2015 Fiscal Year Annual Research Report
核外移行機序を標的とした新規インフルエンザ薬の同定
| Project/Area Number |
15F15416
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
間 陽子 国立研究開発法人理化学研究所, 分子ウイルス学特別研究ユニット, ユニットリーダー (50182994)
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| Co-Investigator(Kenkyū-buntansha) |
CHUTIWITOONCHAI NOPPORN 国立研究開発法人理化学研究所, 分子ウイルス学特別研究ユニット, 外国人特別研究員
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| Project Period (FY) |
2015-10-09 – 2018-03-31
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| Keywords | Influenza A virus / NP / NXT1 / CRM1 / nuclear export |
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| Outline of Annual Research Achievements |
1.Identification and functional study of a new host factor, NXT1 that interacts with NP protein of influenza A virus. We succeeded to identify and proof that NXT1 promotes replication of influenza virus by stimulating nuclear export of viral NP protein via the CRM1-dependent pathway. In addition, NXT1 binds to the C-terminal region of NP and forms complex with CRM1 protein. The data of NXT1/NP interaction is useful for comprehensive understanding of viral nuclear export mechanism and for further identification of the virus-specific drug with minimal cytotoxicity effect.
2.Preparation and submission of NXT1 manuscript for publication. We are now submitting the manuscript of NXT1 study to the Viruses journal.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
NXT1, a new host binding partner of influenza nucleoprotein (NP) was identified and confirmed for the NXT1/NP interaction by; (1) immunoprecipitation using HA-NXT1/NP-FLAG transfected cell lysate and anti-FLAG agarose beads, and (2) pull-down assays using HA-NXT1 immobilized agarose beads and NP-FLAG protein. NXT1 gene was knockdowned by siRNA and infected with influenza A/WSN/33 (H1N1) virus for replication assay. The NXT1-knockdowned cells showed lower replication kinetic when compared with control cells. In addition, immunofluorescent stain and fluorescence in situ hybridization showed delaying of the nuclear exports of NP and viral RNA (vRNA) in the NXT1-knockdowned and infected cells. By contrast, NXT1 overexpression promoted nuclear export of NP which was inhibited by a CRM1 inhibitor, LMB. This indicates NXT1 stimulates NP nuclear export via CRM1-dependent pathway. In addition, pull-down assay showed increasing of NXT1/NP binding by CRM1 protein which may suggest a complex formation of NXT1/NP/CRM1. Identification of NXT1 binding site on NP was performed using pull-down assay with the N-terminal or C-terminal partial NP proteins and HA-NXT1 immobilized agarose beads. The results indicated that the C-terminal region of NP is required for NXT1 binding. Altogether reveals that NXT1 promotes nuclear exports of influenza NP and vRNA via CRM1-dependent pathway.
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| Strategy for Future Research Activity |
Oure recent publication showed high throughput screening method to discover small chemical compounds that inhibits nuclear export of influenza NP-NES3 protein and reduces viral replication (Kakisaka M, et al. 2016. Virus Res 217:23-31.). The identified compound and its analogs will be tested for the inhibitory effect (IC50) against different subtypes of influenza A virus including A/Massachusetts/3740/1965(H6N2), A/Ontario/6118/1968(H8N4), A/Turkey/Wisconsin/1966 (H9N2), A/Mallard/Astrakhan/263/1982 (H14N5), and A/duck/Australia/341/1983 (H15N8). In addition, cytotoxicity (CC50) of these compounds will be examined by WST-1 cell proliferation assay. The inhibitory mechanism of compounds will be investigated and validated by; 1) immunofluorescent stain of viral NP/RNA, 2) pull-down assay using photo-cross-linked compound beads and recombinant NP, CRM1, or NXT1 proteins, and 3) docking models of compound and NP, CRM1, or NXT1 proteins. The in vivo inhibitory effect of compound will be evaluated in the infected Balb/c mice model. A broad spectrum effect of compound will be evaluated against other viruses that use the cellular nuclear export machinery such as HIV-1 (via Rev/CRM1/NXT1).
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