A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.
A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.
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一种基于替代RNA运输机制的慢病毒包装系统，用于表达辅助RNA和基因转移载体RNA，及其用于研究颗粒形成和基因递送的辅助蛋白的需求。
DOI：
10.1128/jvi.73.11.9589-9598.1999
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发表时间：
1999
影响因子：
5.4
通讯作者：
Schuening,FG
中科院分区：
文献类型：
--
作者：
Srinivasakumar,N;Schuening,FG
设计了一种基于慢病毒的包装系统，通过使用Mason-Pfizer猴病毒的组成型转运元件（CTE）表达病毒蛋白和Rev-Rev反应元件（RRE）组合表达基因转移载体，降低辅助和基因转移载体序列之间重组的机会。使用这种方法，我们评估了一系列的人类免疫缺陷病毒1型包装结构，表达一种或多种辅助蛋白（Vif，Vpr和Vpu），除了Gag和Pol蛋白，颗粒形成和病毒储备生产的基因转移。也表达Vpr或Vpr和Vpu两者的构建体比不含这些序列的质粒产生更多的颗粒，如通过p24测定所测量的。反式激活实验表明，编码Vpr或Vpr和Vpu的包装质粒也表达功能性单外显子达特蛋白。对于这些构建体，可以在不存在共转染的Tat表达质粒的情况下制备高滴度病毒原液。用所有包装构建体制备的两性包膜假型病毒原液，无论是否共表达任何辅助蛋白，在转导生长停滞的HeLa细胞中同样有效。将组合/混合包装系统与基于单独CTE或Rev和RRE的系统进行比较，以表达包装质粒以及基因转移载体。组合/混合包装系统与用于生产病毒储备液的其他系统相当，表明该设计可能被证明对于用于治疗目的的慢病毒载体的最终部署更安全。
A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.