BACKGROUND/AIMS Recombinant adeno-associated virus (rAAV) is now in the clinic, yet production of rAAV remains problematic. We previously determined that human papillomavirus type 16 (HPV16) E1 protein boosts rAAV yields and E1 enhances AAV Rep78's replication-related biochemistries. Here, we deletion-mapped the helper domain within E1 to help glean its mechanism of action. METHODS Rep78-E1 interaction was analyzed by Gal4-based yeast two-hybrid (Y2H)-cDNA assay. rAAV DNA replication was studied by AAV/helper plasmid transfection into HEK293 cells and Southern blot. Gene expression analysis was made of AAV and E1 plasmid transfection, cDNA generation, and then quantitative polymerase chain reaction. NCBI protein BLAST was used for the homology analysis. RESULTS Gal4-Y2H- cDNA assay found in vivo Rep78-E1-binding activity across E1, but the carboxyl-third (amino acids [aa] 421-649) of E1 contained the predominant DNA replication helper domain. The amino-half of E1 (aa 1-337) inhibited transcription of rep (p5 promoter) and cap (p40, trending lower) from non-replicating helper plasmid by quantitative (q)RT-PCR. CONCLUSIONS The aa 421-649 helper domain of HPV16 E1 includes the ATP-binding/helicase region of E1 which boosts rAAV production and has homology with the analogous region of parvovirus NS-1/Rep78 by NCBI protein BLAST, suggesting these biochemistries are responsible for the mechanism of action in E1 helper function.

中文翻译：

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HPV16 E1羧基域为腺相关病毒复制提供了辅助功能。

背景技术/ AIMS重组腺相关病毒（rAAV）目前正在临床中，但生产rAAV仍然存在问题。我们先前确定，人类乳头瘤病毒16型（HPV16）E1蛋白可提高rAAV产量，而E1可增强AAV Rep78的复制相关生物化学。在这里，我们删除了E1中的辅助域的缺失映射，以帮助收集其作用机制。方法采用基于Gal4的酵母双杂交（Y2H）-cDNA分析法分析Rep78-E1的相互作用。通过AAV /辅助质粒转染入HEK293细胞和Southern印迹研究了rAAV DNA复制。对AAV和E1质粒转染，cDNA生成，然后定量聚合酶链反应进行基因表达分析。NCBI蛋白BLAST用于同源性分析。结果Gal4-Y2H- cDNA检测发现E1体内具有Rep78-E1结合活性，但E1的羧基的第三个羧基（氨基酸[aa] 421-649）包含主要的DNA复制辅助域。E1的氨基半部分（aa 1-337）通过定量（q）RT-PCR抑制了非复制型辅助质粒中rep（p5启动子）和cap（p40，趋于降低）的转录。结论HPV16 E1的aa 421-649辅助结构域包含E1的ATP结合/解旋酶区域，可增强rAAV的产生，并与NCBI蛋白BLAST细小病毒NS-1 / Rep78的类似区域具有同源性，表明这些生物化学负责E1辅助功能的作用机理。通过定量（q）RT-PCR从非复制型辅助质粒中获得更低的趋势）。结论HPV16 E1的aa 421-649辅助结构域包括E1的ATP结合/解旋酶区域，可增强rAAV的产生，并与NCBI蛋白BLAST细小病毒NS-1 / Rep78的类似区域具有同源性，表明这些生物化学负责E1辅助功能的作用机理。通过定量（q）RT-PCR从非复制型辅助质粒中获得更低的趋势）。结论HPV16 E1的aa 421-649辅助结构域包含E1的ATP结合/解旋酶区域，可增强rAAV的产生，并与NCBI蛋白BLAST细小病毒NS-1 / Rep78的类似区域具有同源性，表明这些生物化学负责E1辅助功能的作用机理。