Westerman Karen A, Ao Zhujun, Cohen Eric A, Leboulch Philippe
Brigham and Women's Hospital, Department of Anesthesia (SR157), 75 Francis Street, Boston, MA 02115, USA.
Retrovirology. 2007 Dec 28;4:96. doi: 10.1186/1742-4690-4-96.
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 x 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.
人类免疫缺陷病毒（HIV）的结构蛋白和酶蛋白最初是以两个由重叠阅读框编码的长多聚蛋白形式产生的，一个产生结构蛋白（Gag），另一个产生结构蛋白和酶蛋白（Gag-Pol）。Gag与Gag-Pol的比例对于病毒颗粒的正确组装和成熟至关重要。为了将产生具有复制能力的慢病毒（RCL）的风险降至最低，我们开发了一种“超级拆分”慢病毒包装系统，其中通过独立于Gag和Pol在反式中提供蛋白酶（PR），将Gag与Pol分离，同时转导能力损失最小。
在开发这种“超级拆分”包装系统时，我们纳入了几个新的安全特性，包括去除Gag/Gag-Pol移码突变，将Gag、PR和逆转录酶/整合酶（RT/IN）功能分别拆分到不同的质粒上，并大幅减少载体与Gag之间以及Gag与Pol之间的核苷酸序列重叠。作为构建这个新系统的一部分，我们使用了辅助蛋白Vpr的截短形式，它与Gag的P6区域结合，作为一种载体，将PR和RT/IN作为融合蛋白递送到病毒组装和出芽位点。我们还用活性稍低的T26S PR突变体取代野生型PR，以防止与野生型PR相关的过早加工和细胞毒性。这种新型的“超级拆分”包装系统产生的慢病毒滴度与完整提供Gag-Pol的传统慢病毒包装系统产生的滴度相当（未浓缩时为1.0×106 TU/ml）。
在这里，我们能够创建一个真正的“拆分功能”慢病毒包装系统，该系统有潜力用于基因治疗应用。这个新系统纳入了许多新的安全特性，同时保持了高滴度。此外，由于PR是在反式中提供的，这个独特的系统还可能为研究病毒蛋白加工和成熟提供机会。
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