Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Virus
2.2. Plasmid Construction
2.3. Yeast Two Hybrid (Y2H) Assays
2.4. Co-IP Assays
2.5. Western Blot Analysis
2.6. Fluorescence Microscopy
2.7. Quantitative Analysis of RGV Genomic DNA
3. Results
3.1. Interaction between RGV-63R and RGV-91R was First Screened by Y2H
3.2. Confirmation of Interaction between RGV-63R and RGV-91R by Co-IP Followed by Western Blotting
3.3. Localization of RGV-63R and RGV-91R in Cells of Different Species
3.4. Detection of RGV-63R and RGV-91R Expressions by Western Blotting
3.5. RGV-63R and RGV-91R Promote RGV Genome Replication
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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Name | Sequence (5’ to 3’) a | Usage |
---|---|---|
63R-F | GGGAATTCCATATGGATCTCTTTGTGTACCAGTG (NdeI) | pGBKT7-63R/Y2H |
63R-R | CCGGAATTCTTACTTTTTCTTGAACGACA (EcoRI) | |
1R-F | CCGGAATTCATGGCATTCTCGGCAGAAGA (EcoRI) | pGADT7-1R/Y2H |
1R-R | CCGCTCGAGTCATAGGGGGGTAAACTTCC (XhoI) | |
2L-F | CCGGAATTCATGTCCATCATCGGAGCGAC (EcoRI) | pGADT7-2L/Y2H |
2L-R | CGCGGATCCTTACCATCTCACTGTAGAGA (BamHI) | |
9R-F | GGGAATTCCATATGGAAATGTTTGCATCTAAATC (NdeI) | pGADT7-9R/Y2H |
9R-R | CCGGAATTCTCATCGCCACTCAAAGGATT (EcoRI) | |
10L-F | CCGGAATTCATGGACACATCACCCTACGA (EcoRI) | pGADT7-10L/Y2H |
10L-R | CCGCTCGAGTCAGGCAAACTTGCCCCTCC (XhoI) | |
13L-F | GGGAATTCCATATGTGCTCCAAACTCGTAGAGAT (NdeI) | pGADT7-13L/Y2H |
13L-R | CCGGAATTCTTAGAAACCCATGGTCTCGA (EcoRI) | |
16R-F | CCGGAATTCATGGAACAAGTACCCATAAA (EcoRI) | pGADT7-16R/Y2H |
16R-R | CCGCTCGAGCTAATCGTCCAAGTCCGACT (XhoI) | |
21R-F | GGGAATTCCATATGGCTACAAATTACTGTGACGA (NdeI) | pGADT7-21R/Y2H |
21R-R | CCGGAATTCTTACATCGTGAAGCTCTCAA (EcoRI) | |
23L-F | CCGGAATTCATGGAAACCATAGTGCTGGT (EcoRI) | pGADT7-23L/Y2H |
23L-R | CCGCTCGAGTTACGACGAGGACCCAAATG (XhoI) | |
24R-F | CCGGAATTCATGGCTAACGCTACCATAAA (EcoRI) | pGADT7-24R/Y2H |
24R-R | CGCGGATCCCTACTCTTGCTGCTCGGCTC (BamHI) | |
29R-F | GGGAATTCCATATGGCCAATTTTCTACAAGATGT (NdeI) | pGADT7-29R/Y2H |
29R-R | CCGGAATTCTCAATGACGCTCCTTGGCCC (EcoRI) | |
40R-F | CCGGAATTCATGCAAGTTTTTCTAGATTT (EcoRI) | pGADT7-40R/Y2H |
40R-R | CGCGGATCCTCACCTCCTCCTGCTCCTGC (BamHI) | |
44R-F | GGGAATTCCATATGAGAGTCGTGGTAAACGCAAA (NdeI) | pGADT7-44R/Y2H |
44R-R | CCGGAATTCTCACATCAGAAGAGACACGT (EcoRI) | |
53R-F | CCGGAATTCATGGGAGCAGCGGAATCTAT (EcoRI) | pGADT7-53R/Y2H |
53R-R | CCGCTCGAGTTAACCCCTGTGGGCCGGAA (XhoI) | |
60R-F | GGGAATTCCATATGGCAATGGTTTCCAACGTAAA (NdeI) | pGADT7-60R/Y2H |
60R-R | CCGGAATTCCTACAGGCTCTTTAGGATAA (EcoRI) | |
63R-F | GGGAATTCCATATGGATCTCTTTGTGTACCAGTG (NdeI) | pGADT7-63R/Y2H |
63R-R2 | CCGGAATTCTTACTTTTTCTTGAACGACA (EcoRI) | |
65L-F | CGCGGATCCTGTCCAGGGGCATGACTACC (BamHI) | pGADT7-65L/Y2H |
65L-R | CCGCTCGAGTCACTTGAAGGCTATGGAAA (XhoI) | |
73L-F | CCGGAATTCATGTTTCCTCACGTCACCAT (EcoRI) | pGADT7-73L/Y2H |
73L-R | CGCGGATCCTTAGATGTCCAGGGGTTCGT (BamHI) | |
87L-F | CCGGAATTCATGGAAGGTTGGTTGGGAAA (EcoRI) | pGADT7-87L/Y2H |
87L-R | CCGCTCGAGCTAGACTCCCTTGGCATGAA (XhoI) | |
88R-F | CCGGAATTCATGTCTTTTCAGAGAGATTA (EcoRI) | pGADT7-88R/Y2H |
88R-R | CCGCTCGAGCTACCTGGTCCACCTCTTGC (XhoI) | |
91R-F | GGGAATTCCATATGCTGTGGGAAGCCGTAACAGA (NdeI) | pGADT7-91R/Y2H |
91R-R | CCGGAATTCTTAGCCCTCAAAGAGAGTCA (EcoRI) | |
92R-F | GGGAATTCCATATGAGCATCCCTACAGTCATAGC (NdeI) | pGADT7-92R/Y2H |
92R-R | CCGGAATTCTTACCGCACATTTCTAGACA (EcoRI) | |
95R-F | CCGGAATTCATGCACGGTTGCAATTGTAA (EcoRI) | pGADT7-95R/Y2H |
95R-R | CCGCTCGAGTCAGTTAAAAGTGCTCGTAT (XhoI) | |
97R-F | CCGGAATTCATGTCTTCTGTAACTGGTTC (EcoRI) | pGADT7-97R/Y2H |
97R-R | CCGCTCGAGGACCCATGACGGAAAAGACT (XhoI) | |
98R-F | CCGGAATTCATGGCAAACTTTGTGACAGA (EcoRI) | pGADT7-98R/Y2H |
98R-R | CCGCTCGAGTTAGGCTCTGACCACAAACA (XhoI) | |
101L-F | CCGGAATTCATGGATCCAGAAGGAATGAT (EcoRI) | pGADT7-101L/Y2H |
101L-R | CCGCTCGAGTCACAGCACCTTTCTCAGGT (XhoI) | |
102R-F | CCGGAATTCATGGGCATAAAAGGACTGAA (EcoRI) | pGADT7-102R/Y2H |
102R-R | CCGCTCGAGTCACTTGCGCTTGCACTTCT (XhoI) | |
63R-F2 | CCCAAGCTTATGGATCTCTTTGTGTACCA (HindIII) | pcDNA3.1-63R-3Flag |
63R-3Flag-R | CCGGAATTCTTACTTATCGTCGTCATCCTTGTAATCGATCTTATCGTCGTCATCCTTGTAATCTCCCTTATCGTCGTCATCCTTGTAATCCTTTTTCTTGAACGACACAA (EcoRI) | |
91R-F2 | CCCAAGCTTATGCTGTGGGAAGCCGTAAC (HindIII) | pcDNA3.1-91R-HA |
91R-HA-R | CCGGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTACATGCCCTCAAAGAGAGTCACGG (EcoRI) | |
63R-R3 | CCGGAATTCGACTTTTTCTTGAACGACAC (EcoRI) | pEGFP-63R/ colocalization |
91R-F3 | CCCAAGCTTCAATGCTGTGGGAAGCCGTA (HindIII) | pDsRed2-91R/ colocalization |
91R-R2 | ACGCGTCGACTTAGCCCTCAAAGAGAGTCA (SalI) | |
91R△NLS1-R | CCCATGAGCCTCAGCGTCACGTAGCTGGTAAAGACCGATG | pDsRed2-91R△NLS1 |
91R△NLS1-F | CATCGGTCTTTACCAGCTACGTGACGCTGAGGCTCATGGG | |
91R△NLS2-R | GAAAAGGGTCCCATCTTGACCACTCCTCCGGACGCCACCA | pDsRed2-91R△NLS2 |
91R△NLS2-F | TGGTGGCGTCCGGAGGAGTGGTCAAGATGGGACCCTTTTC | |
MCP-F | ATGGTTGTGGAGCAGGTG | qPCR |
MCP-R | TGACGCAGGTGTAATTGGAG |
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Zeng, X.-T.; Zhang, Q.-Y. Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species. Viruses 2019, 11, 416. https://doi.org/10.3390/v11050416
Zeng X-T, Zhang Q-Y. Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species. Viruses. 2019; 11(5):416. https://doi.org/10.3390/v11050416
Chicago/Turabian StyleZeng, Xiao-Tao, and Qi-Ya Zhang. 2019. "Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species" Viruses 11, no. 5: 416. https://doi.org/10.3390/v11050416