| 研究実績の概要 |
At the late stage of the replication cycle, influenza A virus (IAV) has to correctly package its genome with eight different segments of negative strand RNA into progeny virions, which then bud at the plasma membrane of host cells. These critical processes ensure the production and infectivity of progeny virions, however, details of the mechanism behind and the involvement of cellular machineries remain largely unresolved. In the first year of this project, we aimed for systematically identifying the cellular factors that contribute to the genome assembly and budding of IAV. We developed image-based, quantitative assays to monitor and analyze viral RNA segments in infected cells. We employed the established assays to screen a series of cellular proteins, and examined how the depletion of these factors could influence IAV infection. Next, we intend to further resolve viral RNA interaction with the surrounding cellular machineries. Therefore, in the second year of this project, we attempted a nanoscopic approach that combines a cell unroofing method and freeze-etching electron microscopy. This approach provides a panoramic view of viral RNA in its native environment inside of the cell, with the molecular-level resolution. By using immunogold labeling to identify various interesting cellular proteins found in the previous screen, we hope to verify the key host factor(s) and how they interact with viral RNA to facilitate the viral genome assembly and budding. Our ultimate goal is to resolve the underlying mechanisms that mediate these essential steps in the virus life cycle.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
By using the single-cell based quantitative assays developed in the first year, we have screened a series of host factors that may play important roles in the late stage of IAV infection, and found several interesting hits. In the second year, we introduced a new nanoscopic approach that allows us to observe viral genome directly in its native form with superior resolution. Employing the new method, we have started to examine the role of cellular machinery of interest in IAV egress.
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| 今後の研究の推進方策 |
For the next step, we will continue to verify the interesting and promising host targets revealed in the previous screen. Combining the newly introduced nanosopy approach and the fluorescent microscopy and molecular virology assays, we hope that we can uncover the cellular machinery involved in the influenza virus genome packaging and budding.
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