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We designed a screening strategy, illustrated in Supplementary Figure S1 to investigate host cellular genes essential for SFTSV infection. GeCKO lentivirus library was generated by transfecting HEK293FT cells with lentiCRISPR-v2 library together with lentiviral helper plasmids (pMD2.G and psPAX2). CRISPR–Cas9 gene knockout screening was performed in 96-well plates. HeLa cells were infected with the lentivirus library at a low MOI to ensure that the majority of cells received only one genetic perturbation and were diluted and calculated to nearly a single cell per well, and then seeded into 96-well plates after a puromycin selection, followed by SFTSV infection. Supernatants of each well were detected for viral infection. Finally, we selected cells from 8 wells with low viral nucleoprotein found in the supernatants, and their OD450 values ranged from 0.148 to 0.239, compared to the positive control (OD450 value = 2.155) (Table 1). Then, the integrated sgRNA fragments of the selected cells were PCR amplified. Amplified products were subjected to deep sequencing, and candidate genes were identified and ranked using model-based analysis of the genome-wide CRISPR/Cas9 knockout (MAGeCK) program. Results found that the majority of the cells from selected wells in which the cells were resistant to SFTSV infection presented multiple sgRNAs inserts, and only one well (Sample 1) showed a dominant gene for SNX11 with a percentage of 99.79% (Table 1). Because the relationship between SFTSV infection and the genes detected in these wells had not yet been clarified, we selected only SNX11, which was closely related to the endocytic pathway for further functional analysis in this study. In order to verify the SFTSV resistance of cells with the SNX11 knockout, we cloned SNX11 sgRNA into the backbone plasmid vector of lentiCRISPR v2 and then transfected the HeLa cells. A single SNX11 knockout cell line (SNX11-KO) was established by utilizing the CRISPR/cas9 system, and then the sequence was analyzed. As depicted in Fig. 1A, the sgRNA target site of the SNX11 gene was fully clipped and disrupted the complete gene. Knockout efficiency was confirmed with Western blotting (Fig. 1B) and immunofluorescence (Fig. 1C), and both showed a lack of SNX11 protein expression in SNX11-KO cells. We performed a cell viability assay in order to investigate whether the knockout of SNX11 affected cell viability and no significant differences in cell viability were found between WT and SNX11-KO cells (Supplementary Figure S2).
Sample Anti-NP ELISA (OD450 value) Total sgRNA reads Genes sgRNA reads Percentage 1 0.204 2, 705, 450 SNX11 2, 700, 017 99.79 2 0.148 862, 251 KRIT1 691, 567 80.20 LRRC31 129, 920 15.07 3 0.186 201, 987 SRRD 105, 551 52.26 DAND5 56, 433 27.94 4 0.152 767, 552 ZBTB6 448, 375 58.42 BOC 144, 551 18.83 5 0.239 1, 222, 401 PTPN11 497, 070 40.66 PRSS37 496, 690 40.63 6 0.201 561, 712 KRTAP5-3 234, 977 41.83 Hsa-mir-5589 215, 497 38.36 7 0.148 635, 352 AHR 421, 470 66.34 PLA2R1 126, 433 19.90 8 0.196 1, 129, 315 CLDN19 391, 174 34.64 DHPS 371, 174 32.87 MIXL1 243, 098 21.53 Table 1. Results of deep sequencing of sgRNAs in SFTSV negative cells.
Figure 1. SNX11 knockout efficiency. A Sequence analysis of the SNX11-KO cell line. SNX11-KO represents one SNX11 knockout cell line. The target and a protospacer adjacent motif (PAM) sequence are highlighted in black boxes. B Western blotting confirmed the efficiency of the SNX11 knockout. β-Actin was used as the loading control. C Immunofluorescence assay (IFA) indicated that there was no expression of SNX11 in SNX11-KO cells.
Further, we cloned the SNX11 gene into the expression vector pcDNA3.1, and transfected and expressed the SNX11 gene into the SNX11-KO cell line; then protein expression was confirmed with Western blotting (Fig. 2A). Cells of the wild-type, SNX11-KO, and SNX11-KOPlus were infected with SFTSV followed by detection of viral RNA in infected supernatants with real-time PCR at 96, 120, and 144 h post infection. These results showed that SFTSV RNA load significantly decreased in the SNX11-KO cell supernatants and was recovered in the SNX11-KOPlus cells (Fig. 2B). SFTSV NP protein expression also significantly decreased in the SNX11-KO cells and was recovered in the SNX11-KOPlus cells via ELISA detection (Fig. 2C). Flow cytometry was also used to compare the infection rates, and the results showed that in wild-type cells, the SFTSV infection rate was 0.01%, 3.37%, 24.43%, 36.21% at 0, 96, 120, 144 h post-infection respectively (Fig. 2D, left panel), which decreased to 0.03%, 0.28%, 0.88%, 5.55% in the SNX11-KO cells (Fig. 2D, middle panel). During re-expression of SNX11, the infection rates recovered to 0.01%, 2.51%, 13.02%, 32.28% in the SNX11-KOplus cells (Fig. 2D, right panel). Fluctuating infection rates caused by the knockout and re-expression of SNX11 indicated that SNX11 was required in order to establish this viral infection within the host.
Figure 2. Confirmation of the reduced and rescued SFTSV infection in the SNX11-KO cell line. A Western blotting for SNX11 expression in the wildtype (WT). SNX11-KO, and SNX11-KOplus cells with triplicates. Cell lysates were collected, and equal amounts of protein were loaded onto an SDS-PAGE gel. β-Actin was used as the loading control. B Real-time PCR analysis for SFTSV RNA titer in the supernatant of the infected cells at 96, 120, and 144 h postinfection. C ELISA for SFTSV NP in the supernatant of the infected cells was performed at 96, 120, and 144 h postinfection in the wild-type (WT), SNX11-KO, and SNX11-KOplus cells. D Flow-cytometric analysis of the SFTSV-positive rate in wild-type, SNX11-KO, and SNX11-KOplus cells at 96, 120, and 144 h post-infection. The dots in the gates represent SFTSV-positive cells.
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To investigate the precise position where SFTSV infection was blocked, we performed a confocal-microscopic analysis to investigate the colocalization between SFTSV and various organelle markers in the SNX11-KO and the wildtype cells. First, we infected two kinds of cells with SFTSV and fixed the cells at 48 h post infection. Cells were permeabilized and blocked. Next, we chose a glycoprotein (GP) of SFTSV required throughout the life cycle of this virus. Then, the sample was stained with mouse monoclonal antibody directed against SFTSV GP and rabbit antibodies against a series of organelle markers and stained with a secondary antibody. Our results found that the infection rate of the SNX11-KO cells was lower than that of the wild-type cells, which was consistent with the validation results above, and therefore, only the cells that were SFTSV GP positive were utilized for confocal microscopic analysis. Our results showed that in wild-type cells, SFTSV GP colocalized with RAB5a+ early endosome (Fig. 3A, upper panel), RAB7a+ late endosome (Fig. 3B, upper panel), LAMP1? endolysosome, or the lysosome (Fig. 3C, upper panel), suggesting that normally SFTSV would enter the early endosome, late endosome, endolysosome, or lysosome. Furthermore, in wild-type cells, SFTSV GP could colocalize with protein disulfide isomerase (PDI), an ER marker (Fig. 3D, upper panel), and receptor binding cancer antigen expressed in SiSo cells (RCAS1), a Golgi apparatus marker (Fig. 3E, upper panel), which suggested that SFTSV could penetrate the endolysosome into the cytoplasm. During RNA replication, synthesis and dimerization of Gn and Gc in the ER and glycosylation of the viral protein in the Golgi apparatus proceeded. However, in SNX11-KO cells, SFTSV GP could only colocalize with RAB5a (Fig. 3A, lower panel), RAB7a (Fig. 3B, lower panel), and LAMP1 (Fig. 3C, lower panel), but could not colocalize with PDI (Fig. 3D, lower panel) and RCAS1 (Fig. 3E, lower panel), which indicated that the knockout of SNX11 did not affect trafficking of SFTSV from the early endosome to the endolysosome but affected the penetration of SFTSV into the cytoplasm and affected the process of SFTSV GP synthesis as well as maturation in the ER. Since SNX11 was primarily an intracellular trafficking protein which mostly localized in the endosomal membrane, we speculated that the knockout of SNX11 could block the penetration of SFTSV into the cytoplasm.
Figure 3. The colocalization of SFTSV GP with series of organelle markers in the wildtype (WT) and SNX11-KO cells. A, B Colocalization of SFTSV GP with early endosome and late endosome, respectively. C Colocalization of SFTSV GP with endolysosome/lysosome. D. E Colocalization of SFTSV GP with ER and Golgi apparatus, respectively. Images were acquired with a 63 × objective.
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We were interested in how the knockout of SNX11 blocked the penetration of SFTSV. Thus, we added a fluorescent acidotropic BB cell Probe P02 (Bestbio, China), which was labeled with fluorescein and the fluorescence intensity increased with acidification of the endosomal compartments of the wild-type and SNX11-KO HeLa cells. Then, we analyzed them with dual-fluorescence flow cytometry. The ratio (R value) of fluorescein emissions when the cells were excited by light at wavelengths of 540 nm (F540) and 440 nm (F440) were inversely proportional to the pH value. This experiment used the R value to measure pH. Our results indicated that the F540/F440 ratio of wild-type cells was 5.79, while it was 4.57 in the SNX11-KO cells (Fig. 4A), which suggested that the pH inside the endosomal compartments of the SNX11-KO cells was higher than in those of the wild-type cells. To examine the specific pH values in both wild-type and SNX11-KO cells, a standard curve of the ratio of F540/F440 (Y axis) versus pH values (X axis), was made with a standard pH calibration buffer in a pH range of 2.8–5.8 (Lin et al. 2001), and the pH values were calculated from the linear relationship (correlation coefficient 0.9934) between F540/F440 and the pH value. Results showed that the pH of the endosomal compartments in the wild-type cells was 5.33, whereas it was 5.56 in SNX11-KO cells (Fig. 4B), suggesting that the knockout of SNX11 led to a pH increase of 0.23 in the endosomal compartments of the SNX11-KO cells. We used confocal microscopy to explore the interaction between SNX11 and V-ATPase, major proton pumps involved in proton homeostasis of the intracellular compartments. Our results showed that SNX11 colocalized with V-ATPase (Fig. 4C), which provided insight into the mechanism by which SNX11 affected acidification of the endosomal compartments.
Figure 4. Increased pH in the endosomal compartments of the SNX11-KO cells. A Flowcytometric analysis of the ratio (R-value) of fluorescein emission when excited by 540 and 440 nm light (F540/F440) in the wild-type (WT) and SNX11-KO cells. B The linear relationship between F540/F440 and pH range 2.8–5.8. "R" represents the coefficient of correlation. C SNX11 colocalized with V-ATPase.
To determine whether the knockout of SNX11 affected the formation of early endosome, late endosome, endolysosome, or lysosome, we performed RNA sequencing analysis to compare the mRNA transcription of RAB5a, RAB7a, and LAMP1 from the wild-type and SNX11-KO cells, which showed no significant differences in the levels of RAB5a or RAB7a in the cells (Fig. 5A, left and middle). LAMP1 mRNA was elevated by nearly 50% in the SNX11-KO cells (Fig. 5A, right). Further, we used flow cytometric analysis to compare the protein expression of RAB5a, RAB7a, and LAMP1 in the two kinds of cells, and we found that a significant increase in LAMP1 was also detected in SNX11-KO cells (Fig. 5B, right). A Western blotting analysis supported these findings (Fig. 5C). This increase in LAMP1 expression suggested that the knockout of SNX11 might be caused by changes in the late endosomal compartment, which was closely associated with the cytoplasmic penetration of bunyaviruses (Albornoz et al. 2016). Therefore, we performed confocal microscopy to investigate the colocalization of SNX11 with RAB5a, RAB7a, and LAMP1, and the results showed that SNX11 was mainly colocalized with RAB7a and LAMP1, while it seldom colocalized with RAB5a (Fig. 5D), which indicated that the late endosomal compartments could be where SNX11 functioned. Further, we used electron microscopy to examine changes in the vesicles by the SNX11-KO cells. The results showed that the endolysosomes had increased within the SNX11-KO cells (Fig. 6).
Figure 5. Increased LAMP1 in SNX11-KO cells. A RNA sequencing result for mRNA transcription of RAB5a (left), RAB7a (middle) and LAMP1 (right), in SNX11-KO and wildtype cells (WT), **P < 0.01. B Flow-cytometric histograms of the expression of RAB5a (left), RAB7a (middle), and LAMP1 (right). Proteins in SNX11-KO (red) and wild-type cells (blue). FITC-A represented the expression level of the specific protein. Secondary antibody control is also shown (black). C Western blotting results for the expression of RAB5a (left), RAB7a (middle), and LAMP1 (right) in two kinds of cells, β-actin was added as the loading control. D Colocalization of SNX11 with RAB5a, RAB7a, and LAMP1, respectively.