clii fc  (New England Biolabs)

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 1. A pooled genome-scale CRISPR screen identifies Lrp1 and Lrp1-associated proteins RAP and Grp94 as critical proteins for RVFV infection (A) Schematic of the CRISPR/Cas9 screen in BV2 cells. (B) Light microscope images (43) of BV2 cells before infection and at 3 different time points post-infection. (C) At 18 dpi, surviving cells were reseeded into new flasks for reinfection on 19 dpi and imaged at 43 by light microscopy. (D) Volcano plot analysis of the BV2 screen results of surviving cells from the initial infection at an MOI 0.1. (E) Summary of key interactions that modulate Lrp1 surface presentation, including RAP and Grp94. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: CRISPR, Infection, Light Microscopy

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 2. Lrp1 is essential for RVFV infection of BV2 cells (A) Western blot of BV2 Lrp1 knockout clones (Lrp1KO C3, Lrp1KO R1, Lrp1KO R2, Lrp1KO R4, Lrp1KO R5, and Lrp1KO R6), and partial knockout (Lrp1PKO R3) generated using either single gRNA or dual gRNA CRISPR/Cas9 approaches, as described in STAR Methods. (B) BV2 wild-type (WT) and Lrp1KO clones were infected with RVFV ZH501 at a MOI of 0.1. After 18 h, the cells were harvested for RNA extraction and subjected to RT-qPCR analysis. Data shown are viral RNA (vRNA) titers normalized to wild-type BV2 cells. (C) Microscopic images showing the WT and LRP1KO R4 cells infected with RVFV MP12GFP (MOI 5 for 6 h) in fluorescence images (top panels) and DAPI-stained images (bottom panels). Images were taken at 203 magnification (for quantification, refer to Figure S2H). (D) Flow cytometry of WT, Lrp1PKO R3, and Lrp1KO R4 cells infected with RVFV MP12GFP. (E) Corresponding analysis of flow cytometry histograms in (D). (F) Western blot of mouse embryonic fibroblasts (MEFs) from Lrp+/+ and Lrp1F/F mice infected with AdCre. (G) Representative flow cytometry of MEFs Lrp1+/+ and LrpF/F cells infected for 5 days with AdCre and then infected with RVFV-MP12GFP at MOI of 1 for 15 h. (H) Corresponding analysis of flow cytometry histogram data in (G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: Infection, Western Blot, Knock-Out, Clone Assay, Generated, CRISPR, RNA Extraction, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 4. LRP1 binds RVFV glycoprotein Gn (A) LRP1 is comprised of four clusters, CLI, CLII, CLIII, and CLIV, and the cytoplasmic and transmembrane domains (left). Mini-domains of CLI, CLII, CLIII, and CLIV were generated as -Fc fusions (top right). Lentiviruses carrying either pLVX-empty vector or pLVX-expressing HA-tagged minidomains mini-LRP1 CLI, CLII, CLIII, and CLIV were also generated (bottom right).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: Generated, Plasmid Preparation, Expressing

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 1. A pooled genome-scale CRISPR screen identifies Lrp1 and Lrp1-associated proteins RAP and Grp94 as critical proteins for RVFV infection (A) Schematic of the CRISPR/Cas9 screen in BV2 cells. (B) Light microscope images (43) of BV2 cells before infection and at 3 different time points post-infection. (C) At 18 dpi, surviving cells were reseeded into new flasks for reinfection on 19 dpi and imaged at 43 by light microscopy. (D) Volcano plot analysis of the BV2 screen results of surviving cells from the initial infection at an MOI 0.1. (E) Summary of key interactions that modulate Lrp1 surface presentation, including RAP and Grp94. See also Figure S1.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in kinetics buffer (Phosphate Buffer Saline (PBS) containing 0.02% Tween-20, 1 mg/mL BSA) for 15 min. Recombinant human LRP1 CLIV-Fc Chimera (R&D SYSTEMS, #5395-L4-050), recombinant human LRP1 CLII-Fc chimera (R&D SYSTEMS, #2368-L2-050), or recombinant human IgG1 Fc (R&D SYSTEMS, #110-HG-100) were loaded at 200 nM in buffer for 600 s prior to baseline equilibration for 300 s. Association of RVFVGn ormRAPD3 at various concentrations (0.5, 1, 2, 4, 8, and 12 mg/mL) was carried out for 900 s prior to dissociation for 900 s. Data were baseline subtracted to the buffer only controls.

Techniques: CRISPR, Infection, Light Microscopy

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 2. Lrp1 is essential for RVFV infection of BV2 cells (A) Western blot of BV2 Lrp1 knockout clones (Lrp1KO C3, Lrp1KO R1, Lrp1KO R2, Lrp1KO R4, Lrp1KO R5, and Lrp1KO R6), and partial knockout (Lrp1PKO R3) generated using either single gRNA or dual gRNA CRISPR/Cas9 approaches, as described in STAR Methods. (B) BV2 wild-type (WT) and Lrp1KO clones were infected with RVFV ZH501 at a MOI of 0.1. After 18 h, the cells were harvested for RNA extraction and subjected to RT-qPCR analysis. Data shown are viral RNA (vRNA) titers normalized to wild-type BV2 cells. (C) Microscopic images showing the WT and LRP1KO R4 cells infected with RVFV MP12GFP (MOI 5 for 6 h) in fluorescence images (top panels) and DAPI-stained images (bottom panels). Images were taken at 203 magnification (for quantification, refer to Figure S2H). (D) Flow cytometry of WT, Lrp1PKO R3, and Lrp1KO R4 cells infected with RVFV MP12GFP. (E) Corresponding analysis of flow cytometry histograms in (D). (F) Western blot of mouse embryonic fibroblasts (MEFs) from Lrp+/+ and Lrp1F/F mice infected with AdCre. (G) Representative flow cytometry of MEFs Lrp1+/+ and LrpF/F cells infected for 5 days with AdCre and then infected with RVFV-MP12GFP at MOI of 1 for 15 h. (H) Corresponding analysis of flow cytometry histogram data in (G).

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in kinetics buffer (Phosphate Buffer Saline (PBS) containing 0.02% Tween-20, 1 mg/mL BSA) for 15 min. Recombinant human LRP1 CLIV-Fc Chimera (R&D SYSTEMS, #5395-L4-050), recombinant human LRP1 CLII-Fc chimera (R&D SYSTEMS, #2368-L2-050), or recombinant human IgG1 Fc (R&D SYSTEMS, #110-HG-100) were loaded at 200 nM in buffer for 600 s prior to baseline equilibration for 300 s. Association of RVFVGn ormRAPD3 at various concentrations (0.5, 1, 2, 4, 8, and 12 mg/mL) was carried out for 900 s prior to dissociation for 900 s. Data were baseline subtracted to the buffer only controls.

Techniques: Infection, Western Blot, Knock-Out, Clone Assay, Generated, CRISPR, RNA Extraction, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 4. LRP1 binds RVFV glycoprotein Gn (A) LRP1 is comprised of four clusters, CLI, CLII, CLIII, and CLIV, and the cytoplasmic and transmembrane domains (left). Mini-domains of CLI, CLII, CLIII, and CLIV were generated as -Fc fusions (top right). Lentiviruses carrying either pLVX-empty vector or pLVX-expressing HA-tagged minidomains mini-LRP1 CLI, CLII, CLIII, and CLIV were also generated (bottom right).

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in kinetics buffer (Phosphate Buffer Saline (PBS) containing 0.02% Tween-20, 1 mg/mL BSA) for 15 min. Recombinant human LRP1 CLIV-Fc Chimera (R&D SYSTEMS, #5395-L4-050), recombinant human LRP1 CLII-Fc chimera (R&D SYSTEMS, #2368-L2-050), or recombinant human IgG1 Fc (R&D SYSTEMS, #110-HG-100) were loaded at 200 nM in buffer for 600 s prior to baseline equilibration for 300 s. Association of RVFVGn ormRAPD3 at various concentrations (0.5, 1, 2, 4, 8, and 12 mg/mL) was carried out for 900 s prior to dissociation for 900 s. Data were baseline subtracted to the buffer only controls.

Techniques: Generated, Plasmid Preparation, Expressing