The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures and Transfections
2.2. FLAG Affinity Purification
2.3. Coimmunoprecipitation and Glutathione S transferase (GST) pull-down assay
2.4. Western Blot
2.5. Immunofluorescence
2.6. Ubiquitination Assay
2.7. HBV DNA Analysis
2.8. Southern Blot Assay
2.9. Statistical Analysis
2.10. Plasmid Construction
2.11. RNA Preparation and Reverse Transcription Quantitative PCR (RT-qPCR)
3. Results
3.1. TRIM21 Interacts with HBV DNA Pol
3.2. TRIM21 Interacts with the TP Domain of HBV DNA Pol via its SPRY Domain
3.3. TRIM21 Negatively Regulates the Stability of HBV DNA Pol
3.4. TRIM21 Promotes the Degradation of HBV DNA Pol through the Ubiquitin Proteasome Pathway via Its RING Domain
3.5. TRIM21 Mediates the K48-Linked Ubiquitination of HBV DNA Pol at K260 and K283
3.6. TRIM21 Restricts HBV DNA Replication, and the SPRY Domain is Crucial for the Antiviral Role of TRIM21
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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Usage | Primer Name | Sequence (5′–3′) |
---|---|---|
Construction for HBV DNA Pol | HBV-DNA- Pol-F | GCCGGTACCGCCATGCCCCTATCTTATCCACAC |
HBV-DNA- Pol-R | ATAAGAATGCGGCCGCTCACGGGGGTCTCCATGCGAC | |
Construction for HBV DNA Pol-mut | HBV-DNA-Pol-K260R-F | CCAGCAGATCCGCCTCCTGCCTCCACCAAT |
HBV-DNA-Pol-K260R-R | GCAGGAGGCGGATCTGCTGGCAAAGTTTGT | |
HBV-DNA-Pol-K283R-F | TGAGAGACACTCATCCTCAGGCCATGCAGT | |
HBV-DNA-Pol-K283R-R | CTGAGGATGAGTGTCTCTCAAAGGTGGAGA | |
Construction for HBV DNA Pol- del-mut | HBV-P-del- TP-mut-F | GCCGGTACCATGTCTACAGCATGGGGCAGAATC |
HBV-P-del- RT-mut-F | CTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGCCAGGTCTGTGCCAAGTGTTTG | |
HBV-P-del- RT-mut-R | CTGGCTCGAGAAGATTGACGATAAGGGAGAGGCAGTAG | |
HBV-P-del- RH-mut-R | ATAAGAATGCGGCCGCTTACCGTTGCCGGGCAACGGGGTA | |
Construction for pHBV1.3 | pHBV1.3 | GGGTTTCGCCACCTCTGACT |
Construction for TRIM21 | TRIM21-F | GCGGAATTCATGGCTTCAGCAGCACGCTTG |
TRIM21-R | GCTCACCTCGAGTAGTCAGTGGATCCTTGTGATC | |
Construction for shR-TRIM21 | shR-TRIM21-F | GATCCGCAGCACGCTTGACAATGATGCTCGAGCATCATTGTCAAGCGTGCTGCTTTTTGA |
shR-TRIM21-R | AGCTTCAAAAAGCAGCACGCTTGACAATGATGCTCGAGCATCATTGTCAAGCGTGCTGCG | |
Construction for TRIM21 -del-mut | TRIM21-del-RING-F | GCGGAATTCATGCTGCTCAAGAATCTCCGGCCC |
TRIM21-del-CC-F | ACGCCATGGTCCCTCTTGAGGAGGAGCTCATCTCAGAGCTAGATCGAAGGTGC | |
TRIM21-del-CC-R | GCACCTTCGATCTAGCTCTGAGATGAGCTCCTCCTCAAGAGGGACCATGGCGT | |
TRIM21-del-PRY-F | AAGATGCTGAGGACATGTGCACAGCACTTTCACTCTGGAAAACATTACTGG | |
TRIM21-del-PRY-R | CCAGTAATGTTTTCCAGAGTGAAAGTGCTGTGCACATGTCCTCAGCATCTT | |
TRIM21-del-SPRY-R | GCTCACCTCGAGTCACTGGGCACCCAGGACCATAGG | |
TRIM21-del-PRY-SPRY-R | GCTCACCTCGAGTCACTGTAGGGCCTGGCTCTGCTG | |
Construction for Ubiquitin | Ubiquitin-F | GACGAATTCATGCAGATCTTCGTGAAAAC |
Ubiquitin-R | GCTCACCTCGAGCTAACCACCTCTCAGACGCAGGAC | |
Construction for Ubiquitin -mut | Ubiquitin- K48R-F | CTCATCTTTGCAGGCAGGCAGCTGGAAGAT |
Ubiquitin- K48R-R | ATCTTCCAGCTGCCTGCCTGCAAAGATGAG | |
Ubiquitin- K63R-R | GCACCTCGAGCTAACCACCTCTCAGACGCAGGACCAGGTGCAGGGTCGACTCCCTCTGGA | |
RT-qPCR for TRIM21 | TRIM21-qRT-F | CAGTCCTGGTTTCAATGATG |
TRIM21-qRT-R | TGGGCCAAATATAAGGAGGC | |
RT-qPCR for HBV DNA Pol | Pol-qRT-F | CTCTCTTTACGCGGACTCCC |
Pol-qRT-R | TCTTCTAGGGGACCTGCCTC | |
qPCR for HBV DNA copies | HBV DNA copies -F | TCTTGCCTTACTTTTGGAAG |
HBV DNA copies -R | AGTTCTTCTTCTAGGGGACC | |
RT-qPCR for β-actin | β-actin-F | CGTGACATTAAGGAGAAGCTG |
β-actin-R | CTAGAAGCATTTGCGGTGGAC |
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Mu, T.; Zhao, X.; Zhu, Y.; Fan, H.; Tang, H. The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation. Viruses 2020, 12, 346. https://doi.org/10.3390/v12030346
Mu T, Zhao X, Zhu Y, Fan H, Tang H. The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation. Viruses. 2020; 12(3):346. https://doi.org/10.3390/v12030346
Chicago/Turabian StyleMu, Ting, Xiaoqing Zhao, Yanan Zhu, Hongxia Fan, and Hua Tang. 2020. "The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation" Viruses 12, no. 3: 346. https://doi.org/10.3390/v12030346