Department of Obstetrics and Gynecology, Showa University School of Medicine, Tokyo, Japan.
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
J Virol. 2018 May 29;92(12). doi: 10.1128/JVI.00017-18. Print 2018 Jun 15.
Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied by the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here, we explored within-host genetic diversity of HPV by performing deep-sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52, and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) and were deep sequenced. After constructing a reference viral genome sequence for each specimen, nucleotide positions showing changes with >0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, = 56; CIN2/3, = 68; ICC, = 27), with various numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the trinucleotide context encompassing substituted bases revealed that TpCpN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions. HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep-sequencing analyses, we show for the first time a comprehensive snapshot of the within-host genetic diversity of high-risk HPVs during cervical carcinogenesis. Quasispecies harboring minor nucleotide variations in viral whole-genome sequences were extensively observed across different grades of CIN and cervical cancer. Among the within-host variations, C-to-T transitions, a characteristic change mediated by cellular APOBEC cytosine deaminases, were predominantly detected throughout the whole viral genome, most strikingly in low-grade CIN lesions. The results strongly suggest that within-host variations of the HPV genome are primarily generated through the interaction with host cell DNA-editing enzymes and that such within-host variability is an evolutionary source of the genetic diversity of HPVs.
持续性感染致癌型人乳头瘤病毒（HPV）可导致宫颈癌，同时宿主基因组中也会积累体细胞突变。在病毒感染过程中，HPV 基因组也会发生伴随的遗传变化；然而，人们对其与宫颈癌发生的相关性知之甚少。在这里，我们通过对来自宫颈上皮内瘤变（CIN）和浸润性宫颈癌（ICC）患者宫颈脱落细胞中总细胞 DNA 进行 HPV 全基因组序列的深度测序分析，探索了 HPV 体内的遗传多样性。通过针对每种标本的特异性 PCR 扩增 HPV 16、52 和 58 型的全基因组，并对其进行深度测序。在为每个标本构建参考病毒基因组序列后，确定了与参考序列相比，频率 >0.5%的核苷酸位置发生变化的个体样本。总共在 151 个样本（CIN1， = 56；CIN2/3， = 68；ICC， = 27）的 HPV 基因组中检测到 1052 个核苷酸变异位置，每个样本的数量各不相同。总体而言，所有组织学分级中均以 C 到 T 和 C 到 A 取代为主。虽然 C 到 T 转换主要在 CIN1 中检测到，但在 CIN2/3 中其发生率降低，并且在 ICC 中低于 C 到 A 颠换。对包含取代碱基的三核苷酸上下文的分析表明，TpCpN，细胞 APOBEC 胞嘧啶脱氨酶的首选靶序列，是 HPV 基因组中 C 到 T 取代的主要部位。这些结果强烈表明，APOBEC 蛋白是 HPV 基因组突变的驱动因素，尤其是在 CIN1 病变中。HPV 表现出惊人的高水平遗传多样性，包括每种病毒基因型中大量的次要基因组变异。在这里，通过进行深度测序分析，我们首次展示了高危型 HPV 在宫颈癌发生过程中体内遗传多样性的全面快照。在不同分级的 CIN 和宫颈癌中，广泛观察到携带病毒全基因组中次要核苷酸变异的准种。在体内变异中，C 到 T 转换，一种由细胞 APOBEC 胞嘧啶脱氨酶介导的特征性变化，在整个病毒基因组中均有显著检测，在低级别 CIN 病变中最为明显。结果强烈表明，HPV 基因组的体内变异主要是通过与宿主细胞 DNA 编辑酶的相互作用产生的，并且这种体内变异性是 HPV 遗传多样性的进化来源。
