Shao Yunting, Wang Peng, Zheng Yunji, Cui Hongtu, Lou Zhangrong, Li Shanhu, Huang Fang, Wu Chengjun
School of Biomedical Engineering, Dalian University of Technology, Dalian, China.
Department of Cell Engineering, Beijing Institute of Biotechnology, Beijing, China.
Front Microbiol. 2023 Aug 30;14:1259510. doi: 10.3389/fmicb.2023.1259510. eCollection 2023.
In past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function.
We initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity.
We successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7.
The successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.
在过去几十年中，高危型人乳头瘤病毒（HR-HPV）感染在癌症发病机制中的作用已得到广泛研究。癌前细胞中表达的病毒E7蛋白已被确定为免疫干预的理想靶点。然而，HPV的培养仍然是一项重大挑战，同时缺乏表达HPV E7蛋白和产生具有复制能力的重组病毒颗粒的方法，这对进一步探索E7癌蛋白的功能和致癌机制构成了主要障碍。因此，迫切需要研究构建表达HPV E7蛋白的具有复制能力的重组病毒颗粒的新方法，以促进对其功能的研究。
我们利用ccdB-Kan正向/反向筛选系统结合Red/ExoCET重组系统启动重组病毒颗粒的构建。我们用获得的重组病毒感染C33A细胞，以使HPV16 E7持续表达。之后，提取总RNA并使用RNA-Seq技术进行转录组测序，以鉴定与HPV诱导的致癌性相关的差异表达基因。
我们成功建立了稳定持续表达HPV16 E7的复制性重组病毒颗粒。用重组病毒颗粒感染C33A细胞以实现E7蛋白的过表达。随后，进行RNA-Seq分析以评估宿主细胞基因表达的变化。结果显示CD36基因上调，其与HPV诱导的致癌途径相关，包括PI3K-Akt和p53信号通路。qRT-PCR分析进一步确定，HPV16 E7的表达导致CD36基因上调。
HPV16 E7在细胞中的成功表达表明，复制的重组病毒保留了Ad4的复制和感染能力，同时还上调了参与PI3K-Akt信号传导和p53途径的CD36基因，从而促进细胞增殖。本研究结果提供了新的视角，为进一步探索HPV相关致癌作用以及开发能够诱导针对HPV的保护性免疫的复制性HPV重组疫苗奠定了坚实基础。
