Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection
Abstract
:1. Introduction
2. Results
2.1. PRV pUL56 Interacted and Co-Localized with BAG3
2.2. PRV pUL56 Interacted with BAG3 through the PPxY Motifs
2.3. PRV pUL56 Re-Localized BAG3 to Golgi
2.4. BAG3 Was Downregulated by the pUL56 C-Terminus
2.5. BAG3 Degradation Was Induced by pUL56 C-Terminal 181L-185L via the Lysosome Pathway in a Golgi Retention-Dependent Manner
2.6. Endo-BAG3 Was Cleaved by PRV Infection in a Species-Specific Manner
2.7. BAG3 Played a Negative Regulation Role during PRV Lytic Infection
3. Discussion
4. Materials and Methods
4.1. Cells and Viruses
4.2. Plasmid Construction
4.3. Generation of ΔUL56 PRV Using the CRISPR/Cas9 System
4.4. Antibodies
4.5. Co-IP
4.6. Viral Stocks and Titers
4.7. Animals and Tissue Preparation
4.8. RNAi
4.9. Indirect Immunofluorescence Assay and Confocal Microscopy
4.10. Western Blot
4.11. Protein Degradation Pathway Inhibition Assays
4.12. Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Primers | Sequence (5′-3′) a | Constructs |
---|---|---|
mBAG3 HA F | GCGAATTCGGATGAGCGCCGCCACCCAATCGCC (Eco RI) | pCMV-HA-mBAG3 |
mBAG3 HA R | GCGGTACCCTAGGGAGCCACCAGGTTGCCAG (Kpn I) | pCMV-HA-mBAG3 |
UL26 Flag F | GCAAGCTTATGGGGCCCGTGTACGTGTCCGGC (Hin dIII) | p3×Flag-UL26 |
UL26 Flag R | GCGGATCCTCATCGGTGGGACATCATCTGGG (Bam HI) | p3×Flag-UL26 |
UL26.5 Flag F | GCAAGCTTATGGCAGCCCCGCCGAGCGCAGCG (Hin dIII) | p3×Flag-UL26.5 |
UL26.5 Flag R | GCGGATCCTCATCGGTGGGACATCATCTGGG (Bam HI) | p3×Flag-UL26.5 |
gD Flag F | GCAAGCTTATGCTGCTCGCAGCGCTATTGGC (Hin dIII) | p3×Flag-gD |
gD Flag R | GCGGATCCCTACGGACCGGGCTGCGCTTTTAG (Bam HI) | p3×Flag-gD |
UL41 Flag F | GCAAGCTTATGGGGCTCTTTGGCCTTTTAAAG (Hin dIII) | p3×Flag-UL41 |
UL41 Flag R | GCGGATCCTTATTTTCTCCTATGGGCGTTAC (Bam HI) | p3×Flag-UL41 |
UL36 PY Flag F | GCAAGCTTGAGTTCGCCGAGCGCATCGACGC (Hin dIII) | p3×Flag-UL36 PY domain |
UL36 PY Flag R | GCGAATTCGCGGTGGCGCACGAAGCGCGCGAAGG (Eco RI) | p3×Flag-UL36 PY domain |
UL56 KO F | GCGTACCCGATCGGTTGAGGTG | |
UL56 KO R | GGGCGGGCGAGCCGAGTTTATTG |
Primers | Sequence (5′-3′) a |
---|---|
UL56 PYM1 | CGGCGCCCCCGGCTCCAGGGCGGCCTCCTACGGGGACGTCGTCCGCGTCG |
UL56 PYM2 | CGCTGTTTGCCCGGGGCCCGGCCGCGTCCTACTCGGAGACGCTCCTGTTCG |
UL56 PYM3 | GGAGACGCTCCTGTTCGACGCGGCCGCGTACGCGGTGACCATCCCGGACC |
UL56 PYM4 UL56 L181A| UL56 L185A UL56 L181/5A | GCGGTGACCATCCCGGACGCGGCGGCGTACGAGCCCACCGTCATCGGGC CCGATGGTGCTCGTGGGCTTCGCCTGGGGGGGACTGCTCCTGCTGGTG CGTGGGCTTCCTCTGGGGGGGAGCGCTCCTGCTGGTGGGCCTCGTGTTTC CGTGGGCTTCGCCTGGGGGGGAGCGCTCCTGCTGGTGGGCCTCGTGTTTC |
Oligos | Sequence (5′-3′) | Constructs |
---|---|---|
UL56 sgRNA1 F | CACCGGCCCCGCGGGCTCGTTGTGG | pX330-UL56 sgRNA1 |
UL56 sgRNA1 R | AAACCCACAACGAGCCCGCGGGGCC | |
UL56 sgRNA2 F | CACCGACGACGGACTCCCGGAGCAC | pX330-UL56 sgRNA2 |
UL56 sgRNA2 R | AAACGTGCTCCGGGAGTCCGTCGTC |
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Lyu, C.; Li, W.-D.; Wang, S.-W.; Peng, J.-M.; Yang, Y.-B.; Tian, Z.-J.; Cai, X.-H. Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection. Int. J. Mol. Sci. 2020, 21, 3148. https://doi.org/10.3390/ijms21093148
Lyu C, Li W-D, Wang S-W, Peng J-M, Yang Y-B, Tian Z-J, Cai X-H. Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection. International Journal of Molecular Sciences. 2020; 21(9):3148. https://doi.org/10.3390/ijms21093148
Chicago/Turabian StyleLyu, Chuang, Wei-Dong Li, Shu-Wen Wang, Jin-Mei Peng, Yong-Bo Yang, Zhi-Jun Tian, and Xue-Hui Cai. 2020. "Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection" International Journal of Molecular Sciences 21, no. 9: 3148. https://doi.org/10.3390/ijms21093148