El Mehdaoui S, Touzé A, Laurent S, Sizaret P Y, Rasschaert D, Coursaget P
Laboratoire de Virologie Moléculaire, EMI-U Protéases et Vectorisation No. 00-10 and USC INRA, Faculté des Sciences Pharmaceutiques, Tours, France.
J Virol. 2000 Nov;74(22):10332-40. doi: 10.1128/jvi.74.22.10332-10340.2000.
The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.
本研究的目的是制备由包装在具有不同细胞嗜性的病毒样颗粒（VLP）中的质粒DNA组成的基因转移载体。为此，我们将兔出血病病毒（RHDV）N端截短的VP60衣壳蛋白与预期足以赋予嵌合VLP DNA包装和基因转移特性的序列融合。人乳头瘤病毒16型（HPV - 16）主要L1和次要L2衣壳蛋白的两个假定DNA结合序列分别在截短的VP60蛋白的N端融合。在昆虫细胞中表达的两种重组嵌合蛋白自组装成大小和外观与天然RHDV病毒粒子相似的VLP。嵌合蛋白获得了结合DNA的能力。因此，这两种嵌合VLP能够包装质粒DNA。然而，只有含有L1蛋白DNA包装信号的嵌合VLP能够以与乳头瘤病毒L1 VLP相似的速率有效地将基因转移到Cos - 7细胞中。用含有L2结合序列的嵌合RHDV衣壳只能转染非常有限数量的RK13兔细胞。含有L1结合序列的嵌合RHDV衣壳将基因转移到兔和野兔细胞中的速率高于HPV - 16 L1 VLP。然而，在人细胞系中未观察到基因转移。本研究结果表明，将DNA包装序列插入不能包裹DNA的VLP中可将该衣壳转化为可作为基因转移载体的人工病毒。这种可能性为通过将此类DNA包装序列插入其他病毒的主要衣壳蛋白中来设计具有不同细胞嗜性的新载体开辟了道路。
