plpro protein  (Viva Biotech)

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, PLpro proteolytic activity from the supernatant of SARS-CoV-2 or mock infected Calu-3 cells 24 and 48 HPI, measured by the fluorescence of cleaved substrate, normalized across samples from the same day. One-way ANOVA: p=0.014, F(3,8)=6.790; Holm-Šídák’s multiple comparisons, p mock vs. infected 24h =0.244, p mock vs. infected 48h =0.030, n =3 wells per group. b, SARS-CoV-2 transcripts from Calu-3 cells infected with SARS-CoV-2 (multiplicity of infection = 1) for 24 and 48 hours post infection (HPI), transcripts per million (TPM), n =3 wells. c , Interpolated concentration of PLpro in the supernatant of Calu-3 cells 48 hours post infection, line represents mean.

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Activity Assay, Infection, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, Schematic of in vivo imaging preparation. b, Image of trigeminal ganglion overlaid with the regions of interest of neurons that respond to intranasal perfusion (10 µL) of vehicle (Veh), 10 µM PLpro, and 100 µM capsaicin (Cap). c, Representative calcium transients in response to intranasal perfusion of Veh, 10 µM PLpro, 1 mM AITC, 100 µM Cap. d, Venn diagram of neurons responsive to vehicle (112/307) and PLpro (116/307), n =307 neurons from 6 mice. PLpro activates a subset of vehicle-sensitive neurons (32/112). e, Percent of recorded neurons that respond to each stimulus. f, Vehicle and PLpro activate a subset of TRPV1+ (capsaicin-responsive) neurons and TRPA1+ (AITC-responsive) neurons. Top Left, percent of TRPV1+ neurons responsive to vehicle only: 13.8%, both vehicle and PLpro: 9.7%, PLpro only: 15.9%, n =145 neurons from 6 mice; Top Right, percent of TRPV1+ neurons responsive to the first delivery of vehicle only: 17.8%, both first and second (Veh 2 ) deliveries of vehicle: 2.7%, second delivery of vehicle only: 2.7%, n =73 neurons from 3 mice, Chi-square test χ 2 = 112.5, df = 3, p<0.0001. Bottom Left, percent of TRPA1+ neurons responsive to vehicle only: 18.4%, both vehicle and PLpro: 17.2%, PLpro only: 18.4%, n =87 neurons from 4 mice; Bottom Right, percent of TRPA1+ neurons responsive to the first delivery of vehicle only: 25.0%, both deliveries of vehicle: 27.3%, second delivery of vehicle only: 9.1%, n =44 neurons from 3 mice, Chi-square test χ 2 = 14.2, df = 3, p = 0.003.

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: In Vivo Imaging

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, Sequential images of a sneeze following intranasal treatment of 10 µM PLpro (10 µL). b, Raster plot of sneezes from individual mice treated with 10 µM PLpro or vehicle identified from audio recordings. c, Average cumulative sneeze count of 10 µM PLpro and vehicle treated mice over 2 minutes d, Sequential images of a nose rub following intranasal treatment e, 10 µM PLpro treated mice display a shorter latency to the first nose rub than vehicle treated mice, t-test: t=2.469, df=29, p=0.020. f, Fraction of mice that elicited a rub and sneeze within the first 30 seconds after treatment. (Vehicle: 9.0%, PLpro (100 nM): 41.7%, PLpro (10 µM): 55.0%). Fisher’s exact test (p vehicle vs. 100nM PLpro =0.252, p vehicle vs. 10µM PLpro =0.020). For all data in this figure, vehicle ( n =11), 100 nM PLpro ( n =12), and 10 µM PLpro ( n =20). Error bars and shading represent the mean ± standard error of the mean (SEM), n = biological replicates (animals).

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques:

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a-c, Sneeze events are identified by audio waveforms and correlate to dye on the bottom of the chamber. a, Representative audio waveform during a sneeze b, Left, images during the same sneeze, arrows indicate dye from the sneeze on the bottom of the chamber. Right, image of dye expulsion from the sneeze event on the bottom of the chamber. c, The number of sneezes over 2 minutes are significantly correlated to the amount of dye at the bottom of the chamber. d, Duration of individual nose rub and face wipe events. e, Image sequence of a nose rub and face wipe. f, Raster plot of rubs, wipes, and body-licking events. g, Average cumulative rub and wipe counts from individual mice treated with 10 µM PLpro ( n =20) or vehicle ( n =11). i-l, Mean and standard error of the sneeze and rub latency and for individual mice treated with j, vehicle ( n =11), k , PLpro (100 nM, n =12), and l , PLpro (10 µM, n =20), n = biological replicates (animals).

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Sequencing

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, PLpro elicited a shorter latency to the first wipe (One-way ANOVA: p=0.003, F(2,18)=9.983; Tukey’s multiple comparisons, p Veh vs. 10µM PLpro =0.049, p Veh vs. 50µM PLpro =0.003, p 10µM PLpro vs. 50µM PLpro =0.789) and b , a dose-dependent increase in the number of wipes in the first minute. (Kruskal-Wallis: p=0.0116, χ 2 =8.920, Dunn’s multiple comparisons, p Veh vs. 10µM PLpro =0.134, p Veh vs. 50µM PLpro =0.021, p 10µM PLpro vs. 50µM PLpro > 0.999), vehicle ( n =17), PLpro (10 µM, n =5), PLpro (50 µM, n =10). c-d, PLpro does not elicit a shorter latency to scratching or any more scratching than vehicle injection, vehicle ( n =11), PLpro (50 µM, n =10). Error bars represent the mean ± SEM. n = biological replicates (animals).

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Injection

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, Representative calcium transients from PLpro responders from cultured neonatal mouse trigeminal ganglia (TG). b, PLpro activates subsets of neurons from adult mouse dorsal root ganglia (DRG; Veh: 2.9%, 1 nM: 4.2%, 10 nM: 6.8%, 100 nM: 11.9%, n =5) and adult mouse nodose and jugular ganglia (NJG; Veh: 5.5%, 1 nM: 21.7%, 10 nM: 23.6%, 100 nM: 33.9%, n =8). c, Venn diagram of PLpro, AITC, and Cap-responsive neurons in DRG and NJG. Of the neurons that respond to these 3 stimuli, Left, in the DRG, 20% responded to PLpro, an additional 48% responded to AITC, and 32% responded to Cap alone. Right, in the NJG, 38% responded to PLpro, an additional 34% responded to AITC, and 28% responded to Cap alone. d, Representative calcium transients evoked by PLpro and capsaicin (Cap) in human iPSC-derived neurons. e, PLpro induces significantly more calcium influx (area under the curve, AUC) than vehicle, t-test: t=2.953, df=6, p=0.026, n =4 wells each. f, Percent of neonatal mouse TG neurons that respond to 250nM PLpro in control neurons from wild-type C57BL/6N mice in physiological extracellular calcium (2 mM Ca 2+ , 5.5%, n =17) is greater than in the absence of extracellular calcium (EGTA, 1.2%, n =3) and in neurons from Trpv1 KO (2.4%, n =27) and Trpa1 KO (1.9%, n =12) mice, and in wild-type mice in the presence of TRPA1 antagonist (HC, 1.7%, n =4). One-way ANOVA: p=0.003, F(4,58)=2.803; Holm-Šídák’s multiple comparisons, p Control vs. EGTA =0.035, p Control vs. Trpv1 KO =0.004, p Control vs. Trpa1 KO =0.004, p Control vs. EGTA =0.035. g-i, SARS-CoV-2 and SARS, but not MERS PLpro activate subsets of neonatal mouse TG neurons in a dose-dependent manner. g , SARS-CoV-2 PLpro: 1nM: 3.8%, 10 nM: 7.2%, 100 nM: 9.3%, n =5. h, SARS PLpro: 1 nM: 1.4%, 10 nM: 3.4%, 100nM: 6.1%, n =5. i, MERS PLpro: 1nM: 1.8%, 10nM: 2.5%, 100nM: 2.7%, n =6. *p <0.05, **p<0.01. Error bars represent the mean ± SEM, n indicates replicates (wells).

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Cell Culture, Derivative Assay, Control

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: PLpro responders are of smaller diameter compared to vehicle responders and the population overall in a. neonatal mouse trigeminal ganglia (Veh: 22.6 µm, PLpro: 15.0 µm, AITC: 15.7 µm, Cap: 16.3 µm, High K+: 16.3 µm) b, adult DRG (Veh: 22.6 µm, PLpro: 16.2 µm, AITC: 22.1 µm, Cap: 19.8 µm, High K+: 21.0 µm) and c, adult NJG neurons (Veh: 22.5 µm, PLpro: 18.2 µm, AITC: 21.4 µm, Cap: 20.9 µm, High K+: 21.5 µm). d, PLpro from independent suppliers R&D Systems (#E-611, 5.5%, n =17) and Cayman (#31817, 5.7%, n =12) display no difference in the percent of responsive neurons in cultured TG, t-test: t=0.13, df=27, p=0.898. Error bars represent the mean ± SEM.

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, SARS-CoV-2 viral transcript expression 48 hours after infection in cultured adult trigeminal ganglia, mock infected: n= 4 wells, SARS-CoV-2 infected: n= 3 wells. b, Infection alters gene sets associated with nociception but not mechanoreceptors and proprioceptors. The median absolute log 2 fold change from each custom gene set was tested against 100,000 permutations of randomly selected gene sets of the same size, size of each gene set listed in parenthesis. c, Heatmap of the most highly differentially expressed nociception-associated transcripts (log 2 fold change of infection vs mock). d, PLpro elicits CGRP release from cultured TG. t-test: t=3.928, df=6, p=0.008, n = 4 per group, biological replicates (animal) e, PLpro does not elicit the release of Substance P from cultured TG, One-way ANOVA: p=0.673, F(2,21)=0.215, vehicle ( n =10), 100 nM PLpro ( n =10), and 1 µM PLpro ( n =4) replicates (wells from pooled animals) f, PLpro but not vehicle increases CGRP release from baseline measurements in an ex vivo trachea preparation. Baseline vs. vehicle: one sample t-test: t=0.909, df=5, p=0.405, baseline vs. 1µM PLpro: one sample t-test: t=6.80, df=5, p=0.001, n = 6, biological replicates (animal) per group. *p <0.05, **p<0.01, ***p<0.001. Bar represents mean and error bars represent the mean ± SEM.

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Expressing, Infection, Cell Culture, Ex Vivo

Journal: bioRxiv

Article Title: SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain

doi: 10.1101/2024.01.10.575114

Figure Lengend Snippet: a, 50 µM PLpro induces a reduction in the paw withdrawal threshold (PWT) for force 3, but not 24 hours post injection in PLpro injected paws, but not vehicle injected paws compared to baseline. (24 hours pre-injection). Two-way ANOVA: p time = 0.008, F(1.793, 64.56) = 5.477; Holm-Šídák’s multiple comparisons, Veh: p-adjusted Baseline vs. 3 hours =0.361, p-adjusted Baseline vs. 24 hours =0.079; PLpro: p-adjusted Baseline vs. 3 hours =0.008, p-adjusted Baseline vs. 24 hours =0.117, n = 19. b , Wild-type C57BL/6J (WT) and Trpv1 KO mice, but not Trpa1 KO mice develop mechanical hypersensitivity 3 hours post injection. WT: one sample t-test: t=2.746, df=19, p=0.013, n = 11; Trpv1 KO: one sample t-test: t=3.001, df=20, p=0.011, n = 13; Trpa1 KO: one sample t-test: t=0.039, df=10, p=0.970, n = 11. c, 50 µM PLpro injection does not change the latency to respond to the radiant heat stimulation, paired t-test: t=0.233, df=7, p=0.822, n = 8. d, Intranasal treatment of 10 µM PLpro triggers more sneezing in Top Left, WT and Top Right, Trpv1 KO mice than Bottom Right, Trpa1 KO mice or Bottom Left, WT mice treated with vehicle. e, Proposed model: PLpro released from infected cells in the airway epithelium and in the sensory ganglia activate Trpa1-expressing neurons to drive sneeze, pain, and the release of CGRP into the airways. Error bars and shading represent the mean ± standard error of the mean (SEM), n = biological replicates (animals).

Article Snippet: Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration.

Techniques: Injection, Infection, Expressing