HPV16 and HPV18 type-specific APOBEC3 and integration profiles in different diagnostic categories of cervical samples

Abstract

Human papillomavirus (HPV) 16 and 18 are the most predominant types in cervical cancer. Only a small fraction of HPV infections progress to cancer, indicating that additional factors and genomic events contribute to the carcinogenesis, such as minor nucleotide variation caused by APOBEC3 and chromosomal integration. We analysed intra-host minor nucleotide variants (MNVs) and integration in HPV16 and HPV18 positive cervical samples with different morphology. Samples were sequenced using an HPV whole genome sequencing protocol TaME-seq. A total of 80 HPV16 and 51 HPV18 positive samples passed the sequencing depth criteria of 300× reads, showing the following distribution: non-progressive disease (HPV16 n = 21, HPV18 n = 12); cervical intraepithelial neoplasia (CIN) grade 2 (HPV16 n = 27, HPV18 n = 9); CIN3/adenocarcinoma in situ (AIS) (HPV16 n = 27, HPV18 n = 30); cervical cancer (HPV16 n = 5). Similar numbers of MNVs in HPV16 and HPV18 samples were observed for most viral genes, with the exception of HPV18 E4 with higher numbers across clinical categories. APOBEC3 signatures were observed in HPV16 lesions, while similar mutation patterns were not detected for HPV18. The proportion of samples with integration was 13% for HPV16 and 59% for HPV18 positive samples, with a noticeable portion located within or close to cancer-related genes.

Differences in integration frequencies and APOBEC3 profiles of five high-risk HPV types adheres to phylogeny

“…However, this decrease is not observed in HPV31 and HPV33 positive samples, suggesting this is not an Alpha-9 specific tendency, but rather a feature of HPV16 carcinogenesis. Our results are in corroboration with previous studies that have observed that the number of APOBEC3-related nucleotide substitutions decreases with lesion severity in HPV16 positive samples [ 17 , 20 , 43 ].…”

“…Yet, contrary to current understanding it cannot be established that APOBEC3-induced mutagenesis in viral genomes is a general detectable feature in high-risk HPV infections. In HPV16 positive samples, the number of APOBEC3-related nucleotide substitutions decreases with lesion severity, which also has been shown in previous studies [ 17 , 20 , 43 ]. However, this decrease is not observed in HPV31 and HPV33 positive samples, suggesting this is not an Alpha-9 specific tendency, but rather a feature of HPV16 carcinogenesis.…”

“…Validation of integration sites for HPV16 and HPV18 is previously described [ 43 ]. The Illumina reads from the respective HPV31, HPV33 and HPV45 sequencing reactions were used to make in silico DNA templates for design of integration-targeting primers suited for PCR and Sanger sequencing.…”

“…By comparison, the remaining carcinogenic HPV-types are understudied. HPV16 and HPV18 have shown differences in integration frequencies and APOBEC3 interaction, suggesting that HPV-induced cancer development follow dissimilar type-dependent routes [ [43] , [44] , [45] ]. Within the Alpha-PVs, HPV16 sorts under Alpha-9 together with HPV31 and HPV33, while HPV18 and HPV45 sort under Alpha-7.…”

“…In this study, we aim to investigate genomic variability and chromosomal integration in cervical cell samples with different morphologies positive for HPV31, HPV33, and HPV45 utilizing the TaME-seq protocol [ 46 ]. Additionally, this study will include comparisons to reanalysed HPV16 and HPV18 data from a previous study [ 43 ] to gain a more comprehensive understanding of specific characteristics of the distinct clades Alpha-7 (HPV18 and HPV45) and Alpha-9 (HPV16, HPV31 and HPV33). A study going deeper into the nature of genomic events in these lesser studied carcinogenic HPV types allows for a phylogenetic approach to better understand the molecular mechanisms of host-responses to infections and those responsible for HPV-induced carcinogenesis.…”