RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses, Cells and Antibodies
2.2. Mapping of the AP mRNA Accessible Regions in Cells
2.3. In Vitro Ribozyme Studies
2.4. Construction of Ribozyme-Expressing Cell Lines
2.5. Studies of Viral Gene Expression and Growth in M1GS-Expressing Cells
2.6. Determination of the HCMV DNA Level
2.7. Statistical Analysis
3. Results
3.1. Ribozyme-Mediated Cleavage of the AP mRNA Sequence in Vitro
Enzyme | (kcat/Km)s (µM−1·min−1) | Kd (nM) |
---|---|---|
M1-A | 0.22 ± 0.05 | 0.31 ± 0.05 |
V718-A | 13.5 ± 2.5 | 0.35 ± 0.06 |
M1-C | <5 × 10−6 | 0.32 ± 0.06 |
V718-C | <5 × 10−6 | 0.34 ± 0.05 |
M1-TK | <5 × 10−6 | ND |
3.2. Ribozyme Expression in the Cultured Cells
3.3. Ribozyme-Mediated Reduction of HCMV AP and PR Expression
Viral Gene Class | Ribozymes | ||||||
---|---|---|---|---|---|---|---|
U251 | M1-C | V718-C | M1-A | V718-A | M1-TK | ||
IE2 mRNA | α | 0% | 0% | 0% | 0% | 0% | 0% |
US2 mRNA | β | 0% | 0% | 0% | 0% | 0% | 0% |
AP mRNA | γ | 0% | 3% | 6% | 78% ± 8% | 99% ± 7% | 0% |
PR mRNA | γ | 0% | 3% | 7% | 75% ± 7% | 98% ± 7% | 0% |
IE2 protein | α | 0% | 0% | 0% | 0% | 0% | 0% |
UL44 protein | β, γ | 0% | 0% | 0% | 0% | 0% | 0% |
UL83 protein | γ | 0% | 0% | 0% | 0% | 0% | 0% |
AP protein | γ | 0% | 3% | 6% | 75% ± 7% | 99% ± 8% | 0% |
PR protein | γ | 0% | 3% | 6% | 72% ± 7% | 98% ± 8% | 0% |
3.4. Inhibition of HCMV Replication Mediated by the Ribozymes
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Yang, Z.; Reeves, M.; Ye, J.; Trang, P.; Zhu, L.; Sheng, J.; Wang, Y.; Zen, K.; Wu, J.; Liu, F. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins. Viruses 2015, 7, 3345-3360. https://doi.org/10.3390/v7072775
Yang Z, Reeves M, Ye J, Trang P, Zhu L, Sheng J, Wang Y, Zen K, Wu J, Liu F. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins. Viruses. 2015; 7(7):3345-3360. https://doi.org/10.3390/v7072775
Chicago/Turabian StyleYang, Zhu, Michael Reeves, Jun Ye, Phong Trang, Li Zhu, Jingxue Sheng, Yu Wang, Ke Zen, Jianguo Wu, and Fenyong Liu. 2015. "RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins" Viruses 7, no. 7: 3345-3360. https://doi.org/10.3390/v7072775