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Proteintech rabbit anti ly6e
Rabbit Anti Ly6e, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti ly6e/product/Proteintech
Average 93 stars, based on 6 article reviews

rabbit anti ly6e - by Bioz Stars, 2026-06

93/100 stars

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Journal: Nature

Article Title: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

doi: 10.1038/s41586-023-06322-y

Figure Lengend Snippet: a , Expression profile of PLSCR1 across cell types. Data were extracted from the Human Protein Atlas database. b , Western blot showing the protein expression level in hTEpiCs overexpressing the indicated proteins. Related to Fig. . c , Left, quantification of intracellular SARS-CoV-2 RNA in Calu-3 cells (MOI = 1, 24 hpi, n = 4) or SARS-CoV-2-mNG infection in HaCaT-ACE2 (MOI = 1, 24 hpi, n = 3), and Tonsil-ACE2 (MOI = 1, 24 hpi, n = 3) cells. Right, western blot showing the expression level of the indicated proteins in Calu-3, HaCaT-ACE2 or Tonsil-ACE2 cells. d , Left, representative images showing the infectivity of SARS-CoV-2-mNG in HeLa-ACE2 of indicated genotypes. Right, western blot showing the expression level of the indicated proteins in HeLa-ACE2. e , Quantification of intracellular SARS-CoV-2 RNA in heLa-ACE2 in d (MOI = 0.2, 24 hpi, n = 3) cells. f , Western blot showing the expression level in Huh7.5 cells stably overexpressing the indicated ISGs. Related to Fig. . g , Western blot showing the endogenous expression levels of PLSCR1 and LY6E in A549-ACE2 single- or double-KO cells. Related to Fig. . h , Bar graph showing the average restriction ratio (−IFNγ/+ IFNγ) in A549-ACE2 cells after SARS-CoV-2 infection of the indicated genotypes in the presence or absence of IFNγ (100 U ml −1 ) 48 hpi (MOI = 0.2) ( n = 6). Related to Fig. . Data are mean ± s.d. P values were calculated using two-sided Student’s t -test in c (middle and right), two-sided Student’s t -test with Welch’s correction in c (left) or one-way ANOVA followed by Tukey’s multiple comparison test in e . Scale bar in b , f : 500 μm. Experiments in this figure were performed three times.

Article Snippet: Rabbit anti-LY6E polyclonal antibody (ab300399) was purchased from Abcam.

Techniques: Expressing, Western Blot, Infection, Stable Transfection

Fig. 4. LY6E efficiently suppresses human coronavirus spike protein-mediated entry. (A) 780

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/jvi.00562-20

Figure Lengend Snippet: Fig. 4. LY6E efficiently suppresses human coronavirus spike protein-mediated entry. (A) 780

Article Snippet: Rabbit 372 polyclonal antibody against human LY6E was obtained from proteintech (Cat.No.

Techniques:

Fig. 5 LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and 798

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/jvi.00562-20

Figure Lengend Snippet: Fig. 5 LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and 798

Article Snippet: Rabbit 372 polyclonal antibody against human LY6E was obtained from proteintech (Cat.No.

Techniques: Infection

Fig. 6. Identification of critical structure motifs essential for LY6E to restrict human 814

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/jvi.00562-20

Figure Lengend Snippet: Fig. 6. Identification of critical structure motifs essential for LY6E to restrict human 814

Article Snippet: Rabbit 372 polyclonal antibody against human LY6E was obtained from proteintech (Cat.No.

Techniques:

Fig. 7. LY6E inhibits TMPRSS2 enhanced entry of human coronaviruses. Flp-In T-Rex 293- 835

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/jvi.00562-20

Figure Lengend Snippet: Fig. 7. LY6E inhibits TMPRSS2 enhanced entry of human coronaviruses. Flp-In T-Rex 293- 835

Article Snippet: Rabbit 372 polyclonal antibody against human LY6E was obtained from proteintech (Cat.No.

Techniques:

IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

LY6E efficiently suppresses human coronavirus spike-protein-mediated entry. (A) Levels of Ly6E, GILT, and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT, or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at 24 h after the addition of tet. The cellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). **, P < 0.001 compared to the control cells expressing CAT.

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E efficiently suppresses human coronavirus spike-protein-mediated entry. (A) Levels of Ly6E, GILT, and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT, or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at 24 h after the addition of tet. The cellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). **, P < 0.001 compared to the control cells expressing CAT.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and lung cancer (A549) cells. (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of cellular LY6E expression was determined by Western blotting using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E-specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). Differences in viral RNA between scramble or LY6E-specific shRNA-expressing cells were analyzed statistically (**, P < 0.001; Student’s t test). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a Western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red); cell nuclei were visualized by DAPI staining (D and F).

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and lung cancer (A549) cells. (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of cellular LY6E expression was determined by Western blotting using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E-specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). Differences in viral RNA between scramble or LY6E-specific shRNA-expressing cells were analyzed statistically (**, P < 0.001; Student’s t test). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a Western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red); cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Identification of critical structural motifs essential for LY6E to restrict human coronavirus entry. (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is presented, in which the “three finger-fold” structure is highlighted with black boxes. The conserved L36 as well as the GPI anchor and N99 glycosylation sites are indicated. (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT or wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E. (D) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in medium with indicated concentrations of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (E) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E (L36A) were cultured with or without the indicated concentrations of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E.

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Identification of critical structural motifs essential for LY6E to restrict human coronavirus entry. (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is presented, in which the “three finger-fold” structure is highlighted with black boxes. The conserved L36 as well as the GPI anchor and N99 glycosylation sites are indicated. (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT or wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E. (D) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in medium with indicated concentrations of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (E) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E (L36A) were cultured with or without the indicated concentrations of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

LY6E inhibits TMPRSS2-enhanced entry of human coronaviruses. Flp-In T-Rex 293-derived cell lines expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. (A) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the absence of tet. (B) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the presence of tet. (C) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation ( n = 4); **, P < 0.001 compared to cells transfected with the pCAGGS vector.

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits TMPRSS2-enhanced entry of human coronaviruses. Flp-In T-Rex 293-derived cell lines expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. (A) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the absence of tet. (B) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the presence of tet. (C) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation ( n = 4); **, P < 0.001 compared to cells transfected with the pCAGGS vector.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Activity Assay, Standard Deviation

Amphotericin B treatment compromises IFITM3 inhibition of human coronavirus entry, but have no impact on Ly6E inhibition of human coronavirus entry. Flp-In T-Rex 293-derived cell lines expressing IFITM3 (A) or LY6E (B) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1 μM AmphoB. Luciferase activity was measured at 48 h postinfection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mock treatment.

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Amphotericin B treatment compromises IFITM3 inhibition of human coronavirus entry, but have no impact on Ly6E inhibition of human coronavirus entry. Flp-In T-Rex 293-derived cell lines expressing IFITM3 (A) or LY6E (B) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1 μM AmphoB. Luciferase activity was measured at 48 h postinfection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mock treatment.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Inhibition, Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay

a , Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs. b , HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector. c-h , Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E ( c ), HCoV-OC43 ( d ), MERS-CoV ( e ), SARS-CoV ( f ), SARS-CoV-2 ( g ), MHV-Gluc ( h ). i , Western blot of WT A549 or two LY6E KO clones (#3, #4). j , HCoV-229E infection of WT or LY6E KO A549. k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty). l , HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549. Viral replication readouts included: titer determination by plaque assay ( c ), infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ), limiting dilution assay ( e,f,g ), luciferase activity shown as relative light units (RLU) ( h,j,l ). Data represent average of independent biological replicates, n=3 ( c-d,f-h ), n=4 ( e , j,l ). In b , scale bars are 200 µM. Statistical significance was determined by unpaired student’s t-test with Welch’s correction ( c-d , f-g ), one-tailed Mann-Whitney U test ( e ), ratio paired student’s t-test ( g ), 2way ANOVA followed by Sidak’s or Dunnett’s multiple comparison test ( j , l ). Error bars: SD. P values: c , ** p=0.0088; d , *** p=0.0001; e , * p=0.0143; f , * p=0.0286; g , * p=0.0101; h , *** p=0.0010; j , ** p=0.0073, p=0.0033; l , ns=0.0740, ** p=0.0013, *** p=0.0001.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a , Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs. b , HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector. c-h , Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E ( c ), HCoV-OC43 ( d ), MERS-CoV ( e ), SARS-CoV ( f ), SARS-CoV-2 ( g ), MHV-Gluc ( h ). i , Western blot of WT A549 or two LY6E KO clones (#3, #4). j , HCoV-229E infection of WT or LY6E KO A549. k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty). l , HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549. Viral replication readouts included: titer determination by plaque assay ( c ), infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ), limiting dilution assay ( e,f,g ), luciferase activity shown as relative light units (RLU) ( h,j,l ). Data represent average of independent biological replicates, n=3 ( c-d,f-h ), n=4 ( e , j,l ). In b , scale bars are 200 µM. Statistical significance was determined by unpaired student’s t-test with Welch’s correction ( c-d , f-g ), one-tailed Mann-Whitney U test ( e ), ratio paired student’s t-test ( g ), 2way ANOVA followed by Sidak’s or Dunnett’s multiple comparison test ( j , l ). Error bars: SD. P values: c , ** p=0.0088; d , *** p=0.0001; e , * p=0.0143; f , * p=0.0286; g , * p=0.0101; h , *** p=0.0010; j , ** p=0.0073, p=0.0033; l , ns=0.0740, ** p=0.0013, *** p=0.0001.

Article Snippet: LY6E was detected using anti-LY6E rabbit monoclonal antibody GEN-93-8-1 (Genentech) at a dilution of 1:5,000 and secondary Peroxidase-AffiniPure Donkey Anti-Rabbit IgG (Jackson Immunoresearch, 711-035-152) at a dilution of 1:10,000.

Techniques: Infection, Expressing, Plasmid Preparation, Stable Transfection, Western Blot, Clone Assay, CRISPR, Plaque Assay, Staining, Quantitation Assay, Flow Cytometry, Limiting Dilution Assay, Luciferase, Activity Assay, One-tailed Test, MANN-WHITNEY

a , Duplicate screens depicting HCoV-229E infection (48 hours post-infection) in Huh7 cells expressing ISGs. b , Multiple sequence alignment of Ly6e amino acid sequences of different species. c , Phylogenetic tree of mammalian LY6E orthologues. d , HCoV-229E infection of Huh7.5 cells expressing vector control or human ( H. sapiens ), camel ( C. dromedarius ), bat ( P. alecto ), mouse ( M. musculus ), or rhesus ( M. mulatta ) LY6E orthologues. e , Stable LY6E or empty vector expressing Huh7.5 cells infected with MERS-CoV in a limiting dilution assay. f , Stable Huh7.5 cells expressing either LY6E or containing empty vector infected with HCoV-229E, HCV, CHIKV, hPIV-3, RSV, SINV, VEEV, WNV, ZIKV, YFV, and DENV. g , HCoV-229E-Rluc infection of naïve or stably transduced Huh7 cells expressing empty vector control or LY6/uPAR family members. h , HCoV-229E-Rluc infection of Huh7 cells transduced with lentiviruses expressing firefly luciferase (Fluc) control, LY6E WT, LY6E HA or specific block mutants . Data represent average of independent biological replicates, n=3 ( d ), n=3 ( e ), n=3 ( f ), n=3 ( g ), n=3 ( h ). Statistical significance was determined by ordinary one-way ANOVA with Dunnett’s correction for multiple comparison ( d, e, g, h ), 2way ANOVA followed by Sidak’s multiple comparisons test ( f ). Error bars: SD. P values: d , **** p=<0.0001, ** p=0.0030; e , * p=0.0118, ** p=0.0013; f , **** p=>0.0001, ** p=0.0095, ns= >0.9999 (CHIKV) >0.9999 (PIV) >0.9999 (RSV) >0.9999 (SINV) >0.9999 (VEEV) 0.9679 (WNV) 0.8794 (ZIKV) 0.3172 (YFV) 0.7667 (DENV) (For CHIKV one outlier was removed in the control cells); g , **** p=<0.0001, ns p=0.9993 (GPFHBPI) 0.9998 (LY6H) 0.9991 (PSCA) 0.9990 (Ly6G6D) 0.9997 (CD59) >0.9999 (LYPD1) 0.9994 (LYPD2) 0.9997 (GML) 0.9997 (LY6D) 0.9992 (LY6K); h , **** p=<0.0001, <0.0001, *** p=0.0005, ns p=0.8472, **** p=<0.0001, *** p=0.0003, **** p=<0.0001, <0.0001, <0.0001, *** p=0.0001, **** p=<0.0001.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a , Duplicate screens depicting HCoV-229E infection (48 hours post-infection) in Huh7 cells expressing ISGs. b , Multiple sequence alignment of Ly6e amino acid sequences of different species. c , Phylogenetic tree of mammalian LY6E orthologues. d , HCoV-229E infection of Huh7.5 cells expressing vector control or human ( H. sapiens ), camel ( C. dromedarius ), bat ( P. alecto ), mouse ( M. musculus ), or rhesus ( M. mulatta ) LY6E orthologues. e , Stable LY6E or empty vector expressing Huh7.5 cells infected with MERS-CoV in a limiting dilution assay. f , Stable Huh7.5 cells expressing either LY6E or containing empty vector infected with HCoV-229E, HCV, CHIKV, hPIV-3, RSV, SINV, VEEV, WNV, ZIKV, YFV, and DENV. g , HCoV-229E-Rluc infection of naïve or stably transduced Huh7 cells expressing empty vector control or LY6/uPAR family members. h , HCoV-229E-Rluc infection of Huh7 cells transduced with lentiviruses expressing firefly luciferase (Fluc) control, LY6E WT, LY6E HA or specific block mutants . Data represent average of independent biological replicates, n=3 ( d ), n=3 ( e ), n=3 ( f ), n=3 ( g ), n=3 ( h ). Statistical significance was determined by ordinary one-way ANOVA with Dunnett’s correction for multiple comparison ( d, e, g, h ), 2way ANOVA followed by Sidak’s multiple comparisons test ( f ). Error bars: SD. P values: d , **** p=<0.0001, ** p=0.0030; e , * p=0.0118, ** p=0.0013; f , **** p=>0.0001, ** p=0.0095, ns= >0.9999 (CHIKV) >0.9999 (PIV) >0.9999 (RSV) >0.9999 (SINV) >0.9999 (VEEV) 0.9679 (WNV) 0.8794 (ZIKV) 0.3172 (YFV) 0.7667 (DENV) (For CHIKV one outlier was removed in the control cells); g , **** p=<0.0001, ns p=0.9993 (GPFHBPI) 0.9998 (LY6H) 0.9991 (PSCA) 0.9990 (Ly6G6D) 0.9997 (CD59) >0.9999 (LYPD1) 0.9994 (LYPD2) 0.9997 (GML) 0.9997 (LY6D) 0.9992 (LY6K); h , **** p=<0.0001, <0.0001, *** p=0.0005, ns p=0.8472, **** p=<0.0001, *** p=0.0003, **** p=<0.0001, <0.0001, <0.0001, *** p=0.0001, **** p=<0.0001.

Techniques: Infection, Expressing, Sequencing, Plasmid Preparation, Limiting Dilution Assay, Stable Transfection, Transduction, Luciferase, Blocking Assay

a , Stable LY6E expressing or empty control Huh7 cells were incubated with HCoV-229E and binding analyzed immediately (t=0 h) or after incubation for 24 hours (t=24 h) by RT-qPCR. b , Stable LY6E or empty vector expressing Huh7 cells were assessed for surface CD13 expression by flow cytometry. c , d, VSV pseudoparticles (PP) harboring VSV-G were inoculated on stable LY6E or empty vector Huh7 cells ( c ) or VeroE6 cells ( d ). e-i , Stable LY6E expressing or control Huh7 cells were mock-infected or infected with HCoV-229E-Rluc. Cell lysates were harvested at the indicated time points and intracellular viral RNA was extracted, and viral replication detected via qRT-PCR ( e ). Cell supernatant was harvested and extracellular viral RNA was extracted and viral replication detected via qRT-PCR ( f ). Cell lysates were harvested and intracellular Renilla luciferase activity was detected upon cell lysis ( g ). Cells were subjected to 3 rounds of freeze/thaw cycles. Cell debris was removed and the supernatant titrated on naïve Huh7 cells. Intracellular infectivity was determined ( h ). Supernatant was harvested and titrated on naïve Huh7 cells to determine extracellular infectivity ( i ). Data represent averages of independent biological replicates, n=3 ( a ), n=3 ( b ), n=9 ( c ), n=6 ( d ), n=3 ( e-i ). Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons test ( a ), unpaired student’s t-test with Welch’s correction ( b, c, d ). Error bars: SD. P values: a , ns p=0.9448 0.0752; b , ns p=0.5188; c , ns p=0.3857; d , * p=0.0112.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a , Stable LY6E expressing or empty control Huh7 cells were incubated with HCoV-229E and binding analyzed immediately (t=0 h) or after incubation for 24 hours (t=24 h) by RT-qPCR. b , Stable LY6E or empty vector expressing Huh7 cells were assessed for surface CD13 expression by flow cytometry. c , d, VSV pseudoparticles (PP) harboring VSV-G were inoculated on stable LY6E or empty vector Huh7 cells ( c ) or VeroE6 cells ( d ). e-i , Stable LY6E expressing or control Huh7 cells were mock-infected or infected with HCoV-229E-Rluc. Cell lysates were harvested at the indicated time points and intracellular viral RNA was extracted, and viral replication detected via qRT-PCR ( e ). Cell supernatant was harvested and extracellular viral RNA was extracted and viral replication detected via qRT-PCR ( f ). Cell lysates were harvested and intracellular Renilla luciferase activity was detected upon cell lysis ( g ). Cells were subjected to 3 rounds of freeze/thaw cycles. Cell debris was removed and the supernatant titrated on naïve Huh7 cells. Intracellular infectivity was determined ( h ). Supernatant was harvested and titrated on naïve Huh7 cells to determine extracellular infectivity ( i ). Data represent averages of independent biological replicates, n=3 ( a ), n=3 ( b ), n=9 ( c ), n=6 ( d ), n=3 ( e-i ). Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons test ( a ), unpaired student’s t-test with Welch’s correction ( b, c, d ). Error bars: SD. P values: a , ns p=0.9448 0.0752; b , ns p=0.5188; c , ns p=0.3857; d , * p=0.0112.

Techniques: Expressing, Incubation, Binding Assay, Quantitative RT-PCR, Plasmid Preparation, Flow Cytometry, Infection, Luciferase, Activity Assay, Lysis

a-d , VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E ( a ), MERS-CoV ( b ), SARS-CoV ( c ), SARS-CoV-2 ( d ) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity. e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector. f , Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). g , Schematic depiction of the cell-cell fusion assay created with BioRender. h , Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11). Data represent averages of independent biological replicates, n=8 ( a,b ), n=6 ( c,d ), n=3 ( f ), n=4 ( h ). Statistical significance was determined by unpaired student’s t-test with Welch’s correction ( a-d ) and 2way ANOVA followed by Sidak’s multiple comparison test ( f , h ). Outliers were removed using Grubbs outlier test ( a ). In e , scale bar is 20 µM. Error bars: SD. P values: a , **** p=<0.0001; b , **** p=<0.0001; c , **p=0.0066; d , **** p=<0.0001; f , **** p=<0.0001; h , ns p=0.9975, ** p=0.0054, ns p=0.1520, 0.9892.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-d , VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E ( a ), MERS-CoV ( b ), SARS-CoV ( c ), SARS-CoV-2 ( d ) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity. e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector. f , Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). g , Schematic depiction of the cell-cell fusion assay created with BioRender. h , Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11). Data represent averages of independent biological replicates, n=8 ( a,b ), n=6 ( c,d ), n=3 ( f ), n=4 ( h ). Statistical significance was determined by unpaired student’s t-test with Welch’s correction ( a-d ) and 2way ANOVA followed by Sidak’s multiple comparison test ( f , h ). Outliers were removed using Grubbs outlier test ( a ). In e , scale bar is 20 µM. Error bars: SD. P values: a , **** p=<0.0001; b , **** p=<0.0001; c , **p=0.0066; d , **** p=<0.0001; f , **** p=<0.0001; h , ns p=0.9975, ** p=0.0054, ns p=0.1520, 0.9892.

Techniques: Plasmid Preparation, Expressing, Luciferase, Activity Assay, Immunofluorescence, Microscopy, Cell-Cell Fusion Assay, Transfection, Construct

a , Schematic depiction of the generation of recombinant VSV*ΔG(CoV S) expressing both CoV S protein and GFP reporter protein. The respective CoV S genes were inserted into the genomic VSV*ΔG cDNA and recombinant virus was generated in BHK-G43 cells expressing the VSV G protein. The recombinant virus produced in this way harbored the VSV G protein in the envelope allowing CoV S protein-independent infection of cells. Infection of cells with VSV*ΔG(CoV S) led to the expression of CoV S protein and consequent syncytia formation. b , Heterologous syncytia formation assay. BHK-21 cells were infected with VSV G protein trans-complemented VSV*ΔG(CoV S) viruses or were mock-infected, followed by co-culture with LY6E or empty control Huh7 cells. Syncytia formation was determined. Blue: DAPI, green: GFP, red: TagRFP encoded in SCRPSY vector. c , Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). Data represent averages of independent biological replicates, n=3 ( c ). In b , scale bar is 20 µM. Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons test ( c ). Error bars: SD. P values: c , **** p=<0.0001 <0.0001.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a , Schematic depiction of the generation of recombinant VSV*ΔG(CoV S) expressing both CoV S protein and GFP reporter protein. The respective CoV S genes were inserted into the genomic VSV*ΔG cDNA and recombinant virus was generated in BHK-G43 cells expressing the VSV G protein. The recombinant virus produced in this way harbored the VSV G protein in the envelope allowing CoV S protein-independent infection of cells. Infection of cells with VSV*ΔG(CoV S) led to the expression of CoV S protein and consequent syncytia formation. b , Heterologous syncytia formation assay. BHK-21 cells were infected with VSV G protein trans-complemented VSV*ΔG(CoV S) viruses or were mock-infected, followed by co-culture with LY6E or empty control Huh7 cells. Syncytia formation was determined. Blue: DAPI, green: GFP, red: TagRFP encoded in SCRPSY vector. c , Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). Data represent averages of independent biological replicates, n=3 ( c ). In b , scale bar is 20 µM. Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons test ( c ). Error bars: SD. P values: c , **** p=<0.0001 <0.0001.

Techniques: Recombinant, Expressing, Generated, Produced, Infection, Tube Formation Assay, Co-Culture Assay, Plasmid Preparation

a , Schematic depiction of cell entry routes of CoVs and intervention by selected compounds. b-c , LY6E or empty control-expressing Huh7 cells naïve for ( b ) or ectopically overexpressing TMPRSS2 ( c ) were pre-treated with the indicated compounds before infection with HCoV-229E-Rluc. d , Schematic depiction of the CoV spike (S) protein containing the subunits S1 and S2. Arrows indicate the S1/S2’ cleavage site, a proposed cleavage site for cathepsin L (CTSL’) and the S2’ cleavage site. Amino acid exchanges disrupting the respective cleavage sites are depicted in red . e , Western blot of S cleavage in LY6E or empty control-expressing Huh7 cells transfected with a plasmid encoding for MERS-CoV S protein (S0= uncleaved, S2= S2 subunit). f , MERS CoV S cleavage was analyzed by quantification of S0 and S2 bands. g , LY6E or empty control-expressing Huh7 cells inoculated with CoV-pseudoparticles (PP) harboring MERS-CoV S WT or MERS-CoV S proteins containing various cleavage site mutations. Data represent average of independent biological replicates, n=3 ( b ), n=3 ( c ), n=3 ( e ), n=3 ( f ), n=5 ( g ). Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons ( b-c, g ), unpaired student’s t-test with Welch’s correction ( f ). Error bars: SD. P values: b , **** p=<0.0001, <0.0001, <0.0001, <0.0001, <0.0001, * p=0.0179; c , *** p=0.0007, 0.0005, 0.0006, **** p=<0.0001, <0.0001, ns p=0.1909; f, ns p=0.4968; g , ns p=0.9999 (WT) >0.9999 (NYT) >0.9999 (ASAA) >0.9999 (ASVA) >0.9999 (ASAA+ASVA) >0.9999 (SSVR) 0.9867 (VSV).

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a , Schematic depiction of cell entry routes of CoVs and intervention by selected compounds. b-c , LY6E or empty control-expressing Huh7 cells naïve for ( b ) or ectopically overexpressing TMPRSS2 ( c ) were pre-treated with the indicated compounds before infection with HCoV-229E-Rluc. d , Schematic depiction of the CoV spike (S) protein containing the subunits S1 and S2. Arrows indicate the S1/S2’ cleavage site, a proposed cleavage site for cathepsin L (CTSL’) and the S2’ cleavage site. Amino acid exchanges disrupting the respective cleavage sites are depicted in red . e , Western blot of S cleavage in LY6E or empty control-expressing Huh7 cells transfected with a plasmid encoding for MERS-CoV S protein (S0= uncleaved, S2= S2 subunit). f , MERS CoV S cleavage was analyzed by quantification of S0 and S2 bands. g , LY6E or empty control-expressing Huh7 cells inoculated with CoV-pseudoparticles (PP) harboring MERS-CoV S WT or MERS-CoV S proteins containing various cleavage site mutations. Data represent average of independent biological replicates, n=3 ( b ), n=3 ( c ), n=3 ( e ), n=3 ( f ), n=5 ( g ). Statistical significance was determined by 2way ANOVA followed by Sidak’s multiple comparisons ( b-c, g ), unpaired student’s t-test with Welch’s correction ( f ). Error bars: SD. P values: b , **** p=<0.0001, <0.0001, <0.0001, <0.0001, <0.0001, * p=0.0179; c , *** p=0.0007, 0.0005, 0.0006, **** p=<0.0001, <0.0001, ns p=0.1909; f, ns p=0.4968; g , ns p=0.9999 (WT) >0.9999 (NYT) >0.9999 (ASAA) >0.9999 (ASVA) >0.9999 (ASAA+ASVA) >0.9999 (SSVR) 0.9867 (VSV).

Techniques: Expressing, Infection, Western Blot, Transfection, Plasmid Preparation

a, Strategy used to generate Ly6e ΔHSC mice. Hematopoietic-tissue specific ablation of Ly6e was achieved by crossing Ly6e fl/fl mice with transgenic Vav1-iCre mice to remove the LoxP-flanked exon III and IV, resulting in a 17 amino acid truncated variant of the 130 amino acid full-length protein. Primers used for tissue genotyping and gene expression are also depicted as arrows. b, Gel electrophoresis of tissue genotyping PCR, representing Ly6e fl/fl , Ly6e fl/+ , and Ly6e +/+ mice. c , Ly6e gene expression in bone marrow-derived macrophages (BMDM). d, Infection of BMDM. For c-d , n=9 ( Ly6e fl/fl ) or n=8 ( Ly6e ΔHSC ) mice from three pooled experiments. Data for c-d is shown normalized to Ly6e fl/fl values. Statistical significance was determined by using two-tailed unpaired student’s t-test with Welch’s correction ( c ), two-tailed ratio-paired t-test ( d ). Error bars: SD. P values: c , *** p=0.0010; d , ** p=0.0021.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a, Strategy used to generate Ly6e ΔHSC mice. Hematopoietic-tissue specific ablation of Ly6e was achieved by crossing Ly6e fl/fl mice with transgenic Vav1-iCre mice to remove the LoxP-flanked exon III and IV, resulting in a 17 amino acid truncated variant of the 130 amino acid full-length protein. Primers used for tissue genotyping and gene expression are also depicted as arrows. b, Gel electrophoresis of tissue genotyping PCR, representing Ly6e fl/fl , Ly6e fl/+ , and Ly6e +/+ mice. c , Ly6e gene expression in bone marrow-derived macrophages (BMDM). d, Infection of BMDM. For c-d , n=9 ( Ly6e fl/fl ) or n=8 ( Ly6e ΔHSC ) mice from three pooled experiments. Data for c-d is shown normalized to Ly6e fl/fl values. Statistical significance was determined by using two-tailed unpaired student’s t-test with Welch’s correction ( c ), two-tailed ratio-paired t-test ( d ). Error bars: SD. P values: c , *** p=0.0010; d , ** p=0.0021.

Techniques: Transgenic Assay, Variant Assay, Expressing, Nucleic Acid Electrophoresis, Derivative Assay, Infection, Two Tailed Test

a-b, Female Ly6e fl/fl and Ly6e ΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ). c-n , Mice were assessed at 3 ( c-h ) or 5 ( i-n ) days post-infection for serum ALT ( c,i ), viral titers in liver ( d,j ) and spleen ( e,k ), and liver necrosis and inflammation ( f-h , l-n ). For a-b , n=13 for days 0-14 ( Ly6e fl/fl ), n=11 for day 0-3, n= 10 for days 4-5, n=5 for days 5-6, n=3 for days 6-14 ( Ly6e ΔHSC ) from three pooled experiments. For c-g , n=9 ( Ly6e fl/fl ), n=10 ( Ly6e ΔHSC ), n=6 (PBS) from two pooled experiments. For i-m , n=9 ( Ly6e fl/fl ,), n=7 ( Ly6e ΔHSC ), n=6 (PBS) from two pooled experiments. In h,n , scale bars are 500 µM (4x) and 100 µM (20x). Statistical significance was determined by Mantel-Cox test ( a ), two-tailed unpaired student’s t-test with Welch’s correction ( c-e , i-k ), two-tailed Mann-Whitney U test ( f-g , l-m ). Error bars: SEM ( b ), SD ( c-g , i-m ). P values: a , *** p=0.0002; c, ** p=0.0031; d , ns p=0.2136; e , **** p=5.759 × 10 −7 ; f , ns p=0.3698; g , ** p=0.0054; i , * p=0.0430; j , ns p=0.1321; k , ** p=0.0094; l , ** p=0.0072; m , **** p=8.741 × 10 −5 .

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-b, Female Ly6e fl/fl and Ly6e ΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ). c-n , Mice were assessed at 3 ( c-h ) or 5 ( i-n ) days post-infection for serum ALT ( c,i ), viral titers in liver ( d,j ) and spleen ( e,k ), and liver necrosis and inflammation ( f-h , l-n ). For a-b , n=13 for days 0-14 ( Ly6e fl/fl ), n=11 for day 0-3, n= 10 for days 4-5, n=5 for days 5-6, n=3 for days 6-14 ( Ly6e ΔHSC ) from three pooled experiments. For c-g , n=9 ( Ly6e fl/fl ), n=10 ( Ly6e ΔHSC ), n=6 (PBS) from two pooled experiments. For i-m , n=9 ( Ly6e fl/fl ,), n=7 ( Ly6e ΔHSC ), n=6 (PBS) from two pooled experiments. In h,n , scale bars are 500 µM (4x) and 100 µM (20x). Statistical significance was determined by Mantel-Cox test ( a ), two-tailed unpaired student’s t-test with Welch’s correction ( c-e , i-k ), two-tailed Mann-Whitney U test ( f-g , l-m ). Error bars: SEM ( b ), SD ( c-g , i-m ). P values: a , *** p=0.0002; c, ** p=0.0031; d , ns p=0.2136; e , **** p=5.759 × 10 −7 ; f , ns p=0.3698; g , ** p=0.0054; i , * p=0.0430; j , ns p=0.1321; k , ** p=0.0094; l , ** p=0.0072; m , **** p=8.741 × 10 −5 .

Techniques: Injection, Infection, Two Tailed Test, MANN-WHITNEY

a-b , Male Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ). c-n, Mice were euthanized at 3- ( c-h ) or 5- (i-n ) days post-infection for determination of serum ALT ( c,i ), viral titers in liver ( d,j ) and spleen ( e,k ), and liver necrosis and inflammation ( f-h , l-n ). For a-b , n=15 for days 0-6, n=14 for days 6-14, ( Ly6e fl/fl ), n=10 for days 0-5, n=4 for days 5-6, n=0 for days 6-14 ( Ly6e ΔHSC ) from three pooled experiments. For c-g , n=8 ( Ly6e fl/fl ,), n=6 ( Ly6e ΔHSC ), or n=6 (PBS) from two pooled experiments. For i-m , n=8 ( Ly6e fl/fl ,), n=6 ( Ly6e ΔHSC ), or n=3 (PBS) from two pooled experiments. In h,n , scale bars are 500 µM (4x) and 100 µM (20x). Significance for was tested by Mantel-Cox test ( a ), two-tailed unpaired student’s t-test with Welch’s correction ( c-e , i-k ), two-tailed Mann-Whitney U test ( f-g , l-m ). Error bars: SEM ( b ), SD ( c-g , i-m ). P value: a , **** p=3.946 × 10 −6 ; c, ns p=1144; d , ns p=0.1407; e , ** p=0.0026; f , ns p=0.1708; g , ns p=0.0779; i , ns p=0.6191; j , ns p=0.6842; k , * p=0.0119; l , ns p>0.9999; m , ns p=0.0779.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-b , Male Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ). c-n, Mice were euthanized at 3- ( c-h ) or 5- (i-n ) days post-infection for determination of serum ALT ( c,i ), viral titers in liver ( d,j ) and spleen ( e,k ), and liver necrosis and inflammation ( f-h , l-n ). For a-b , n=15 for days 0-6, n=14 for days 6-14, ( Ly6e fl/fl ), n=10 for days 0-5, n=4 for days 5-6, n=0 for days 6-14 ( Ly6e ΔHSC ) from three pooled experiments. For c-g , n=8 ( Ly6e fl/fl ,), n=6 ( Ly6e ΔHSC ), or n=6 (PBS) from two pooled experiments. For i-m , n=8 ( Ly6e fl/fl ,), n=6 ( Ly6e ΔHSC ), or n=3 (PBS) from two pooled experiments. In h,n , scale bars are 500 µM (4x) and 100 µM (20x). Significance for was tested by Mantel-Cox test ( a ), two-tailed unpaired student’s t-test with Welch’s correction ( c-e , i-k ), two-tailed Mann-Whitney U test ( f-g , l-m ). Error bars: SEM ( b ), SD ( c-g , i-m ). P value: a , **** p=3.946 × 10 −6 ; c, ns p=1144; d , ns p=0.1407; e , ** p=0.0026; f , ns p=0.1708; g , ns p=0.0779; i , ns p=0.6191; j , ns p=0.6842; k , * p=0.0119; l , ns p>0.9999; m , ns p=0.0779.

Techniques: Injection, Infection, Two Tailed Test, MANN-WHITNEY

a-j , Female ( a-e ) and male ( f-g ) Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and assessed for serum ALT ( a , f ), viral titers in liver ( b , g ) and spleen ( c , h ), and liver necrosis and inflammation ( d-e , i-j ). k-n , Female Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and whole livers were imaged. Mock-infected (PBS) Ly6e ΔHSC mice ( k ), mock-infected Ly6e fl/fl mice ( l ), MHV-infected Ly6e ΔHSC mice ( m ), MHV-infected Ly6e fl/fl mice ( n ). For a-e , n=8 ( Ly6e fl/fl ,), n=8 ( Ly6e ΔHSC ), or n=3 (PBS) from two pooled experiments. For f-j , n=9 ( Ly6e fl/fl ,), n=7 ( Ly6e ΔHSC ), or n=3 (PBS) two pooled experiments. For k-n , n=3. Statistical significance was determined by two-tailed unpaired student’s t-test with Welch’s correction ( a-c , f-h ), two-tailed Mann-Whitney U test ( d-e , i-j ). Error bars: SD. P value: a , * p=0.0234; b , * p=0.0428; c , *** p=0.0004; d, * p=0.0210; e , ** p=0.0054; f , ns p=0.1544; g , ns p=0.5322; h , ns p=0.0662; i , ns p=0.0592; j , ns p=0.9064.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-j , Female ( a-e ) and male ( f-g ) Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and assessed for serum ALT ( a , f ), viral titers in liver ( b , g ) and spleen ( c , h ), and liver necrosis and inflammation ( d-e , i-j ). k-n , Female Ly6e fl/fl and Ly6e ΔHSC mice injected with PBS or MHV and whole livers were imaged. Mock-infected (PBS) Ly6e ΔHSC mice ( k ), mock-infected Ly6e fl/fl mice ( l ), MHV-infected Ly6e ΔHSC mice ( m ), MHV-infected Ly6e fl/fl mice ( n ). For a-e , n=8 ( Ly6e fl/fl ,), n=8 ( Ly6e ΔHSC ), or n=3 (PBS) from two pooled experiments. For f-j , n=9 ( Ly6e fl/fl ,), n=7 ( Ly6e ΔHSC ), or n=3 (PBS) two pooled experiments. For k-n , n=3. Statistical significance was determined by two-tailed unpaired student’s t-test with Welch’s correction ( a-c , f-h ), two-tailed Mann-Whitney U test ( d-e , i-j ). Error bars: SD. P value: a , * p=0.0234; b , * p=0.0428; c , *** p=0.0004; d, * p=0.0210; e , ** p=0.0054; f , ns p=0.1544; g , ns p=0.5322; h , ns p=0.0662; i , ns p=0.0592; j , ns p=0.9064.

Techniques: Injection, Infection, Two Tailed Test, MANN-WHITNEY

a-e , Transcriptomic analysis from Ly6e fl/fl (WT) and Ly6e ΔHSC (KO) mice. a-b , Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver ( a ) and spleen ( b ). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement. c , Pathway analysis of KO versus WT. Up-(red) or down-regulated (blue) pathways indicated by activation z-score. Numbers show significantly dysregulated genes as percentage of total gene number included in pathway. d-e , Expression of select genes in liver ( d ) and spleen ( e ). f-g , Immune cell counts from liver ( f ) and spleen ( g ). h , Infection of cultured splenocytes. For a-e , n=3. For f-g , n=8 MHV-injected, n=4 PBS-injected from two pooled experiments. For h , n=3 ( Ly6e fl/fl and Ly6e ΔHSC ) or n=2 ( Ifnar −/− ) from two pooled experiments. Significance for f-h was determined by two-tailed unpaired student’s t-test with Welch’s correction. Error bars: SD ( d-h ). P values (left-right): f , **** p=1.3644 × 10 −6 , ns p=0.7952, ns p=0.0781, ns p=0.7732, ** p=0.0016, ns p=0.6261, ** p=0.0068, ns p=0.9494, * p=0.0141, ns p=0.7639, * p=0.0436, ns p=0.9600, * p=0.0174, ns p=0.3096; g, ** p=0.0052, ns p=0.9447, ns p=0.1579, ns p=0.7769, ns p=0.4104, ns p=0.6924, *** p=1.754 × 10 −4 , ns p=0.9842, ** p=0.0034, ns p=0.9848, * p=0.0169, ns p=0.8591, ** p=0.0076, ns p=0.8623; h, ** p=0.0051, ns p=0.4525, ns p=0.7379, ns p=0.0702, * p=0.0119, ns p=0.6787, ns p=0.4112.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-e , Transcriptomic analysis from Ly6e fl/fl (WT) and Ly6e ΔHSC (KO) mice. a-b , Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver ( a ) and spleen ( b ). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement. c , Pathway analysis of KO versus WT. Up-(red) or down-regulated (blue) pathways indicated by activation z-score. Numbers show significantly dysregulated genes as percentage of total gene number included in pathway. d-e , Expression of select genes in liver ( d ) and spleen ( e ). f-g , Immune cell counts from liver ( f ) and spleen ( g ). h , Infection of cultured splenocytes. For a-e , n=3. For f-g , n=8 MHV-injected, n=4 PBS-injected from two pooled experiments. For h , n=3 ( Ly6e fl/fl and Ly6e ΔHSC ) or n=2 ( Ifnar −/− ) from two pooled experiments. Significance for f-h was determined by two-tailed unpaired student’s t-test with Welch’s correction. Error bars: SD ( d-h ). P values (left-right): f , **** p=1.3644 × 10 −6 , ns p=0.7952, ns p=0.0781, ns p=0.7732, ** p=0.0016, ns p=0.6261, ** p=0.0068, ns p=0.9494, * p=0.0141, ns p=0.7639, * p=0.0436, ns p=0.9600, * p=0.0174, ns p=0.3096; g, ** p=0.0052, ns p=0.9447, ns p=0.1579, ns p=0.7769, ns p=0.4104, ns p=0.6924, *** p=1.754 × 10 −4 , ns p=0.9842, ** p=0.0034, ns p=0.9848, * p=0.0169, ns p=0.8591, ** p=0.0076, ns p=0.8623; h, ** p=0.0051, ns p=0.4525, ns p=0.7379, ns p=0.0702, * p=0.0119, ns p=0.6787, ns p=0.4112.

Techniques: Activation Assay, Expressing, Infection, Cell Culture, Injection, Two Tailed Test

Depicted are fold changes in liver and spleen gene expression from Ly6e ΔHSC mice compared to Ly6e fl/fl mice. RPKM values from RNAseq data for liver ( a ) and spleen ( b ) were used to calculate fold changes. Crossed out genes were not detected in either KO or WT mice at respective conditions.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: Depicted are fold changes in liver and spleen gene expression from Ly6e ΔHSC mice compared to Ly6e fl/fl mice. RPKM values from RNAseq data for liver ( a ) and spleen ( b ) were used to calculate fold changes. Crossed out genes were not detected in either KO or WT mice at respective conditions.

Techniques: Expressing

a-b , Immune cell counts from liver ( a ) and spleen ( b ). c , Infection of cultured splenocytes. For a-b , n=8 (infected Ly6e fl/fl ), n=7 (infected Ly6e ΔHSC ), n=4 PBS-injected from two pooled experiments. For h , n=6 ( Ly6e fl/fl ), n=3 ( Ly6e ΔHSC ), or n=e ( Ifnar −/− ) mice from two pooled experiments. Statistical significance was determined by two-tailed unpaired student’s t-test with Welch’s correction ( a-c ). Error bars: SD. P values (left-right): a , ** p=0.0053, ns p=0.5297, ns p=0.0641, ns p=0.4066, * p=0.0429, ns p=0.8678, * p=0.0163, ns p=0.2261, * p=0.0148, ns p=0.9694, *** p=0.0001, ns p=0.7840, ** p=0.0011, ns p=0.3465; b , * p=0.0284, ns p=0.3291, ns p=0.4421, ns p=0.6188, ns p=0.9412, ns p=0.2802, *** p=0.0082, ns p=0.4336, * p=0.0105, ns p=0.6763, * p=0.0419, ns p=0.9712, ** p=0.0046, ns p=0.6857; c , * p=0.0105, ns p=0.7080, ns p=0.2470, ns p=0.0503, ns p=0.0568, ns p=0.4637, ns p=0.7915.

Journal: bioRxiv

Article Title: LY6E impairs coronavirus fusion and confers immune control of viral disease

doi: 10.1101/2020.03.05.979260

Figure Lengend Snippet: a-b , Immune cell counts from liver ( a ) and spleen ( b ). c , Infection of cultured splenocytes. For a-b , n=8 (infected Ly6e fl/fl ), n=7 (infected Ly6e ΔHSC ), n=4 PBS-injected from two pooled experiments. For h , n=6 ( Ly6e fl/fl ), n=3 ( Ly6e ΔHSC ), or n=e ( Ifnar −/− ) mice from two pooled experiments. Statistical significance was determined by two-tailed unpaired student’s t-test with Welch’s correction ( a-c ). Error bars: SD. P values (left-right): a , ** p=0.0053, ns p=0.5297, ns p=0.0641, ns p=0.4066, * p=0.0429, ns p=0.8678, * p=0.0163, ns p=0.2261, * p=0.0148, ns p=0.9694, *** p=0.0001, ns p=0.7840, ** p=0.0011, ns p=0.3465; b , * p=0.0284, ns p=0.3291, ns p=0.4421, ns p=0.6188, ns p=0.9412, ns p=0.2802, *** p=0.0082, ns p=0.4336, * p=0.0105, ns p=0.6763, * p=0.0419, ns p=0.9712, ** p=0.0046, ns p=0.6857; c , * p=0.0105, ns p=0.7080, ns p=0.2470, ns p=0.0503, ns p=0.0568, ns p=0.4637, ns p=0.7915.

Techniques: Infection, Cell Culture, Injection, Two Tailed Test

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