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オルソボルナウイルスの膜糖蛋白質の発現調整による戦略的粒子産出機構とその応用

酒井, まどか 京都大学 DOI:10.14989/doctor.k23342

2021.03.23

概要

オルソボルナウイルス属に属するボルナ病ウイルス 1 型( BoDV-1 )は、核内で非細胞傷害性に持続感染できる稀な性状を持つ RNA ウイルスである。また、ウイルスゲノムは細胞核内でエピゾーマル RNA として存在しており、分裂細胞においても染色体との相互作用を介して感染を維持することが明らかとなっている。これらの性状から、Bo DV-1 は有用遺伝子を安全にかつ長期に導入できる遺伝子治療用ベクターとして開発が進んでいる。Bo DV-1 を利用したウイルスベクターREVec は、幹細胞を含む多様な細胞への導入が可能であるが、導入力価の高い REVec 粒子を得ることが困難であるという課題があり、REVec の実用化を進めるにあたり克服すべき問題となっている。

本論文は、BoDV-1 を含むオルソボルナウイルス属の膜糖蛋白質( G)の感染性ウイルス粒子産出における役割を詳細に解析し応用することで、REVec の力価の改善を目指した研究である。

申請者は、まず Bo DV- 1 感染細胞において、G 蛋白質の発現が極めて低いことを免疫染色により明らかにし、G 蛋白質の発現量の制御が BoDV-1 の感染維持と感染性粒子の産生に重要であることを突き止めた。そこで、G 遺伝子欠損 REVec( ΔG- REVec)に G 蛋白質を一過性に補完したシュードタイプ REVec を用いて解析したところ、細胞内での G 蛋白質の過剰な発現は、成熟していない G 蛋白質を多く持つ導入効率の低い REVec 粒子の産出をもたらすとともに、REVec ゲノム RNA の粒子への取り込みが阻害されることを明らかにした。さらに、G蛋白質の過剰な発現の副産物である未開裂の G がこれらの原因になることを示した。

申請者は、感染性ウイルス粒子産生に係わるG 蛋白質の発現量の制御が、他のオルソボルナウイルス属ウイルスでも共通して観察されるのかを確認するために、オルソボルナウイルス属ウイルスのG 遺伝子の一過性発現によるシュードタイプ REVec により解析を行った。その結果、カナリアボルナウイルス 1 型( CnBV- 1)の G 蛋白質では、その発現量を増加させた場合においても高力価の REVec を産生できることを突き止めた。そして、Cn BV-1 の G 蛋白質のシグナルペプチド領域が、高い導入効率を示す粒子の産出に関与していることを明らかにした。

これらの知見を基に、CnBV-1 G 遺伝子を持つキメラタイプ REVec の作製し、この REVecが、従来型の REVec と比較して、幹細胞や初代培養細胞を含むヒト由来細胞へ高い遺伝子導入効率を示した。

以上から、オルソボルナウイルスは G 蛋白質の発現を制限することによって、感染性粒子の産生を制御し感染を維持させている可能性が示された。また、G の発現量や種類を改良することで、REVec 産生システムと力価の向上につながることを示した。

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