Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors
Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors
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DOI：
10.1128/jvi.70.5.3227-3234.1996
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发表时间：
1996-05-01
影响因子：
5.4
通讯作者：
Samulski, RJ
中科院分区：
文献类型：
--
作者：
Ferrari, FK;Samulski, T;Samulski, RJ
重组腺相关病毒（AAV）在体外用标记基因转染细胞的能力被发现通过腺病毒的存在而显著增加。用腺病毒基因组DNA的转染实验表明，这种增加不是由腺病毒介导的病毒摄取促进的，而是依赖于腺病毒基因表达。使用各种腺病毒突变体，我们能够映射到早期区域E4开放阅读框6的这一功能。在用重组AAV感染的细胞中，开放阅读框6蛋白的质粒表达使转导增加100倍至1，000倍。转导的增加不依赖于重组AAV基因盒，而是似乎涉及AAV生命周期的立即早期步骤。已经显示诱导野生型AAV的无辅助复制的化学和物理试剂也能够刺激重组AAV转导，表明该现象可能影响AAV DNA复制。进一步的实验表明，病毒脱壳不受影响，限速步骤涉及单链基因组AAV DNA上第二条链的合成。这些数据表明，腺病毒E4区，以及化学和物理试剂，可以在AAV生命周期的立即早期步骤中发挥重要作用，特别是在第二链合成中，并且对基因治疗方案中使用AAV载体具有重要意义。
The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.