ABSTRACT LY6E is an antiviral restriction factor that inhibits coronavirus spike-mediated fusion, but the cell types in vivo that require LY6E for protection from respiratory coronavirus

infection are unknown. Here we used a panel of seven conditional _Ly6e_ knockout mice to define which _Ly6e_-expressing cells confer control of airway infection by murine coronavirus and

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loss of _Ly6e_ in _Lyz2_-expressing cells, radioresistant _Vav1_-expressing cells and non-haematopoietic cells increased

susceptibility to murine coronavirus. Global conditional loss of _Ly6e_ expression resulted in clinical disease and higher viral burden after SARS-CoV-2 infection, but little evidence of

coronaviruses. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access

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CONTENT BEING VIEWED BY OTHERS GENOME-WIDE BIDIRECTIONAL CRISPR SCREENS IDENTIFY MUCINS AS HOST FACTORS MODULATING SARS-COV-2 INFECTION Article Open access 25 July 2022 LY6E IMPAIRS

CORONAVIRUS FUSION AND CONFERS IMMUNE CONTROL OF VIRAL DISEASE Article 23 July 2020 GENOME-WIDE CRISPR SCREENING IDENTIFIES TMEM106B AS A PROVIRAL HOST FACTOR FOR SARS-COV-2 Article 08 March

2021 DATA AVAILABILITY The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files or are available on

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cells. _Science_ 343, 84–87 (2014). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank members of the Schoggins lab for useful discussions. We also thank M. Aufiero

and T. Hohl (Memorial Sloan Kettering Cancer Center) for advice and feedback for bone marrow chimera studies, the lab of L. Hooper (University of Texas Southwestern Medical Center (UTSW))

for CD11c-Cre and LysM-Cre transgenic mice, the labs of J. Pfeiffer (UTSW) and M. Baldridge (Washington University in St. Louis) for _Ifnar__−/−_ and _Ifnlr__−/−_ mice, respectively, the

UTSW Animal Resource Center for training and animal husbandry, the UTSW Metabolic Phenotyping Core for analysis of serum samples for ALT levels and expertise, the UTSW Histo Pathology Core,

the UTSW Preclinical Radiation Core Facility (supported by funding from CPRIT grant RP180770) and the UTSW Immunology Flow Cytometry Core. The authors also acknowledge the Quantitative Light

Microscopy Core, a Shared Resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, 1P30 CA142543-01, and 1S10 RR029731-01 to K. Luby-Phelps.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Katrina B. Mar, Alexandra I. Wells, Marley C.

Caballero Van Dyke, Alexandra H. Lopez, Jennifer L. Eitson, Wenchun Fan & John W. Schoggins * Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA

Natasha W. Hanners * Departments of Pathology and Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USA Bret M. Evers * Department of Internal Medicine, University

of Texas Southwestern Medical Center, Dallas, TX, USA John M. Shelton Authors * Katrina B. Mar View author publications You can also search for this author inPubMed Google Scholar *

Alexandra I. Wells View author publications You can also search for this author inPubMed Google Scholar * Marley C. Caballero Van Dyke View author publications You can also search for this

author inPubMed Google Scholar * Alexandra H. Lopez View author publications You can also search for this author inPubMed Google Scholar * Jennifer L. Eitson View author publications You can

also search for this author inPubMed Google Scholar * Wenchun Fan View author publications You can also search for this author inPubMed Google Scholar * Natasha W. Hanners View author

publications You can also search for this author inPubMed Google Scholar * Bret M. Evers View author publications You can also search for this author inPubMed Google Scholar * John M.

Shelton View author publications You can also search for this author inPubMed Google Scholar * John W. Schoggins View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS K.B.M. and J.W.S. designed the project. K.B.M., M.C.C.V.D., A.H.L. and J.W.S. performed in vivo and in vitro experiments with help from W.F. and N.W.H. A.I.W. performed

HCR-RNA-FISH and immunofluorescence experiments. J.L.E. and J.W.S. performed and analysed HBE experiments. J.L.E. performed in vitro experiments. B.M.E. and J.M.S. contributed to pathology

analysis. K.B.M. analysed remaining data and prepared figures. K.B.M. and J.W.S. wrote the manuscript. All authors reviewed and provided comments on the manuscript. This study was supported

by grants from The Clayton Foundation (to J.W.S.) and NIH (AI158124 to J.W.S. and AI132751 to N.W.H.). J.W.S. holds an Investigators in the Pathogenesis of Infectious Disease Award from the

Burroughs Wellcome Fund. CORRESPONDING AUTHOR Correspondence to John W. Schoggins. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW

INFORMATION _Nature Microbiology_ thanks Yize Li, Olivier Schwartz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 A-F, _Ly6e__fl/fl_

and _Ly6e__ΔVav1_ mice were intraperitoneally infected with 5,000 PFU MHV-A59 and assessed for survival (A), viral burden in brain, lung, spleen, and liver (B), viral burden in heart (C),

viral burden in serum (D), serum alanine aminotransferase (E), and post-mortem spleen weight (F). In A, data represents means from n = 34 _Ly6e__fl/fl_ and n = 23 Ly6eΔVav1; B, D-F, n = 8

_Ly6e__fl/fl_ and n = 15 _Ly6e__ΔVav1_; C, n = 5 _Ly6e__fl/fl_ and n = 10 _Ly6e__ΔVav1_. G, Flow cytometry gating strategy for sorting lymphocytes from the spleen for examining Ly6e gene

expression in _Ly6e__ΔCD4_, _Ly6e__ΔCD8a_, and _Ly6e__ΔCD19_ mice relative to _Ly6e__fl/fl_ littermates in Fig. 1g. H–L, mice were intraperitoneally infected with 5,000 PFU MHV-A59 and

assessed for (H) survival (n = 47 _Ly6e__fl/fl_, n = 15 _Ly6e__ΔLyz2_, n = 14 _Ly6e__ΔCD11c_, n = 14 _Ly6e__ΔCD4_, n = 5 _Ly6e__ΔCD8a_, and n = 16 _Ly6e__ΔCD19_), viral burden in brain,

lung, spleen, and liver (I), viral burden in serum (J), serum alanine aminotransferase (K), and post-mortem spleen weight (L). In I-L, data represents means from n = 30 _Ly6e__fl/fl_, n=13

_Ly6e__ΔLysM_, n = 14 _Ly6e__ΔCD11c_, n = 10 _Ly6e__ΔCD4_, n = 6 _Ly6e__ΔCD8a_, and n = 14 _Ly6e__ΔCD19_. Male and female mice were used at an approximately 1 to 1 ratio for these

experiments. Statistical significance was determined by log-rank (Mantel-Cox) tests (A, H), two-sided Mann-Whitney test (B-D), two-sided unpaired t-test (E-F), Kruskal-Wallis test (I-J), and

one-way ANOVA (K-L). Error bars represent mean ± standard deviation. EXTENDED DATA FIG. 2 A, Flow cytometry gating strategy for sorting lung CD45+ cells and CD31+ cells from _Ly6e__ΔVav1_

mice for determining Ly6e gene expression as shown in Fig. 2c,d. B, Flow cytometry gating strategy for identifying donor (CD45.1+) and recipient (CD45.2+) immune cells. C, Relative

composition of CD45.1+ and CD45.2+ immune cells of compartment total CD45+ cells in lung, spleen, and blood. D, Relative Ly6e mRNA levels in brain, heart, lung, liver, and spleen from _Ly6e_

wildtype, heterozygous, and knockout mice. In C, data represents means from n = 3 _Ly6e__fl/fl__; CD45__.2/.2_, n = 3 _Ly6e__fl/fl__; CD45__.1/.1_, n = 3 _Ly6e__fl→fl_, n = 3

_Ly6e__fl→ΔVav1_; D, n = 3 _Ly6_e wildtype, n = 3 _Ly6e_ heterozygous, n = 3 _Ly6e_ knockout. Error bars represent mean ± standard deviation. EXTENDED DATA FIG. 3 A, _Ly6e_ wildtype and

knockout mice were intranasally infected with 8,700 PFU SARS-CoV-2 and monitored daily for weight loss. Lung viral burden measured by quantitative PCR for _Ly6e_ wildtype and knockout mice

(B) or C57BL/6J, _Ifnar__−/−_, and _Ifnlr__−/−_ mice (C) that were infected with 60,000 PFU SARS-CoV-2 and euthanized 3 days post-infection. D, Lung _Ly6e_ expression from C57BL/6J,

_Ifnar__−/−_, and _Ifnlr__−/−_ mice that were mock treated with PBS or infected with 8,700 PFU SARS-CoV-2 and euthanized the next day. Expression is shown relative to PBS-treated C57BL/6J

mice. Expression of _Ly6e_ (E) and _Mx1_ (F) in lungs from C57BL/6J mice treated intranasally with recombinant human IFNλ or retro-orbitally with recombinant murine IFNβ relative to

untreated (untr). In A, data represents means from n = 5 _Ly6e_ wildtype, n = 5 _Ly6e_ knockout mice; B, n = 13 _Ly6e_ wildtype, n = 13 _Ly6e_ knockout mice; C, n = 8 C57BL6/J, n = 8

_Ifnar__−/−_, and n=8 _Ifnlr__−/−_; D, n = 5 for each group and genotype; E-F, n = 6 untreated, n = 9 IFNλ-treated, n = 8 IFNβ-treated. G, Representative hematoxylin and eosin-stained lung

sections used for analysis shown in Fig. 3e from _Ly6e_ wildtype (n = 6) and _Ly6e_ knockout (n = 6) mice euthanized 3 days after treated with PBS or intranasal infection with 60,000 PFU

SARS-CoV-2. Example of automated image analysis of SARS-CoV-2 infected cells for Fig. 3g–h (H) and of CD45+ cells for Fig. 3i, j (I). In the corresponding markup images, cells without DAB

marker (for example positive stain) are colored blue, and positive cells identified as weak positive, moderate positive, and strong positive for the DAB marker are colored as yellow, orange,

and red respectively. Statistical significance was determined by two-sided unpaired t-test (A), two-sided Mann-Whitney test (B-C), one way ANOVA with Holm-Šídák′s multiple comparisons test

(D–F). Error bars represent mean ± SEM in A and mean ± SD in B-F. Scale bars: 600 μm (G), 1 mm and 100 μm (H-I). EXTENDED DATA FIG. 4 mRNA sequencing of lungs from _Ly6e_ wildtype and _Ly6e_

knockout mice that were intranasally infected with 60,000 PFU P.1 SARS-CoV-2 and euthanized 3 days post-infection. Volcano plot summarization of differentially expressed genes (DEGs)

between uninfected _Ly6e_ wildtype (n = 6) and _Ly6e_ knockout (n = 4) mice (A), uninfected wildtype (n = 6) and SARS-CoV-2 infected wildtype (n = 7) mice (B), and uninfected knockout (n = 

4) and SARS-CoV-2 infected knockout (n=7) mice (C). D, Z-score heatmap of the top 25 DEGs for data summarized in A. E, Gene Ontology (GO) enrichment analysis of transcriptomic data from

SARS-CoV-2 infected _Ly6e_ wildtype and _Ly6e_ knockout mice. The abscissa is the ratio of the number of differential genes linked with the GO term to the total number of differential genes.

The size of a point represents the number of genes annotated to a specific GO term, and the color from red to purple represents the significant level of the enrichment. Male and female mice

were used at an approximately 1 to 1 ratio for these experiments. For A-C, P-values were determined using the negative binomial distribution model and adjusted for multiple hypothesis

testing using the Benjamini and Hochberg’s approach for controlling the false discovery rate. P-values for E were determined using hypergeometric distribution and adjusted testing using the

Benjamini and Hochberg’s approach for controlling the false discovery rate. EXTENDED DATA FIG. 5 Differential expression of select genes highlighted in Fig. 4a between SARS-CoV-2-infected

_Ly6e_ wildtype mice and uninfected _Ly6e_ wildtype mice (A) and SARS-CoV-2-infected _Ly6e_ knockout mice and uninfected _Ly6e_ knockout mice (B). EXTENDED DATA FIG. 6 Mice were infected

with 8,700 PFU P.1 SARS-CoV-2 and euthanized 1 day post-infection. Lung sections were probed for SARS-CoV-2 RNA (green) and _Scgb1a1_ mRNA (red) (A, C) or _Scgb3a1_ mRNA (red) (B, D). Lung

sections from n = 3 _Ly6e_ knockout mice were stained and imaged. Images shown are from two different mice than in Fig. 4. Scale bars: 100 µM (A-B), 25 µM (C-D). EXTENDED DATA FIG. 7 Mice

were infected with 8,700 PFU SARS-CoV-2 and euthanized 1 day post-infection. Lung sections were probed for SARS-CoV-2 RNA (green) and _Sftpc_ mRNA (red) (A, C) or for SARS-CoV-2 nucleocapsid

(green) and acetylated tubulin (red) (B, D). Lung sections from n = 3 _Ly6e_ knockout mice were stained and imaged. Images shown are from different mice than in Fig. 4. Scale bars: 100 µM

(A-B), 25 µM (C-D). EXTENDED DATA FIG. 8 A, Flow cytometry gating strategy used in Fig. 4g–h for detecting SARS-CoV-2 nucleocapsid (N) in SCGB1A1-positive and acetylated tubulin-positive

pulmonary epithelial cells. Representative plots are from uninfected _Ly6e_ knockout mice. EXTENDED DATA FIG. 9 A, Area under the curve analysis of SARS-CoV-2 infectivity in HBE-ACE2 cells

from n = 3 independent experiments, B, Western blot of two independent preparations of control and LY6E knockout cells used in A. Statistical significance was determined by two-sided

unpaired t-test (A). Error bars represent mean ± standard deviation. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Tables 1–5. REPORTING SUMMARY SOURCE DATA SOURCE DATA

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publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mar, K.B., Wells, A.I., Caballero Van Dyke, M.C. _et al._ LY6E is a pan-coronavirus

restriction factor in the respiratory tract. _Nat Microbiol_ 8, 1587–1599 (2023). https://doi.org/10.1038/s41564-023-01431-w Download citation * Received: 07 July 2022 * Accepted: 19 June

2023 * Published: 13 July 2023 * Issue Date: August 2023 * DOI: https://doi.org/10.1038/s41564-023-01431-w SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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