human lrp1 cluster iv  (R&D Systems)

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Lack of Lrp1 reduces JCV binding, internalization, and infection of murine cells. ( A, B ) Virus infectivity was determined by infecting WT and Lrp1 KO cells at an MOI of 0.1 with either ( A ) JCV or ( B ) ZIKV and quantifying viral RNA in the supernatant at 24 hpi (JCV) or 48 hpi (ZIKV). ( C, D ) JCV-N antigen (pink) counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. ( E, F ) Binding and internalization assays after surfen treatment using ( E ) JCV and ( F ) ZIKV as a control. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test ( A, E, F ) or Welch’s t -test ( B ). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Binding Assay, Infection, Virus, Inverted Microscopy, Control

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: CL IV of human LRP1 binds and neutralizes JCV. ( A–C ) FRNT assays were performed by mixing virus with increasing concentrations of LRP1 CL II -Fc, LRP1 CL IV -Fc, or an Fc control protein prior to infection of Vero E6 cells. Twenty-four hours later, foci were visualized by immunostaining, imaged using a Cytation 5, and quantified using ImageJ. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. ( D ) Biolayer interferometry sensograms displaying association and dissociation of JCV virions to immobilized LRP1 CL IV -Fc-His (blue), CL II -Fc-His (red), or recombinant human IgG1 Fc (gray). Data were baseline subtracted using sensors in buffer alone. BLI experiments were performed in triplicate. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Virus, Control, Infection, Immunostaining, Recombinant

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Replication kinetics of JCV in primary rat neurons. Primary rat neurons were infected with JCV at the indicated MOI. ( A ) Viral RNA or ( B ) infectious virus was quantified at 24, 36, 48, and 60 hpi time points. ( C ) Infected or mock-infected coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 20× using a Nikon A1 confocal microscope. Scale bar = 250 µm. ( D ) Immunofluorescent microscopy of neurons after 4 days of culture. Coverslips were stained for Lrp1 (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Infection, Virus, Staining, Microscopy, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Pre-treatment with a high-affinity Lrp1 binding protein reduces JCV infection. Primary rat neurons were pre-treated with different concentrations of mRAP D3 for 45 min, followed by infection with JCV ( A ) or ZIKV MR766 ( B ) at an MOI of 0.1. Supernatant was collected for quantification of virus at 24 hpi (JCV) or 48 hpi (ZIKV). ( C ) Coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Binding Assay, Infection, Virus, Staining, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Specificity of Lrp1 for JCV entry. ( A ) BV2 WT and LRP1 KO cells infected with either JCV, OROV, or RVFV. ( B ) BV2 WT cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. ( C ) BV2 Lrp1 KO cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Infection

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Lack of Lrp1 reduces JCV binding, internalization, and infection of murine cells. ( A, B ) Virus infectivity was determined by infecting WT and Lrp1 KO cells at an MOI of 0.1 with either ( A ) JCV or ( B ) ZIKV and quantifying viral RNA in the supernatant at 24 hpi (JCV) or 48 hpi (ZIKV). ( C, D ) JCV-N antigen (pink) counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. ( E, F ) Binding and internalization assays after surfen treatment using ( E ) JCV and ( F ) ZIKV as a control. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test ( A, E, F ) or Welch’s t -test ( B ). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in PBS (pH = 7.4) supplemented with 1 mg/mL BSA and 0.05% Tween-20 for 15 min. Recombinant human LRP1 CL IV -FcHis, recombinant human LRP1 CL II -FcHis, or recombinant human IgG1 Fc (R&D Systems, #110-HG-100) were loaded at 100 nM for 500 seconds prior to baseline equilibration for 300 seconds.

Techniques: Binding Assay, Infection, Virus, Inverted Microscopy, Control

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: CL IV of human LRP1 binds and neutralizes JCV. ( A–C ) FRNT assays were performed by mixing virus with increasing concentrations of LRP1 CL II -Fc, LRP1 CL IV -Fc, or an Fc control protein prior to infection of Vero E6 cells. Twenty-four hours later, foci were visualized by immunostaining, imaged using a Cytation 5, and quantified using ImageJ. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. ( D ) Biolayer interferometry sensograms displaying association and dissociation of JCV virions to immobilized LRP1 CL IV -Fc-His (blue), CL II -Fc-His (red), or recombinant human IgG1 Fc (gray). Data were baseline subtracted using sensors in buffer alone. BLI experiments were performed in triplicate. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, no significance.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in PBS (pH = 7.4) supplemented with 1 mg/mL BSA and 0.05% Tween-20 for 15 min. Recombinant human LRP1 CL IV -FcHis, recombinant human LRP1 CL II -FcHis, or recombinant human IgG1 Fc (R&D Systems, #110-HG-100) were loaded at 100 nM for 500 seconds prior to baseline equilibration for 300 seconds.

Techniques: Virus, Control, Infection, Immunostaining, Recombinant

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Replication kinetics of JCV in primary rat neurons. Primary rat neurons were infected with JCV at the indicated MOI. ( A ) Viral RNA or ( B ) infectious virus was quantified at 24, 36, 48, and 60 hpi time points. ( C ) Infected or mock-infected coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 20× using a Nikon A1 confocal microscope. Scale bar = 250 µm. ( D ) Immunofluorescent microscopy of neurons after 4 days of culture. Coverslips were stained for Lrp1 (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in PBS (pH = 7.4) supplemented with 1 mg/mL BSA and 0.05% Tween-20 for 15 min. Recombinant human LRP1 CL IV -FcHis, recombinant human LRP1 CL II -FcHis, or recombinant human IgG1 Fc (R&D Systems, #110-HG-100) were loaded at 100 nM for 500 seconds prior to baseline equilibration for 300 seconds.

Techniques: Infection, Virus, Staining, Microscopy, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Pre-treatment with a high-affinity Lrp1 binding protein reduces JCV infection. Primary rat neurons were pre-treated with different concentrations of mRAP D3 for 45 min, followed by infection with JCV ( A ) or ZIKV MR766 ( B ) at an MOI of 0.1. Supernatant was collected for quantification of virus at 24 hpi (JCV) or 48 hpi (ZIKV). ( C ) Coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ****, P < 0.0001; ns, no significance.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in PBS (pH = 7.4) supplemented with 1 mg/mL BSA and 0.05% Tween-20 for 15 min. Recombinant human LRP1 CL IV -FcHis, recombinant human LRP1 CL II -FcHis, or recombinant human IgG1 Fc (R&D Systems, #110-HG-100) were loaded at 100 nM for 500 seconds prior to baseline equilibration for 300 seconds.

Techniques: Binding Assay, Infection, Virus, Staining, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Specificity of Lrp1 for JCV entry. ( A ) BV2 WT and LRP1 KO cells infected with either JCV, OROV, or RVFV. ( B ) BV2 WT cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. ( C ) BV2 Lrp1 KO cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: Anti-Human IgG Fc Capture biosensors were hydrated in PBS (pH = 7.4) supplemented with 1 mg/mL BSA and 0.05% Tween-20 for 15 min. Recombinant human LRP1 CL IV -FcHis, recombinant human LRP1 CL II -FcHis, or recombinant human IgG1 Fc (R&D Systems, #110-HG-100) were loaded at 100 nM for 500 seconds prior to baseline equilibration for 300 seconds.

Techniques: Infection

Journal: Nature Communications

Article Title: Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses

doi: 10.1038/s41467-023-41171-3

Figure Lengend Snippet: A , B Using BLI, we loaded his-tagged Gn onto HIS1K biosensors and then associated human antibodies or RVFV-429 Fab. Following this, we then bound A LRP1 Cluster II or B LRP1 Cluster IV. The values shown are a percentage of maximal signal from LRP1 Cluster II or IV binding relative to the no-antibody control. A similarly prepared human mAb to another bunyavirus designated CCHF-245 was used as the negative control mAb. The values are indicated by the gray scale. Competition was defined as less than 33% residual binding (dark gray), partial competition was greater than 33% but less than 66% residual binding (medium gray), and non-competitive percentages were greater than 66% residual binding of receptor fragments relative to the no-antibody control (light gray). Values shown are representative of three technical replicates from two independent experiments. RVFV-401, RVFV-296, RVFV-379, RVFV-436, RVFV-426, RVFV-268, RVFV-405 bind Gn domain A, RVFV-226 binds Gn domain B, and RVFV-429 binds an epitope away from the other antibodies where K197 was identified as a critical residue. Critical residues to map these antibodies were previously described . Data are presented as mean values +/− SD and statistical test was done by using an ordinary one-way ANOVA test corrected for multiple comparisons. C Neutralization of RVFV strain MP-12 by IgG1 or Fab forms of RVFV-268 or RVFV-140. Serial dilutions of IgG1 or Fab molecules were mixed with 100 infectious units of MP-12 and allowed to incubate on Vero cells for 3 days. The Fab molecule starting dilution was 2/3 of that of IgG1 to normalize the molarity of binding sites. IC 50 values were calculated using a three-parameter nonlinear fit. Curves reflect data from duplicate wells in three independent experiments. Data are presented as mean values +/− SEM. D RVFV-268 F(ab′)2 retains potent activity like IgG1. Similarly, Fab and F(ab′)2 dilutions began at 2/3 of the IgG1 starting concentrations. IC 50 values were calculated using a three-parameter nonlinear fit. Curves reflect data from duplicate wells in three independent experiments. Data are presented as mean values +/− SD. Source data are provided as a Source data file.

Article Snippet: Following this, his-tagged Gn was loaded on the biosensor at 20 μg/mL for 1000 s. A brief 30 s baseline step was used, and following this, human mAbs or RVFV-429 Fab were allowed to associate to Gn at 40 μg/mL for 150 s. Without using another baseline step, LRP1 Cluster II (Recombinant human LRP1 cluster II Fc Chimera Protein, CF (2368-L2-050)) or LRP1 Cluster IV (Recombinant human LRP1 cluster IV Fc Chimera Protein, CF (5395-L4-050), R&D Biosystems) was allowed to associate to the Gn-antibody complex for 1500 s at 40 μg/mL.

Techniques: Binding Assay, Control, Negative Control, Residue, Neutralization, Activity Assay