Efficient intracellular assembly of papillomaviral vectors
Efficient intracellular assembly of papillomaviral vectors
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DOI：
10.1128/jvi.78.2.751-757.2004
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发表时间：
2004-01-01
影响因子：
5.4
通讯作者：
Schiller, JT
中科院分区：
文献类型：
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作者：
Buck, CB;Pastrana, DV;Schiller, JT
虽然乳头瘤病毒结构蛋白L1和L2可以自发地共组装形成病毒样颗粒，但是目前可用于生产能够将报告质粒转导到哺乳动物细胞中的L1/L2颗粒的方法在技术上要求很高并且产量相对较低。在这份报告中，我们描述了一个简单的293细胞转染方法，有效的细胞内生产的乳头状瘤病毒为基础的基因转移载体携带报告质粒。使用牛乳头瘤病毒1型（BPV 1）和人乳头瘤病毒16型作为模型乳头瘤病毒，我们已经开发了一个系统，用于生产乳头瘤病毒载体股票与几十亿转导单位每毫升的滴度。这些载体的生产需要L1和L2，并且可以通过乳头瘤病毒中和抗体来阻止转导。可以通过碘克沙醇（OptiPrep）梯度离心程序纯化原液，该程序比标准氯化铯梯度纯化实质上更有效。虽然早期的数据表明，病毒早期蛋白E2的潜在作用，我们发现，E2蛋白的表达并没有提高BPV 1载体的细胞内生产。也可以将缺乏BPV 1 DNA序列的报告质粒进行酶切。BPV 1载体生产效率受包装的靶质粒大小的显著影响。使用6-kb靶质粒导致BPV 1载体产量高于使用接近乳头瘤病毒基因组的天然7.9-kb大小的靶质粒的产量。结果表明，乳头瘤病毒结构蛋白的异源报告质粒周围的细胞内组装是令人惊讶的混杂，并可能主要由大小歧视机制驱动。
Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.