A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8.
A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8.
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DOI：
10.1038/sj.mt.6300053
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发表时间：
2007-03
期刊：
影响因子：
--
通讯作者：
Kay MA
中科院分区：
文献类型：
--
作者：
Akache B;Grimm D;Shen X;Fuess S;Yant SR;Glazer DS;Park J;Kay MA
基于腺相关病毒不同血清型的载体，基于其独特的组织嗜性和不同的免疫学特性，为人类基因治疗带来了巨大的希望。一个特别令人感兴趣的候选者是AAV8，它可以在体内有效和快速地覆盖广泛的组织。为了进一步阐明AAV8转导背后的机制，我们使用酵母双杂交分析来筛选小鼠肝脏互补DNA文库中能够与病毒衣壳蛋白相互作用的细胞蛋白。我们总共回收了大约700个克隆，包括300多个独立的基因。序列分析揭示了超过100个基因的多重命中，包括两个编码内体半胱氨酸蛋白酶组织蛋白酶B和L的基因。值得注意的是，这两种蛋白酶也与酵母中的AAV 2衣壳的相应部分物理相互作用，但不与AAV 5相互作用。我们证明了组织蛋白酶B和L对于有效的AAV 2和AAV 8介导的哺乳动物细胞的转导是必需的，并且记录了纯化的组织蛋白酶B和L蛋白在体外结合和切割完整的AAV 2和AAV 8颗粒的能力。这些数据表明，组织蛋白酶介导的切割可以引发AAV衣壳用于随后的核脱壳，并表明在我们的筛选中回收的其他基因的分析可能有助于进一步阐明AAV8和相关血清型转导背后的机制。
Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.
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影响因子：
5.4
作者：
Akache, Bassel;Grimm, Dirk;Kay, Mark A.
通讯作者：
Kay, Mark A.
DOI：
10.1074/jbc.m508381200
发表时间：
2006-02-10
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Huang IC;Bosch BJ;Li F;Li W;Lee KH;Ghiran S;Vasilieva N;Dermody TS;Harrison SC;Dormitzer PR;Farzan M;Rottier PJ;Choe H
通讯作者：
Choe H
影响因子：
4.8
作者：
Grimm, D
通讯作者：
Grimm, D
影响因子：
82.9
作者：
Qing, K;Mah, C;Srivastava, A
通讯作者：
Srivastava, A
影响因子：
3.7
作者：
Pager, CT;Craft, WW;Dutch, RE
通讯作者：
Dutch, RE