APOBEC3 (A3) mutation signatures have been observed in a variety of human cancer genomes, including those of cervical and head and neck cancers caused by human papillomavirus (HPV) infection. However, the driving forces that promote off-target A3 activity remain mostly unclear. Here, we report a mechanism for the dramatic increase of A3A protein levels in HPV-positive keratinocytes. We show that expression of the viral protein E7 from high-risk HPVs, but not E7 from low-risk HPVs, significantly prolongs the cellular half-life of A3A protein in human keratinocytes and HPV-positive cancer cell lines. We have mapped several residues within the cullin 2 (CUL2) binding motif of HPV16 E7 as being important for mediating A3A protein stabilization. Furthermore, we provide direct evidence that both A3A and HPV16 E7 interact with CUL2, suggesting that the E7-CUL2 complex formed during HPV infection may regulate A3A protein levels in the cell. Using an in vitro cytidine deaminase assay, we show that E7-stabilized A3A remains catalytically active. Taken together, our findings suggest that the HPV oncoprotein E7 dysregulates endogenous A3A protein levels and thus provides novel mechanistic insight into cellular triggers of A3 mutations in HPV-positive cancers.

IMPORTANCE Human papillomavirus (HPV) is causally associated with over 5% of all human malignancies. Several recent studies have shown that a subset of cancers, including HPV-positive head and neck and cervical cancers, have distinct mutational signatures potentially caused by members of the APOBEC3 cytidine deaminase family. However, the mechanism that induces APOBEC3 activity in cancer cells is poorly understood. Here, we report that the HPV oncoprotein E7 stabilizes the APOBEC3A (A3A) protein in human keratinocytes by inhibiting ubiquitin-dependent protein degradation in a cullin-dependent manner. Interestingly, the HPV E7-stabilized A3A protein maintains its deaminase activity. These findings provide a new insight into cancer mutagenesis enhanced by virus-induced A3A protein stabilization.

APOBEC3 (A3) 突变特征已在多种人类癌症基因组中观察到，包括由人乳头瘤病毒 (HPV) 感染引起的宫颈癌和头颈癌。然而，促进 A3 活性脱靶的驱动力仍不清楚。在这里，我们报告了 HPV 阳性角质形成细胞中 A3A 蛋白水平急剧增加的机制。我们发现，来自高危 HPV 的病毒蛋白 E7 的表达，而非来自低危 HPV 的 E7，可显着延长人角质形成细胞和 HPV 阳性癌细胞系中 A3A 蛋白的细胞半衰期。我们已经绘制了 HPV16 E7 的 cullin 2 (CUL2) 结合基序内的几个残基，这些残基对于介导 A3A 蛋白稳定非常重要。此外，我们提供了 A3A 和 HPV16 E7 与 CUL2 相互作用的直接证据，表明 HPV 感染期间形成的 E7-CUL2 复合物可能调节细胞中的 A3A 蛋白水平。使用体外胞苷脱氨酶测定，我们表明 E7 稳定的 A3A 仍然具有催化活性。总而言之，我们的研究结果表明，HPV 癌蛋白 E7 失调内源性 A3A 蛋白水平，从而为 HPV 阳性癌症中 A3 突变的细胞触发因素提供了新的机制见解。

重要性人乳头瘤病毒 (HPV) 与超过 5% 的人类恶性肿瘤有因果关系。最近的几项研究表明，一部分癌症，包括 HPV 阳性头颈癌和宫颈癌，具有可能由 APOBEC3 胞苷脱氨酶家族成员引起的独特突变特征。然而，人们对癌细胞中诱导 APOBEC3 活性的机制知之甚少。在此，我们报道 HPV 癌蛋白 E7 通过以 cullin 依赖性方式抑制泛素依赖性蛋白降解来稳定人角质形成细胞中的 APOBEC3A (A3A) 蛋白。有趣的是，HPV E7 稳定的 A3A 蛋白保持其脱氨酶活性。这些发现为病毒诱导的 A3A 蛋白稳定增强癌症诱变提供了新的见解。