Design of a trans protease lentiviral packaging system that produces high titer virus.
Design of a trans protease lentiviral packaging system that produces high titer virus.
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DOI：
10.1186/1742-4690-4-96
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发表时间：
2007-12-28
期刊：
影响因子：
3.3
通讯作者：
Leboulch, Philippe
中科院分区：
文献类型：
--
作者：
Westerman, Karen A.;Ao, Zhujun;Cohen, Eric A.;Leboulch, Philippe
人类免疫缺陷病毒（HIV）的结构蛋白和酶蛋白最初产生为由重叠的阅读框编码的两个长的多聚蛋白，一个产生结构蛋白（Gag），第二个产生结构蛋白和酶蛋白（Gag-Pol）。Gag与Gag-Pol的比率对于病毒颗粒的适当组装和成熟至关重要。为了最大限度地降低产生可复制慢病毒（RCL）的风险，我们开发了一种“超级分裂”慢病毒包装系统，其中通过独立于Gag和Pol提供反式蛋白酶（PR），将Gag与Pol分离，转导损失最小。在开发这种“超级分裂”包装系统时，我们纳入了几个新的安全性特征，包括去除Gag/Gag-Pol移码，将Gag、PR和逆转录酶/整合酶（RT/IN）功能分离到单独的质粒上，并大大减少载体和Gag之间以及Gag和Pol之间的核苷酸序列重叠。作为该新系统构建的一部分，我们使用了辅助蛋白Vpr的截短形式，其结合Gag的P6区，作为载体将PR和RT/IN作为融合蛋白递送到病毒组装和出芽的位点。我们还用活性稍低的T26 S PR突变体取代了wt PR，以防止与wt PR相关的过早加工和细胞毒性。这种新型“超分裂”包装系统产生的慢病毒滴度与传统慢病毒包装产生的滴度相当，其中Gag-Pol完整供应（1.0 × 106 TU/ml，未浓缩）。在这里，我们能够创建一个真正的“分裂功能”慢病毒包装系统，有可能用于基因治疗应用。这种新型系统在保持高滴度的同时结合了许多新的安全特性。此外，由于PR是反式提供的，这种独特的系统也可以提供检查病毒蛋白质加工和成熟的机会。
The structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol. In developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 × 106 TU/ml, unconcentrated). Here, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.
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影响因子：
5.4
作者：
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通讯作者：
LANDAU, NR
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作者：
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