Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada.
J Virol. 2014 Apr;88(7):3653-63. doi: 10.1128/JVI.03263-13. Epub 2014 Jan 8.
Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis.
This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.
使用质谱分析法，我们鉴定出人乳头瘤病毒 16（HPV16）E7 的结合伴侣 p190RhoGAP（p190）。p190 属于 GTP 酶激活蛋白（GAP）家族，是 RhoA 的主要 GAP 之一。GAPs 刺激 Rho 蛋白的内在 GTP 酶活性，导致 Rho 失活，并影响众多生物学过程。RhoA 是研究最为透彻的 Rho 蛋白之一，特异性参与形成黏附斑和应力纤维，从而调节细胞迁移和细胞铺展。由于这是 E7 与 p190 结合的首次报道，我们进行了详细的相互作用研究。我们发现，其他 HPV 类型的 E7 蛋白也与 p190 结合。此外，我们发现 E7 的保守区 3（CR3）和 p190 的中间结构域对这种相互作用很重要。更具体地说，我们鉴定出 E7 的 CR3 中的两个残基是与 p190 结合所必需的，并使用具有这些残基突变的 E7 突变体来确定 E7-p190 相互作用的生物学后果。我们的数据表明，E7 与 p190 的相互作用使这种 GAP 失活，并改变了肌动蛋白细胞骨架。我们还发现，这种相互作用负调节细胞在纤维连接蛋白底物上的铺展，因此可能对 HPV 生命周期或 HPV 诱导的肿瘤发生的重要方面有贡献。
本研究鉴定出 p190RhoGAP 是 HPV E7 蛋白的一种新型细胞结合伴侣。我们的研究表明，大量不同的 HPV E7 蛋白与 p190RhoGAP 结合，并且鉴定出 E7 和 p190RhoGAP 中对相互作用发生很重要的区域。本研究还强调了 E7-p190RhoGAP 相互作用可能与感染细胞中肌动蛋白组织有重要的生物学后果的可能性。这些变化可能是 HPV 感染细胞中病毒生命周期和向癌症进展的重要贡献者。重要的是，这项工作还强调了在 HPV 生命周期和 HPV 诱导转化方面，需要进一步研究这一大部分尚未探索的领域。
