Li K J, Garoff H
Karolinska Institute, Department of Bioscience at Novum, Huddinge, Sweden.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11658-63. doi: 10.1073/pnas.93.21.11658.
We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.
我们描述了一种用于生产感染性重组莫洛尼鼠白血病病毒颗粒的异源辛德毕斯病毒（SFV）驱动的包装系统。将gag-pol和env基因以及重组逆转录病毒基因组（LTR-psi(+)-neoR-LTR）插入到单个SFV1表达质粒中。具有复制能力的RNA在体外转录，并通过电穿孔导入BHK-21细胞的细胞质中。表达的莫洛尼鼠白血病病毒结构蛋白产生细胞外病毒样颗粒。在这些颗粒中，gag前体被加工成成熟产物，表明颗粒中含有活性蛋白酶。在使用大量Pr65gag的反式互补试验中，gag-pol融合蛋白的蛋白酶也显示出活性。此外，在体外试验中测得颗粒具有逆转录酶（RT）活性。用所有三种SFV1构建体共转染BHK-21细胞，在5小时的孵育期内产生了转导能力为4×10(6)集落形成单位（cfu）/ml的颗粒。总共从约4×10(6)个转染细胞中获得了2.9×10(7)个具有转导能力的颗粒。因此，该系统代表了第一个用于生产感染性逆转录病毒颗粒的基于RNA的包装系统。在病毒储备中未检测到辅助病毒，并且携带嗜异性包膜的颗粒与携带亲嗜性包膜的颗粒能够以相似的效率产生，这些事实使得该系统在基因治疗方面非常有吸引力。
