Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression.

中文翻译：

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hnRNP L以Akt激酶依赖性方式控制HPV16 RNA多聚腺苷酸化和剪接

通过降低HPV16早期多腺苷酸化和激活HPV16晚期L1 mRNA剪接，抑制Akt激酶可激活HPV16晚期基因表达。我们在早期polyA信号和HPV16晚期mRNA的剪接位点上确定了RNA结合蛋白的“热点”。我们观察到hnRNP L与所有HPV16晚期剪接位点和早期polyA信号的序列相关。Akt激酶抑制导致hnRNP L去磷酸化，并减少hnRNP L与HPV16 mRNA的关联。这伴随着U2AF65和Sam68与HPV16 mRNA的结合增加。此外，siRNA敲低hnRNP L或Akt诱导了HPV16基因表达。用Akt激酶抑制剂治疗HPV16永生化角质形成细胞可减少hnRNP L与HPV16 mRNA的结合并诱导HPV16 L1 mRNA的产生。最后，HPV16亚基因组表达质粒中hnRNP L结合位点的缺失导致HPV16晚期基因表达被激活。总之，Akt激酶通过控制RNA结合蛋白hnRNP L在RNA加工水平上抑制HPV16晚期基因的表达。我们推测Akt激酶活性维持了有利于HPV16早期基因表达的细胞内环境，并抑制了HPV16晚期基因表达。