Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Ethics Statements
2.2. HepG2-hNTCP Overexpressing cGAS and STING
2.3. Hepatitis B Virus Production and Infection
2.4. Other Viruses and Viral Vectors
2.5. Quantitative Reverse Transcription PCR (RT-qPCR)
2.6. Reagents
2.7. Preparation and Quantification of Viral Nucleic Acids for Transfection Experiments
2.8. Transfections
2.9. Western Blots
2.10. IRF3 Nuclear Translocation Assays
2.11. Immunofluorescence
2.12. Microscope Image Acquisition
3. Results
3.1. HBV DNAs but not RNAs are Immunostimulatory
3.2. Virion-Associated HBV DNA is Sensed by the cGAS/STING Pathway
3.3. Hepatocytes Express Low Levels of the DNA Sensors Compared to Immune Cells
3.4. Hepatocytes are Competent for Sensing HBV DNA
3.5. HBV DNA is not Sensed During Productive Infection of Hepatocytes
3.6. HBV Does not Inhibit the Innate Immune Response to Foreign DNA in Hepatocytes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name | Citation | Supplier | Authentication Test Method |
---|---|---|---|
HepG2 | Figure 3 | Gift from Prof S.Urban, Heidelberg, Germany | Cell line authentication performed by Eurofins Genomics, using Applied BiosystemsTM AmpFLSTRTM IdentifilerTM Plus PCR Amplification Kit with 16 markers |
HepG2-hNTCP | Figures 3–6 | Gift from Prof S.Urban, Heidelberg, Germany [33] | Cell line authentication performed by Eurofins Genomics, using Applied BiosystemsTM AmpFLSTRTM IdentifilerTM Plus PCR Amplification Kit with 16 markers |
HepaRG-hNTCP | Figure 3 | Gift from Prof S.Urban, Heidelberg, Germany | Multiplexion cell contamination assay (Heidelberg, Germany) as described [39] |
HepAD38 | Figure 1 (for HBV RNAs and replication intermediate extraction) | Gift from Prof E Hild, Langen, Germany | Release of HBV particles (qPCR + infectivity test) |
THP1 | Figures 2 and 3 | Gift from Prof V. Hornung, Munich, Germany | Cell line authentication by Eurofins Genomics on 24.03.2017 |
THP1 ΔSTING | Figure 2 | Gift from Prof V. Hornung, Munich, Germany | Western blot |
THP1 ΔcGAS | Figure 2 | Gift from Prof V. Hornung, Munich, Germany | Western blot |
THP1 ΔMAVS | Figure 2 | Gift from Prof V. Hornung, Munich, Germany | Western blot |
Name | Sequence (5′–3′) | Supplier | Use |
---|---|---|---|
HBV_RC_F | CACTCTATGGAAGGCGGGTA | Eurofins Genomics | qPCR for quantification of HBV stocks |
HBV_RC_R | TGCTCCAGCTCCTACCTTGT | Eurofins Genomics | qPCR for quantification of HBV stocks |
HBV RNA 3 F | GCTTTCACTTTCTCGCCAAC | Eurofins Genomics | RT-qPCR |
HBV RNA 3 R | GAGTTCCGCAGTATGGATCG | Eurofins Genomics | RT-qPCR |
ISG54_F | GGTGGCAGAAGAGGAAGATT | Eurofins Genomics | RT-qPCR |
ISG54_R | TAGGCCAGTAGGTTGCACAT | Eurofins Genomics | RT-qPCR |
IFNλ-1_F | CGCCTTGGAAGAGTCACTCA | Eurofins Genomics | RT-qPCR |
IFNλ-1_R | GAAGCCTCAGGTCCCAATTC | Eurofins Genomics | RT-qPCR |
cGAS_F | CAAGAAGGCCTGCGCATTCA | Eurofins Genomics | RT-qPCR |
cGAS_R | GAGAAGGATAGCCGCCATGT | Eurofins Genomics | RT-qPCR |
STING_F | GATATCTGCGGCTGATCCTG | Eurofins Genomics | RT-qPCR |
STING_R | GCTGTAAACCCGATCCTTGA | Eurofins Genomics | RT-qPCR |
IFI16_F | CGCTTGAAGACCTGGCTGAA | Eurofins Genomics | RT-qPCR |
IFI16_R | TGACAGTGCTGCTTGTGGAG | Eurofins Genomics | RT-qPCR |
PQBP1_F | TCTGGAGCCTGAACCAGAGGAA | Eurofins Genomics | RT-qPCR |
PQBP1_R | TCCAACCTGGTGGCCTCGTAGT | Eurofins Genomics | RT-qPCR |
RIG-I_F | CCTACCTACATCCTGAGCTACAT | Eurofins Genomics | RT-qPCR |
RIG-I_R | TCTAGGGCATCCAAAAAGCCA | Eurofins Genomics | RT-qPCR |
MAVS_F | GGTGCTCACCAAGGTGTCTG | Eurofins Genomics | RT-qPCR |
MAVS_R | AGGAGGTGCTGGCACTGATG | Eurofins Genomics | RT-qPCR |
MDA5_F | AGAGTGGCTGTTTACATTGCC | Eurofins Genomics | RT-qPCR |
MDA5_R | GCTGTTCAACTAGCAGTACCTT | Eurofins Genomics | RT-qPCR |
TLR3_F | ACCTCCAGCACAATGAGCTA | Eurofins Genomics | RT-qPCR |
TLR3_R | TCCAGCTGAACCTGAGTTCC | Eurofins Genomics | RT-qPCR |
RPL13A_F | CCT GGA GGA GAA GAG GAA AGA GA | Eurofins Genomics | RT-qPCR |
RPL13A_R | TTG AGG ACC TCT GTG TAT TTG TCA A | Eurofins Genomics | RT-qPCR |
TBP_F | GGAGCTGTGATGTGAAGT | Eurofins Genomics | RT-qPCR |
TBP_R | TACGTCITCTTCCTGAATCC | Eurofins Genomics | RT-qPCR |
HBV_quant_F | TGTCAACACTAATATGGGCCTAA | Eurofins Genomics | quantification of HBV RNA and DNA |
HBV_quant_R | AGGGGCATTTGGTGGTCTAT | Eurofins Genomics | quantification of HBV RNA and DNA |
HBV_st | TGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTTATAGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCT | Integrated DNA Technologies, BvBA | standard for quantification of HBV RNA and DNA |
MVAgfp_F | AGCACGACTTCTTCAAGTCC | Eurofins Genomics | quantification of MVAgfp DNA |
MVAgfp_R | GTTGTAGTTGTACTCCAGCTTG | Eurofins Genomics | quantification of MVAgfp DNA |
MVAgfp_st | AGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC | Integrated DNA Technologies, BvBA | standard for quantification of MVAgfp DNA |
SeV_F | TTCATTATCATCCCGTGAGA | Eurofins Genomics | quantification of SeV RNA |
SeV_R | CCAGTGATCCATCATCAATC | Eurofins Genomics | quantification of SeV RNA |
SeV_st | TTCATTATCATCCCGTGAGATCAGGAACCTGAGGGTTATCACAAAAACTTTATTAGACAGGTTTGAGGATATTATACATAGTATAACGTATAGATTCCTCACCAAAGAGATAAAGATTTTGATGAAGATTTTAGGGGCAGTCAAGATGTTCGGGGCCAGGCAAAATGAATACACGACCGTGATTGATGATGGATCACTGG | Integrated DNA Technologies, BvBA | standard for quantification of SeV RNA |
SeV n.ac. from Particles | MVA n.ac. from Particles | HBV n.ac. from Particles | HepaD38 RNA | HBV R.I. | |
---|---|---|---|---|---|
Viral DNA (copies/cell) | 5.0E+03 | 5.0E+03 | 5.0E+03 | ||
Viral RNA (cDNA-equivalent copies/cell) | 2.8E+01 | 2.3E+01 | 1.3E+04 |
Name | Supplier | Cat No. | Clone No. |
---|---|---|---|
Anti phospho S386-IRF3 (rabbit) | Abcam | ab76493 | EPR2346 |
Anti IRF3 (rabbit) | Epitomics | 2241-1 | EP2419Y |
Anti beta-actin (mouse) | Sigma-Aldrich | A5441 | AC-15 |
Anti beta-tubulin (mouse) | Sigma-Aldrich | 075K4875 | TUB 2.1 |
Anti GAPDH (rabbit) | Cell Signaling Technology | 2118 | 14C10 |
Anti MAVS (rabbit) | Abcam | Ab25084 | |
Anti STING (rabbit) | Cell Signaling Technology | 13647 | D2P2F |
Anti cGAS (rabbit) | Cell Signaling Technology | 15102 | D1D3G |
Anti-mouse HRP | Cell Signaling Technology | 7076S | |
Anti-rabbit HRP | Cell Signaling Technology | 7074S |
Name | Citation | Supplier | Cat No. | Clone No. |
---|---|---|---|---|
Anti IRF3 (rabbit) | IRF3 translocation assay, Figure 6 | Cell Signaling Technology | 11904S | D6I4C |
anti HBc (mouse) | IRF3 translocation assay, Figure 6 | gift from Prof. S. Urban, Heidelberg | M312 | |
anti-rabbit Alexa Fluor 488 | IRF3 translocation assay, Figure 6 | Invitrogen | A11008 | |
anti-mouse Alexa Fluor 555 | IRF3 translocation assay, Figure 6 | Thermo Fischer Scientific | A21422 | |
Anti-HBc (rabbit) | Figure S5 | Dako, Agilent | B0586 | |
Goat anti-rabbit IgG(H+L), Alexa Fluor 546 | Figure S5 | Thermo Fischer Scientific | A-11010 |
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Lauterbach-Rivière, L.; Bergez, M.; Mönch, S.; Qu, B.; Riess, M.; Vondran, F.W.R.; Liese, J.; Hornung, V.; Urban, S.; König, R. Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes. Viruses 2020, 12, 592. https://doi.org/10.3390/v12060592
Lauterbach-Rivière L, Bergez M, Mönch S, Qu B, Riess M, Vondran FWR, Liese J, Hornung V, Urban S, König R. Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes. Viruses. 2020; 12(6):592. https://doi.org/10.3390/v12060592
Chicago/Turabian StyleLauterbach-Rivière, Lise, Maïwenn Bergez, Saskia Mönch, Bingqian Qu, Maximilian Riess, Florian W. R. Vondran, Juliane Liese, Veit Hornung, Stephan Urban, and Renate König. 2020. "Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes" Viruses 12, no. 6: 592. https://doi.org/10.3390/v12060592