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Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells
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Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid
Tomoaki Mori1, Kento Nakamura1, Keisuke Masaoka1
1Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Abstract:
Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.
Insights
Researchers developed a novel artificial RNA restriction enzyme using staphylococcal nuclease (SNase) to target and cleave influenza RNA. This new enzyme shows significantly enhanced RNA-cleavage rates, offering a promising strategy for antiviral development.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Viral infections pose significant health risks, necessitating effective antiviral strategies.
- Current methods for preventing viral infections, particularly RNA viruses, are limited.
- Previous research demonstrated success in cleaving DNA virus genomes to inhibit replication.
Purpose of the Study:
- To expand the RNA cleavage methodology to RNA viruses.
- To develop an artificial RNA restriction enzyme with enhanced RNA-cleavage rates.
- To investigate the efficacy of staphylococcal nuclease (SNase) as an RNA-cleavage domain.
Main Methods:
- Constructed an artificial RNA restriction enzyme by fusing SNase to a human Pumilio/fem-3 binding factor (PUF)-based RNA-binding protein.
- Utilized SNase as an alternative RNA-cleavage domain to the PIN domain of human SMG6.
- Evaluated RNA cleavage rates and binding properties using gel shift assays.
Main Results:
- The SNase-fusion nuclease achieved RNA cleavage rates 120-fold greater than the PIN-fusion nuclease for influenzavirus.
- Modifications to the linker moiety did not improve the PIN-fusion nuclease's cleaving ability.
- Gel shift assays indicated suboptimal RNA-binding properties for the PUF derivative used.
Conclusions:
- The SNase-fusion nuclease demonstrates superior RNA-cleavage efficiency compared to previous designs.
- Further improvements in RNA-binding properties could enable targeted RNA cleavage in mammalian cells.
- This technology holds potential for developing novel antiviral therapies against RNA viruses.
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