Publications by authors named "Craig B Wilen"

60 Publications

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy.

bioRxiv 2021 Mar 22. Epub 2021 Mar 22.

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal efficacy afforded by NAbs against SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.22.436337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010726PMC
March 2021

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

Emerg Infect Dis 2021 04;27(4):1146-1150

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2704.204199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007305PMC
April 2021

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

Cell 2021 Mar 11. Epub 2021 Mar 11.

Department of Pathology, Stanford University, Stanford, CA, USA. Electronic address:

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2021.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951565PMC
March 2021

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

PLoS Biol 2021 03 17;19(3):e3001143. Epub 2021 Mar 17.

Department of Laboratory Medicine, Yale University, New Haven, Connecticut, United States of America.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.3001143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007021PMC
March 2021

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

PLoS Pathog 2021 Mar 11;17(3):e1009402. Epub 2021 Mar 11.

Division of Infectious Diseases, Department of Medicine, Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1009402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987144PMC
March 2021

Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms.

Mol Cell 2021 02 1;81(3):584-598.e5. Epub 2021 Jan 1.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA; Department of Chemistry, Yale University, New Haven, CT 06511, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across β-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775661PMC
February 2021

Non-steroidal anti-inflammatory drugs dampen the cytokine and antibody response to SARS-CoV-2 infection.

J Virol 2021 Jan 13. Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). As PGs play diverse biological roles in homeostasis and inflammatory responses, inhibiting PG production with NSAIDs could affect COVID-19 pathogenesis in multiple ways, including: (1) altering susceptibility to infection by modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; (2) regulating replication of SARS-CoV-2 in host cells; and (3) modulating the immune response to SARS-CoV-2. Here, we investigate these potential roles. We demonstrate that SARS-CoV-2 infection upregulates COX-2 in diverse human cell culture and mouse systems. However, suppression of COX-2 by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. In contrast, in a mouse model of SARS-CoV-2 infection, NSAID treatment reduced production of pro-inflammatory cytokines and impaired the humoral immune response to SARS-CoV-2 as demonstrated by reduced neutralizing antibody titers. Our findings indicate that NSAID treatment may influence COVID-19 outcomes by dampening the inflammatory response and production of protective antibodies rather than modifying susceptibility to infection or viral replication.Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19). NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which are critical for the generation of prostaglandins - lipid molecules with diverse roles in homeostasis and inflammation. Inhibition of prostaglandin production by NSAIDs could therefore have multiple effects on COVID-19 pathogenesis. Here, we demonstrate that NSAID treatment reduced both the antibody and pro-inflammatory cytokine response to SARS-CoV-2 infection. The ability of NSAIDs to modulate the immune response to SARS-CoV-2 infection has important implications for COVID-19 pathogenesis in patients. Whether this occurs in humans and whether it is beneficial or detrimental to the host remains an important area of future investigation. This also raises the possibility that NSAIDs may alter the immune response to SARS-CoV-2 vaccination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00014-21DOI Listing
January 2021

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

J Exp Med 2021 03;218(3)

Department of Immunobiology, Yale School of Medicine, New Haven, CT.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20202135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808299PMC
March 2021

UFMylation inhibits the proinflammatory capacity of interferon-γ-activated macrophages.

Proc Natl Acad Sci U S A 2021 01;118(1)

Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2011763118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817147PMC
January 2021

An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2.

Cell Rep 2020 12 1;33(12):108528. Epub 2020 Dec 1.

Department of Microbiology, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address:

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of β coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705358PMC
December 2020

Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity.

Cell Metab 2021 Feb 4;33(2):270-282.e8. Epub 2020 Dec 4.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Recent studies suggest that mitochondria can be transferred between cells to support the survival of metabolically compromised cells. However, whether intercellular mitochondria transfer occurs in white adipose tissue (WAT) or regulates metabolic homeostasis in vivo remains unknown. We found that macrophages acquire mitochondria from neighboring adipocytes in vivo and that this process defines a transcriptionally distinct macrophage subpopulation. A genome-wide CRISPR-Cas9 knockout screen revealed that mitochondria uptake depends on heparan sulfates (HS). High-fat diet (HFD)-induced obese mice exhibit lower HS levels on WAT macrophages and decreased intercellular mitochondria transfer from adipocytes to macrophages. Deletion of the HS biosynthetic gene Ext1 in myeloid cells decreases mitochondria uptake by WAT macrophages, increases WAT mass, lowers energy expenditure, and exacerbates HFD-induced obesity in vivo. Collectively, this study suggests that adipocytes and macrophages employ intercellular mitochondria transfer as a mechanism of immunometabolic crosstalk that regulates metabolic homeostasis and is impaired in obesity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2020.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858234PMC
February 2021

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a conditional knockout ( ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV) In contrast, the nonpersistent MNoV strain CW3 (MNoV) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (), neutrophils (), and dendritic cells () did not affect MNoV viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV; that myelomonocytic cells are a major, but not exclusive, target cell of MNoV; and that STAT1 signaling restricts the cellular tropism of MNoV This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV infection. Mortality associated with the MNoV strain in mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01652-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925115PMC
January 2021

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Cytidine Monophosphate -Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

J Virol 2020 12 22;95(2). Epub 2020 Dec 22.

Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP -acetylneuraminic acid synthetase () and solute carrier family 35 member A1 (), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA and T3SA, were used to evaluate and as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA, which does not engage sialic acid. Furthermore, infectivity of T3SA was diminished to levels similar to those of T3SA in cells lacking and by CRISPR ablation. However, exogenous expression of and into the respective null cells restored sialic acid expression and T3SA binding and infectivity. These results demonstrate that and , which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, and , which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01571-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944449PMC
December 2020

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection.

bioRxiv 2020 Oct 6. Epub 2020 Oct 6.

Department of Pathology, Stanford University, Stanford, CA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.

Highlights: · ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.10.06.327445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553159PMC
October 2020

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication.

bioRxiv 2020 Sep 25. Epub 2020 Sep 25.

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE , one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

Importance: Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.09.24.312769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523115PMC
September 2020

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

bioRxiv 2020 Sep 8. Epub 2020 Sep 8.

Department of Immunobiology, Yale School of Medicine, New Haven, CT 06510, USA.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.06.25.169946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491522PMC
September 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

The Interpretation of SARS-CoV-2 Diagnostic Tests.

Med (N Y) 2020 Dec 21;1(1):78-89. Epub 2020 Aug 21.

Departments of Laboratory Medicine and Immunobiology, Yale University School of Medicine, New Haven, CT 06520 USA.

Testing for SARS-CoV-2 has attracted a tremendous amount of attention as a tool to manage the ongoing COVID-19 pandemic. Although diagnostic laboratory testing is used ubiquitously by physicians and encountered regularly by individuals receiving medical care, several aspects of test interpretation are incompletely understood by medical communities and the general population, creating a significant challenge in minimizing the damage caused by disease spread through informed decision making and proper testing utilization. Here, general principles of test interpretation are reviewed and applied to specific examples, such as whether asymptomatic individuals should be tested, what it means to test positive (or negative), and how to interpret tests for "immunity passports." Unexpectedly, the answers seem to run contrary to many of the popular narratives about testing as a tool for managing COVID-19. Although testing is an important and essential part of managing diseases such as COVID-19, improper utilization can have unintended negative consequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.medj.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441939PMC
December 2020

Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices.

medRxiv 2020 Aug 4. Epub 2020 Aug 4.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.08.03.20165233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418742PMC
August 2020

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

J Exp Med 2020 12;217(12)

Department of Immunobiology, Yale University School of Medicine, New Haven, CT.

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20201241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401025PMC
December 2020

Mouse Model of SARS-CoV-2 Reveals Inflammatory Role of Type I Interferon Signaling.

SSRN 2020 Jun 16:3628297. Epub 2020 Jun 16.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate. Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies.  Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.  This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19. Funding: This study was supported by awards from National Institute of Health grants, 2T32AI007517-16 (to BI), T32GM007205 and F30CA239444 (to ES), AI054359 and AI127429 (to AI), T32AI007019 (to TM),K08 AI128043 (to CBW), as well as Women's Health Research at Yale Pilot Project Program (AI, AR), Fast Grant from Emergent Ventures at the Mercatus Center (AI, ES), Mathers Foundation (AR, CBW, AI), and the Ludwig Family Foundation (AI, AR, CBW). A.I. is an investigator of the Howard Hughes Medical Institute. Conflict of Interest: None of the authors declare interests related to the manuscript. Ethical Approval: All procedures were performed in a BSL-3 facility (for SARS-CoV-2 infected mice) with approval from the Yale Environmental Health and Safety office.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2139/ssrn.3628297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366812PMC
June 2020

CD300LF Polymorphisms of Inbred Mouse Strains Confer Resistance to Murine Norovirus Infection in a Cell Type-Dependent Manner.

J Virol 2020 08 17;94(17). Epub 2020 Aug 17.

Committee on Microbiology, The University of Chicago, Chicago, Illinois, USA

Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC' loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry. MNV is a prevalent model system for studying human norovirus, which is the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele, which functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell type-specific modifiers of CD300LF-dependent MNV entry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00837-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431780PMC
August 2020

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

bioRxiv 2020 May 27. Epub 2020 May 27.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate. Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies. Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.27.118893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302201PMC
May 2020

Single-cell longitudinal analysis of SARS-CoV-2 infection in human bronchial epithelial cells.

bioRxiv 2020 May 7. Epub 2020 May 7.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT, USA.

SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.06.081695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263511PMC
May 2020

CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus.

PLoS Pathog 2020 04 6;16(4):e1008242. Epub 2020 Apr 6.

Departments of Laboratory Medicine and Immunobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162533PMC
April 2020

Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes.

Nat Microbiol 2020 02 20;5(2):272-281. Epub 2020 Jan 20.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens. However, if left uncontrolled, these responses may lead to detrimental inflammation. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0633-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147835PMC
February 2020

The intestinal regionalization of acute norovirus infection is regulated by the microbiota via bile acid-mediated priming of type III interferon.

Nat Microbiol 2020 01 25;5(1):84-92. Epub 2019 Nov 25.

Department of Molecular Genetics & Microbiology, Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA.

Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0602-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925324PMC
January 2020

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Elife 2019 08 12;8. Epub 2019 Aug 12.

Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.46681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739877PMC
August 2019

Bile Salts Alter the Mouse Norovirus Capsid Conformation: Possible Implications for Cell Attachment and Immune Evasion.

J Virol 2019 10 12;93(19). Epub 2019 Sep 12.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA

Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 Å in physiological buffers. Since this unusual feature is shared among, and unique to, the , it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (∼3 Å) and the receptor CD300lf (∼8 Å). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity. Mouse norovirus and several other members of the have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90° rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00970-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744230PMC
October 2019