Differential Expression of HPV16 L2 Gene in Cervical Cancers Harboring Episomal HPV16 Genomes: Influence of Synonymous and Non-Coding Region Variations

Paramita Mandal,Damayanti Das Ghosh,Nidhu Ranjan Mondal,Sharmila Sengupta,Bornali Bhattacharjee,Sudipta Roy,Rahul Roy Chowdhury,Rui Medeiros

doi:10.1371/journal.pone.0065647

Abstract

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4–6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1–2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

Highlights

The association of genital human papillomavirus (HPV) with cervical cancer (CaCx) is strong and independent of other risk factors, as evident from the consistent findings recorded from epidemiologic studies conducted in several countries [1]
Our study exemplifies the biological relevance of synonymous sequence variations as well as those variations that are located within non-coding regions of HPV16 genomes, in CaCx pathogenesis
The L2 gene appears to be the hot-pot of such variations, culminating into multiple routes employed by episomal HPV16 in CaCx cases to sustain L2 expression in a lineage specific manner

Summary

Introduction

The association of genital human papillomavirus (HPV) with cervical cancer (CaCx) is strong and independent of other risk factors, as evident from the consistent findings recorded from epidemiologic studies conducted in several countries [1]. In India HPV16 infection is the most predominant type associated with CaCx [4,5,6] and is the most prevalent type identified in the general populations based on data available from some regions of India [5,6,7,8,9]. The transforming potential of HPVs are likely to be correlated with the potential of deregulating the expression of key viral proteins [12,13,14], as well as, with the ability to avoid immune attack by the host in order to persist within the host cervical epithelium [15]

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Discussion

Conclusion