A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry
Abstract
:1. Introduction
2. Materials and Methods
2.1. CRISPR Activation Screen and Validation of Candidate Gene Upregulation
2.2. Cells, Plasmids, Viruses, and Infections
2.3. RhoV and WWTR1 Knockout (KO) by CRISPR–Cas9
2.4. Generation and Detection of RhoV or WWTR1 Inducible Cell Lines
2.5. Staining and Analysis of ZIKV-Infected Cells
2.6. Viral Entry Assay
2.7. Immunofluorescence
2.8. siRNA Knockdown of Rho GTPases and Their Effector Proteins
2.9. Quantitative Reverse Transcription PCR (RT-qPCR)
3. Results
3.1. CRISPR–Cas9 Activation Screen in ZIKV-Infected Cells
3.2. Both RhoV and WWTR1 Enhance ZIKV Infection in A549 Cells
3.3. RhoV Promotes ZIKV and DENV Infection in SNB-19 Cells
3.4. The GTPase Domain Plays an Important Role in RhoV Proviral Effects
3.5. siRNA Screen of Rho GTPases and Effector Proteins Reveals That Both RhoB and Pak1 Are Proviral Factors for ZIKV
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene Name | Forward (F) and Reverse (R) Primer Sequences |
---|---|
RhoV | F: 5′-CCTCATCGTCAGCTACACCTG-3′ R: 5′-GAACGAAGTCGGTCAAAATCCT-3′ |
RhoU | F: 5′-GCTACCCCACCGAGTACATC-3′ R: 5′-GGCTCACGACACTGAAGCA-3′ |
Cdc42 | F: 5′-CCATCGGAATATGTACCGACTG-3′ R: 5′-CTCAGCGGTCGTAATCTGTCA-3′ |
RhoQ | F: 5′-CCACCGTCTTCGACCACTAC-3′ R: 5′-AGGCTGGATTTACCACCGAGA-3′ |
RhoJ | F: 5′-AGGGGCAACGACGAGAAGA-3′ R: 5′-TTGGCGTAGCTCATCAGCAG-3′ |
Rac1 | F: 5′-ATGTCCGTGCAAAGTGGTATC-3′ R: 5′-CTCGGATCGCTTCGTCAAACA-3′ |
RhoA | F: 5′-GGAAAGCAGGTAGAGTTGGCT-3′ R: 5′-GGCTGTCGATGGAAAAACACAT-3′ |
RhoB | F: 5′-CTGCTGATCGTGTTCAGTAAGG-3′ R: 5′-TCAATGTCGGCCACATAGTTC-3′ |
RhoC | F: 5′-GGAGGTCTACGTCCCTACTGT-3′ R: 5′-CGCAGTCGATCATAGTCTTCC-3′ |
Pak1 | F: 5′-CAGCCCCTCCGATGAGAAATA-3′ R: 5′-CAAAACCGACATGAATTGTGTGT-3′ |
ZIKV | F: 5′-TTGTGGAAGGTATGTCAGGTG-3′ R: 5′-ATCTTACCTCCGCCATGTTG-3′ |
RPS11 | F: 5′-GCCGAGACTATCTGCACTAC-3′ R: 5′-ATGTCCAGCCTCAGAACTTC-3′ |
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Luu, A.P.; Yao, Z.; Ramachandran, S.; Azzopardi, S.A.; Miles, L.A.; Schneider, W.M.; Hoffmann, H.-H.; Bozzacco, L.; Garcia, G., Jr.; Gong, D.; et al. A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry. Viruses 2021, 13, 2113. https://doi.org/10.3390/v13112113
Luu AP, Yao Z, Ramachandran S, Azzopardi SA, Miles LA, Schneider WM, Hoffmann H-H, Bozzacco L, Garcia G Jr., Gong D, et al. A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry. Viruses. 2021; 13(11):2113. https://doi.org/10.3390/v13112113
Chicago/Turabian StyleLuu, Anh Phuong, Zhenlan Yao, Sangeetha Ramachandran, Stephanie A. Azzopardi, Linde A. Miles, William M. Schneider, H.-Heinrich Hoffmann, Leonia Bozzacco, Gustavo Garcia, Jr., Danyang Gong, and et al. 2021. "A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry" Viruses 13, no. 11: 2113. https://doi.org/10.3390/v13112113
APA StyleLuu, A. P., Yao, Z., Ramachandran, S., Azzopardi, S. A., Miles, L. A., Schneider, W. M., Hoffmann, H.-H., Bozzacco, L., Garcia, G., Jr., Gong, D., Damoiseaux, R., Tang, H., Morizono, K., Rudin, C. M., Sun, R., Arumugaswami, V., Poirier, J. T., MacDonald, M. R., Rice, C. M., & Li, M. M. H. (2021). A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry. Viruses, 13(11), 2113. https://doi.org/10.3390/v13112113