Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.1.1. Generation of A549 CRISPR/Cas9 Knockout Cell Lines
2.1.2. Validation of CRISPR/Cas9 Knockouts
Validation by Immunoblotting
Validation of Functional Knockout (Quantitative RT-PCR)
Validation by Sequencing of Genomic Loci
2.1.3. Generation of A549 Overexpression Cell Lines
Validation of IRF1, IRF5, and IRF7 Overexpression by Immunoblotting
Validation of IRF1, IRF5, and IRF7 Overexpression by Quantitative RT-PCR
2.2. Virus Stock Production and Titration
2.2.1. Influenza A SC35M Virus Stock Production
2.2.2. Rift Valley Fever Virus Stock Production
2.2.3. Adenovirus Stock Production
2.2.4. Herpes Simplex Virus 1 Stock Production
2.2.5. Virus Stock Titration by Plaque Assay
2.2.6. Virus Stock Titration by TCID50 Luciferase Assay
2.3. Virus Infections
2.3.1. Rift Valley Fever Virus Infection
2.3.2. Influenza A SC35M Virus Infection
2.3.3. Adenovirus Infection
2.3.4. Herpes Simplex Virus 1 Infection
2.4. IFN Stimulation
2.5. dsRNA Stimulation
2.6. Read-Out Methods
2.6.1. Read-Out by Luciferase Assay
2.6.2. Read-Out by Immunoblot
2.6.3. Read-Out by Quantitative RT-PCR
2.6.4. Read-Out by MSD Electroluminescent Multiplex Assay
2.7. Statistics
3. Results
3.1. Generation of Knockout Cell Lines and Regulation of IRF Expression
3.2. Impact of IRFs on the Induction of Antiviral Response Gene Expression
3.3. Impact of IRFs on the Expression Pattern of Cytokines and Chemokines
3.4. Replication of RNA and DNA Viruses under IRF KO Conditions
3.5. Impact of Increased Expression of IRF1, IRF5 and IRF7
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Forward Sequence (5′-3′) |
---|---|
NT | gtattactgatattggt |
IFNAR1 | gaccctagtgctcgtcgccg |
IFNGR1 | cctggtgctcacccagacgg |
IFNLR | caccggagtaccagatcatgccac |
IRF1 | tggtgagaggtggaagcatc |
IRF2 | gataaactccaacacgatcc |
IRF3 | cggaaattcctcttccaggt |
IRF5 | gccagggcttcagccgcacg |
IRF7 | atcagcagcggctgctatga |
IRF9 | gagggagtcctggagcacag |
RELA | gctcaatgatctccacatag |
RIG-I | tggagctccaggaggaaggc |
Name | Identifier |
---|---|
mouse monoclonal anti-β-Actin | A5441, RRID: AB_476744 |
rabbit polyclonal anti-Calnexin | ADI-SPA-865-F, RRID: AB_11180747 |
rabbit monoclonal anti-IRF1 (D5E4) XP | 8478, RRID: AB_10949108 |
rabbit monoclonal anti-IRF2 [EPR4644(2)] | ab124744, RRID: AB_10974405 |
rabbit monoclonal anti-IRF3 (D6I4C) XP | 11904, RRID: AB_2722521 |
rabbit polyclonal anti-IRF5 | HPA046700, RRID: AB_2679761 |
rabbit polyclonal anti-IRF7 | 4920, RRID: AB_2127551 |
mouse monoclonal anti-IRF9 Clone 6/ISGF3γ | 610285, RRID: AB_397680 |
mouse monoclonal anti-RIG-I (Alme-1) | AG-20B-0009, RRID: AB_2490189 |
mouse monoclonal anti-NFkB p65 (L8F6) | 6956, RRID: AB_10828935 |
Mouse monoclonal anti-HA (Clone HA-7) | H3663, RRID: AB_262051 |
Name | Forward Sequence (5′-3′) | Reverse Sequence (5′-3′) |
---|---|---|
GAPDH | tcggagtcaacggatttggt | ttcccgttctcagccttgac |
IFIT1 | gaatagccagatctcagaggagc | ccatttgtactcatggttgctgt |
IFNB1 | cgccgcattgaccatcta | gacattagccaggaggttctc |
IRF1 | cctgactccagcactgtcg | tgggtgacacctggaagttg |
IRF2 | acggtgaacatcatagtttagga | ggggagatctgcagagggta |
IRF3 | tgggcccccagatctgatta | gcacaaccttgaccatcacg |
IRF5 | aagatctacgaggtctgctccaat | atcctctgcagctcttcctctt |
IRF7 | ttggctcctgagagggca | cagcccaggccttgaagatg |
IRF9 | tcctccagagccagactact | caatccaggctttgcacctg |
MX1 | accattccaaggaggtgcag | tgcgatgtccacttcggaaa |
RANTES | gctgtcatcctcattgctactg | tggtgtagaaatactccttgatgtg |
TNFAIP3 (A20) | tcctcaggctttgtatttgagc | tgtgtatcggtgcatggtttta |
Common Name | Alternative Name(s) | Systematic Name |
---|---|---|
MCP-1 | MCAF, … | CCL2 |
MIP-1α | -- | CCL3 |
MIP-1β | -- | CCL4 |
RANTES | -- | CCL5 |
Eotaxin | -- | CCL11 |
MCP-4 | NCC-1, Ckβ10, … | CCL13 |
TARC | dendrokine, ABCD-2 | CCL17 |
MDC | DC/β-CK, ABCD-1, … | CCL22 |
Eotaxin-3 | MIP-4α, IMAC, TSC-1 | CCL26 |
IL-8 | NAP-1, MDNCF, GCP-1, … | CXCL8 |
IP-10 | CRG-10, IFI10, … | CXCL10 |
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Wüst, S.; Schad, P.; Burkart, S.; Binder, M. Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses. Cells 2021, 10, 2600. https://doi.org/10.3390/cells10102600
Wüst S, Schad P, Burkart S, Binder M. Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses. Cells. 2021; 10(10):2600. https://doi.org/10.3390/cells10102600
Chicago/Turabian StyleWüst, Sandra, Paulina Schad, Sandy Burkart, and Marco Binder. 2021. "Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses" Cells 10, no. 10: 2600. https://doi.org/10.3390/cells10102600