Publications by authors named "Vincent R Graziano"

7 Publications

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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.
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http://dx.doi.org/10.1371/journal.ppat.1009402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987144PMC
March 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 .
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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.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

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.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 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.
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http://dx.doi.org/10.1371/journal.ppat.1008242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162533PMC
April 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.
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http://dx.doi.org/10.1038/s41564-019-0602-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925324PMC
January 2020

Norovirus Attachment and Entry.

Viruses 2019 05 30;11(6). Epub 2019 May 30.

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

Human norovirus is a major human pathogen causing the majority of cases of viral gastroenteritis globally. Viral entry is the first step of the viral life cycle and is a significant determinant of cell tropism, host range, immune interactions, and pathogenesis. Bile salts and histo-blood group antigens are key mediators of norovirus entry; however, the molecular mechanisms by which these molecules promote infection and the identity of a potential human norovirus receptor remain unknown. Recently, there have been several important advances in norovirus entry biology including the identification of CD300lf as the receptor for murine norovirus and of the role of the minor capsid protein VP2 in viral genome release. Here, we will review the current understanding about norovirus attachment and entry and highlight important future directions.
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http://dx.doi.org/10.3390/v11060495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630345PMC
May 2019