Differences in Transcriptional Activity of Human Papillomavirus Type 6 Molecular Variants in Recurrent Respiratory Papillomatosis

Abstract

A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied.

Regulation of HPV transcription

“…Since a significant association between variants of the B1 sublineage and the development of GWs was recently described, it is possible to hypothesize that the increased transcriptional efficiency could impact the increased expression of viral E1 and E2, which can confer B1 variants to have an increased replication potential 106 . Moreover, the results support a crucial role of the ELF1 protein in the lower transcriptional activity observed for the B3 variant 105 . A correlation between the transcriptional activity of specific HPV-11 variants and the clinical aggressiveness of RRP has also been reported 107 .…”

“…Genome variability of low-risk HPVs 6 and 11 has also been evaluated, and molecular variants of both viral types were phylogenetic grouped 102 - 104 . It was reported that a variant from the HPV-6 B1 sublineage is ten times more transcriptionally active than a B3 variant is 105 ( Figure 2B ). Since a significant association between variants of the B1 sublineage and the development of GWs was recently described, it is possible to hypothesize that the increased transcriptional efficiency could impact the increased expression of viral E1 and E2, which can confer B1 variants to have an increased replication potential 106 .…”

Associação de variantes moleculares de HPV-6 com o desenvolvimento de lesões genitais externas em homens participantes no estudo HIM

“…Cabe ressaltar que os achados são independentes da origem geográfica ou idade dos participantes. O número de amostras incluídas e o fato deste estudo acompanhar uma coorte possivelmente explicam por que em outros trabalhos associações clínicas com variantes específicas de HPV-6 não foram encontradas (Heinzel et al,1995;Measso do Bonfim et al, 2015).…”

“…At each biannual visit, 3 different prewetted Dacron swabs were used to sample the external genitalia (coronal sulcus, glans penis, penile shaft) and scrotum of participants; these swabs were further combined to form a single sample [17]. DNA extraction was performed using the Qiagen Media kit (Qiagen, Venlo, The Netherlands), and HPV detection and typing was conducted using the Linear Array HPV Genotyping Test (Roche Molecular Diagnosis, CA) capable of identifying 37 HPV types classified as high risk 18,31,33,35,39,45,51,52,56,58,59,68) or low risk 11,26,40,42,53,54,55,61,62,64,66,67,69,70,71,72,73,81,82,IS39,83,84,89).…”

“…Participants could also attend the clinic for an intervening visit whenever a suspicious lesion was present; 66 of the 102 EGLs collected in this study were collected at these intermediate visits. DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissue biopsies were extracted using the QIAamp DNA FFPE Tissue kit (Qiagen), and HPV detection and genotyping was conducted using the INNO-LiPA HPV Genotyping Extra assay (Fujirebio, Göteborg, Sweden), which detects 28 viral types classified as high risk 18,31,33,35,39,45,51,52,56,58,59,68) or low risk 11,26,40,43,44,53,54,66,69,70,71,73,74,82). DNAs were considered adequate if tested positive for β-globin or any HPV type.…”