Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

Abstract

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

“…Although it was difficult to detect a small population of variants in the host genome using a traditional approach such as PCR, NGS, owing to its depth, enabled us to detect a novel genomic variation (27). NGS has strongly supported the studies of viral genetic diversity, especially in RNA viruses (28,29), whereas the detection by NGS of quasispecies in DNA virus has been reported less frequently (30)(31)(32)(33). Using PCR analysis, the presence of VP1 quasispecies has been reported in polyomavirus BK (BKV) (34).…”

The Biological Impact of Genomic Diversity in Cervical Cancer Development

“…Studies that analyzed the near full HPV16 genome, using deep sequencing, did not observe any deletion event in the invasive cervical cancer samples analyzed [39,48]. However, a significant increase in read depth close to L1 and L2, respectively, compatible with a duplication event of a genome fragment spanning these regions was observed [39].…”

“…Intrahost next-generation sequencing data have shown that HPV16 episomes present in the W12 cell line did not accumulate sequence variation above 0.5% [48]. However, the same authors reported that more than 40% of the clinical samples contained polymorphic sites reaching frequencies up to 5% [48].…”

Genetic Variations of Human Papillomavirus Type 16: Implications for Cervical Carcinogenesis

“…To comprehensively explore the quasispecies status of the HPV16 genome in individual clinical specimens, the mutational landscape in the full-length HPV16 genome was investigated with NGS technology (36). In that study, whole HPV16 genomes were amplified with onestep, long-range PCR from HPV16-positive clinical samples of either CIN1 or ICC, and analyzed by deep sequencing.…”

“…The physiological relevance of HPV16 hypermutation in the viral life cycle and cervical carcinogenesis has yet to be determined, because hypermutation in E2 and the LCR in clinical samples seems to be extremely rare in total viral populations, insofar as deep sequencing analysis was unable to detect hypermutated HPV16 sequences at frequencies of more than 0.5z (36). One intriguing possibility is that hypermutation is involved in the integration of HPV into the host genome, a critical event in the generation of cervical cancer, because E2 is a``hot spot'' for HPV integration and is frequently disrupted by integration (50).…”