Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2013 Apr;87(8):4571-83. doi: 10.1128/JVI.03443-12. Epub 2013 Feb 13.
Interactions with cellular stress pathways are central to the life cycle of many latent viruses. Here, we utilize adeno-associated virus (AAV) as a model to study these interactions, as previous studies have demonstrated that cellular stressors frequently increase transduction of recombinant AAV (rAAV) vectors and may even substitute for helper virus functions. Since several chemotherapeutic drugs are known to increase rAAV transduction, we investigated the effect of arsenic trioxide (As(2)O(3)), an FDA-approved chemotherapeutic agent with known effects on several other virus life cycles, on the transduction of rAAV. In vitro, As(2)O(3) caused a dose-dependent increase in rAAV2 transduction over a broad range of cell lines from various cell types and species (e.g., HEK-293, HeLa, HFF hTERT, C-12, and Cos-1). Mechanistically, As(2)O(3) treatment acted to prevent loss of virions from the perinuclear region, which correlated with increased cellular vector genome retention, and was distinguishable from proteasome inhibition. To extend our investigation of the cellular mechanism, we inhibited reactive oxygen species formation and determined that the As(2)O(3)-mediated increase in rAAV2 transduction was dependent upon production of reactive oxygen species. To further validate our in vitro data, we tested the effect of As(2)O(3) on rAAV transduction in vivo and determined that treatment initiated transgene expression as early as 2 days posttransduction and increased reporter expression by up to 10-fold. Moreover, the transduction of several other serotypes of rAAV was also enhanced in vivo, suggesting that As(2)O(3) affects a pathway used by several AAV serotypes. In summary, our data support a model wherein As(2)O(3) increases rAAV transduction both in vitro and in vivo and maintains perinuclear accumulations of capsids, facilitating productive nuclear trafficking.
许多潜伏病毒的生命周期都与细胞应激途径的相互作用有关。在这里，我们利用腺相关病毒（AAV）作为模型来研究这些相互作用，因为先前的研究表明，细胞应激因子经常会增加重组 AAV（rAAV）载体的转导，甚至可以替代辅助病毒的功能。由于几种化疗药物已知会增加 rAAV 的转导，因此我们研究了三氧化二砷（As(2)O(3)），一种已被批准用于治疗癌症的药物，对几种其他病毒生命周期都有影响，对 rAAV 转导的影响。在体外，As(2)O(3)在广泛的细胞系中，从不同的细胞类型和物种（如 HEK-293、HeLa、HFF hTERT、C-12 和 Cos-1），引起了剂量依赖性的 rAAV2 转导增加。从机制上讲，As(2)O(3)处理可防止病毒粒子从核周区丢失，这与细胞内载体基因组的保留增加相关，并且与蛋白酶体抑制作用不同。为了扩展我们对细胞机制的研究，我们抑制了活性氧的形成，并确定 As(2)O(3)介导的 rAAV2 转导增加依赖于活性氧的产生。为了进一步验证我们的体外数据，我们测试了 As(2)O(3)对体内 rAAV 转导的影响，并确定治疗在转导后 2 天即可启动转基因表达，并将报告基因的表达增加了 10 倍。此外，体内还增强了几种其他血清型 rAAV 的转导，这表明 As(2)O(3)影响了几种 AAV 血清型使用的途径。总之，我们的数据支持了一种模型，即 As(2)O(3)在体外和体内均能增加 rAAV 的转导，并维持衣壳在核周的聚集，促进有效的核运输。
