronavirus EndoU-mediated innate immune evasion.

<p>Following coronavirus infection, the EndoU activity residing in the coronavirus replication complex prevents simultaneous activation of dsRNA sensors Mda5, OAS, and PKR. This strategy allows coronaviruses to efficiently evade antiviral innate host responses such as induction of IFN-I expression, RNase L-mediated RNA degradation, and inhibition of host cell translation.</p

Infection with EndoU-deficient MHV results in increased cytosolic dsRNA.

<p><b>(a-b)</b> Intracellular staining of dsRNA and FACS analysis of MHV-A59 and MHV_H277A infected (MOI = 1) C57BL/6 <b>(a)</b> and IFNAR^-/- <b>(b)</b> macrophages at 4, 6, 9 and 12 h.p.i.. One representative histogram out of two (a) and three (b) is shown for each time point. Cells without virus infection (mock) were used as controls. <b>(c-d)</b> The left panels show cells that were co-stained for MHV-nsp2/3 to control for MHV-A59 and MHV_H277A infection. The right panels display the median fluorescent intensity (MFI) of dsRNA peaks detected in <b>(a-b).</b> The left panels show data from two (c) and three (d) independent experiments. Cells without virus infection (mock) were used as controls. Mean and SEM are depicted. The 95% confidence band is highlighted in grey. Statistically significant comparisons are displayed (**, p< 0.01).</p

Replication of EndoU-deficient MHV is partially restored in IFNAR^-/- macrophages and EndoU mutants display a pronounced sensitivity to IFN-I treatment.

<p>(<b>a</b>) Replication kinetics of MHV-A59 and MHV_H277A (left panel; titers in pfu) and cell-associated viral RNA (right panel; qRT-PCR) following infection of IFNAR^-/- bone marrow-derived macrophages (MOI = 1). Data represent four independent experiments, each performed in two to three replicas. Mean and SEM are depicted. The 95% confidence band is highlighted in grey. The differences in peak levels of viral titers (MHV-A59: 6.0, MHV_H277A: 5.2) and RNA copies (MHV-A59: 9.7, MHV_H277A: 9.3) were statistically significant (p<0.001, p = 0.032, respectively). (<b>b</b>) Expression of IFN-β mRNA (left panel; qRT-PCR) and protein (right panel; ELISA) in IFNAR^-/- macrophages following infection of MHV-A59 and MHV_H277A (MOI = 1). Data represent four (left panel) and three (right panel) independent experiments, each performed in two to three replicas. Median and the 1–99 percentiles are displayed. Dashed line depicts limit of detection (right panel). The difference in peak levels of IFN-β expression (MHV-A59: 9.4, MHV_H277A: 13.8) was statistically significant (p = 0.002). Significance of IFN-β expression was assesses by a Wilcoxon matched-pairs test, * p < 0.05. ND, not detected. (<b>c</b>) Sensitivity of wild type and EndoU-deficient MHV (left panel) and HCoV-229E (right panel) viruses to IFN-I pre-treatment (4 h) in L929 cells (left panel) and MRC-5 cells (right panel) with various dosages of IFN-I (MOI = 1). Virus replication was measured at 24 h.p.i. by plaque assay (MHV) and at 48 h.p.i. by qRT-PCR (HCoV-229E), respectively. Data represent three independent experiments, each performed in two to three replicas. Data are displayed as differences to untreated controls and statistical comparisons between wild type and EndoU-deficient viruses were performed for each concentration. Mean and SEM are displayed. Data points that show significant differences in a two-sided, unpaired Student’s t-test are depicted. * p < 0.05, ** p < 0.01 and *** p < 0001.</p