Fig 1.
Knockdown of JAK/STAT pathway inhibits LGTV infection.
(A, B) Quantification of Dome and STAT gene levels in LGTV-infected nymphs. (C) Schematic of experimental design for dsRNA treatment and LGTV infection via injection. (D, E) Quantification of Dome knocking down efficiency (D) and preM levels (E) by qPCR in dsDome and dsGFP nymphs. (F) Schematic of experimental design for dsRNA treatment and LGTV infection via blood feeding. (G, H) Quantification of preM gene levels in mice (G) and dsRNA-treated nymphs (H). (I, J) Quantification of STAT knocking down efficiency (I) and preM levels (J) by qPCR in dsSTAT and dsGFP nymphs. Each dot represents 2 pooled nymphs in (A), (B), (D), (E), (H), (I), (J), and individual mouse blood sample in (G). Data are presented as mean ± SEM in (D) (n = 11) and (I) (n = 11–12). Horizontal lines represent the mean in (A) (n = 8), (B) (n = 8), (E) (n = 10–12), (G) (n = 6), (H) (n = 7–8), and (J) (n = 10–11). Significance was determined by Student t test in (A), (B), (D), (E), (G), (H), (I), and (J). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data underlying this figure are provided in S1 Raw Data.
Fig 2.
(A)The volcano plot of differentially expressed genes in dsGFP and dsSTAT ticks. Red circles represent 506 significantly up-regulated genes, and green circles represent 333 significantly down-regulated genes (Padj < 0.05). All genes showing significant changes were listed in the S1 Table. (B, C) Quantification of LRP knocking down efficiency (B) and preM levels (C) by qPCR in dsLRP and dsGFP nymphs. (D, E) Quantification of preM gene levels in mice and dsRNA-treated nymphs. (F) The 2-day feeding weights of nymphs treated with dsGFP and dsLRP. (G) Quantification of LRP gene levels in LGTV-infected nymphs. (H) Verification of LRP expression in BHK-21 cells by western blot analysis. The pCAGGS vector served as control. (I, J) Influence of LRP overexpression on LGTV infection in BHK-21 cells. The levels of preM and E protein were accessed by qPCR (I) and immunohistochemistry (J), respectively. Cells were stained with anti-EDⅢ antibody (green). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Each dot represents 2 pooled nymphs in (B), (C), (E), (G), 10 nymphs in (F), individual mouse blood sample in (D), and individual cell well in (I). Data are presented as mean ± SEM in (B) (n = 7–12) and (I) (n = 6). Horizontal lines represent the mean in (C) (n = 10–11), (D) (n = 4–6), (E) (n = 8), (F) (n = 6), and (G) (n = 6). Significance was determined by Student t test in (B), (C), (D), (E), (F), (G), and (I). * p < 0.05, ** p < 0.01. The data underlying this figure are provided in S1 Raw Data. The uncropped blots are included in S1 Raw Images.
Fig 3.
LRP promotes LGTV replication.
(A) Quantification of NS5 levels in LRP-expressed BHK-21 cells. Cells transfected with pCAGGS vector were served as control. (B) Co-immunoprecipitation of LRP-Flag and NS5-HA. The cell lysates were harvested and immunoprecipitated with antibodies against Flag (left) and HA (right). Samples were subjected to western blot analysis using antibodies against the Flag-tag, HA-tag, or ACTIN. (C) Co-localization (left) of LRP (green) with NS5 (red) in BHK-21 cells. Nuclei were stained by DAPI (blue). Scale bar: 25 μm. Quantification (right) was performed using at least 10 cells across three independent experiments. Pearson’s R = 0.90; Manders’ M1 = 0.92; M2 = 1.00; Costes’ P = 1.00. ****p < 0.001 by unpaired Student t test. (D) The effect of LRP overexpression on NS5 in BHK-21 cells. The cell lysates were harvested for western blot analysis using antibodies against the Flag-tag, HA-tag, or ACTIN. (E) Co-immunoprecipitation of LRP and TBEV-NS5. The cell lysates were harvested and immunoprecipitated with antibodies against Flag. Samples were subjected to western blot analysis using antibodies against the Flag-tag, HA-tag, or ACTIN. Each dot represents individual cell well in (A). Data are presented as mean ± SEM in (A) (n = 11–12). Significance was determined by Student t test in (A). * p < 0.05. The data underlying this figure are provided in S1 Raw Data. The uncropped blots are included in S1 Raw Images.
Fig 4.
LRP-mediated LD catabolism facilitates LGTV replication.
(A–C) Immunofluorescence staining of LDs in midguts of dsRNA-treated ticks fed on uninfected mice for two days (A), ticks fed on LGTV-infected mice for two days (B), and dsRNA-treated ticks fed on LGTV-infected mice for two days (C). The dsGFP-injected nymphs (A) and the nymphs bitten DMEM-injected A6 mice (B) were served as controls. The midguts were stained with Bodipy 493/503 (green). Nuclei were stained with DAPI (blue). Quantification of LDs fluorescence intensities was analyzed using ImageJ (right). (D) Lipid profiles following LRP knockdown. LRP deficiency significantly altered the midgut lipidome (right), specifically leading to a shift in sphingolipid and glycerophospholipid levels (left). (E, F) The total ATP levels during LRP knockdown (E) and LGTV infection (F) in F2D nymphs. All lipids showing significant changes are listed in S2 Table. (A) Scale bars, 500 μm. (B, C) Scale bars, 100 μm. Each dot represents individual image in (A–C), 10 midguts of nymphs in (E) and (F). Data are presented as mean ± SEM in (A) (n = 7–8), (B) (n = 11–12), (C) (n = 8–14), (D) (n = 5), (E) (n = 6), and (F) (n = 6). Significance was determined by Student t test in (A), (B), (D), (E), (F), and by one-way ANOVA in (C). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data underlying this figure are provided in S1 Raw Data.
Fig 5.
Lipophagy is essential for LGTV replication.
(A, C, D, E) Western blot (left) and quantification analysis (right) of LC3B in dsGFP/dsLRP-treated nymphs (A), LGTV-infected nymphs for feeding for 2 days (C) and fully engorged (D), and rapamycin-treated ticks (E). ACTIN was used as internal control. (B) Whole-mount staining of LC3B (green) and lysosome (red) in midgut of dsRNA-treated ticks. Nuclei were stained with DAPI (blue). Scale bars, 100 μm. (F, H) Whole-mount staining of LDs in midgut of rapamycin-treated (F) or dsLC3B-treated (H) ticks via Bodipy 493/503 (green). Nuclei were stained with DAPI (blue). Quantification of LDs fluorescence intensity was analyzed using ImageJ (right). Scale bars, 50 μm. (G, I) Quantification of LC3 knocking down efficiency (G) and preM levels (I) by qPCR in dsLC3- and dsGFP-treated nymphs. Each dot represents 2 pooled nymphs in (G), (I), an individual image in (F), (H), and an independent replicates in (A), (C), (D), (E). Data are presented as mean ± SEM in (G) (n = 11), (H) (n = 7–13). Horizontal lines represent the mean in (I) (n = 11–12). Significance was determined by Student t test in (F–I), and Mann–Whitney test in (A, C–E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data underlying this figure are provided in S1 Data. The uncropped blots are included in S1 Raw Images.