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

Iwao Kukimoto,Seiichiro Mori,Makoto Kuroda,Rika Kusumoto-Matsuo,Toshiyuki Yamaji,Yoshiyuki Ishii,Tomohiko Maehama,Takamasa Takeuchi,Fumihiko Takeuchi,Tsuyoshi Sekizuka,Yumiko Ogasawara,Kazunari Kondo,Kentaro Hanada,Robert D Burk

doi:10.1371/journal.pone.0080583

Iwao Kukimoto, Seiichiro Mori + Show 12 more

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https://doi.org/10.1371/journal.pone.0080583

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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.

Highlights

Human papillomaviruses (HPVs) are small DNA viruses having a circular double-stranded DNA genome of approximately 8-kb, some of which can induce benign and malignant hyper-proliferative lesions in the skin or mucosa [1]
Using the primer-pair 1742F/1873R and PrimeSTAR® GXL polymerase, we amplified two DNA fragments from extrachromosomal HPV type 16 (HPV16) DNA isolated from W12 cells that were originally isolated from an low-grade squamous intraepithelial lesion (LSIL) biopsy (Figure 1C)
In this study, utilizing a recently developed long PCR enzyme, PrimeSTAR® GXL polymerase, we have succeeded in amplifying full-length HPV16, HPV52, and HPV58 genome sequences, using DNA isolated from clinical specimens, in a single reaction

Summary

Introduction

Human papillomaviruses (HPVs) are small DNA viruses having a circular double-stranded DNA genome of approximately 8-kb, some of which can induce benign and malignant hyper-proliferative lesions in the skin or mucosa [1]. When infected cells migrate from the basal layer and begin the process of differentiation, viral genome amplification is initiated, followed by the induction of viral capsid expression and the assembly of infectious virions in the upper layers of epithelia [3]. This differentiation-dependent propagation of HPVs can manifest clinically as low-grade squamous intraepithelial lesion (LSIL). In contrast to the episomal state in their normal life-cycle, DNAs of high-risk HPVs are frequently found integrated into the host genome in invasive cervical cancer (ICC) [4,5]. Viral DNA integration leads to enhanced expression of two viral transforming genes, E6 and E7, which play critical roles in carcinogenesis [6,7]

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