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Master's Dissertation
DOI
https://doi.org/10.11606/D.5.2020.tde-27102020-151744
Document
Author
Full name
Amanda Schiersner Caodaglio
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Vettorazzo, Laura Cristina Sichero (President)
Lepique, Ana Paula
Rangel, Maria Cristina Rodrigues
Santos, Tiago Góss dos
Title in Portuguese
Caracterização da atividade transcricional de HPV-6
Keywords in Portuguese
Fatores de transcrição
Genoma viral
Papillomaviridae
Papillomavirus humano 6
Transcrição gênica
Verrugas
Abstract in Portuguese
As verrugas genitais (VG), a papilomatose respiratória recorrente e alguns raros casos de câncer estão associados à infecção pelo HPV-6. Variantes da sublinhagem B1 de HPV-6 estão associadas a um maior risco de desenvolvimento de VGs em homens em comparação às variantes da sublinhagem B3, o que poderia estar em parte associado à maior atividade transcricional da variante B1 quando comparada à variante B3. A transcrição viral é regulada por proteínas celulares e virais que se ligam à LCR e pouco foi descrito sobre a regulação da transcrição do HPV-6. Portanto, os objetivos deste trabalho foram identificar fatores de transcrição (FTs) que afetam a transcrição do HPV-6; identificar FTs que atuam distintamente na transcrição das variantes B1 e B3; determinar quais FTs se ligam à LCR; e determinar o impacto dos FTs sinergicamente na transcrição do HPV-6. Usando a tecnologia GFC-Transfection Array (Origene), mais de 700 plasmídeos de expressão de FTs foram transformados em bactérias E. coli e o DNA plasmidial isolado por minipreparação. Em seguida, células C33 foram co-transfectadas com plasmídeos de expressão de FTs e plasmídeos repórteres recombinantes LCR-Luc. Foi observado que 77 FTs ativaram ou reprimiram a LCR de pelo menos uma das variantes testadas. Entre estes, 9 FTs apresentaram potenciais sítios de ligação na LCR de ambas as variantes, baseado em análise in silico, e foram selecionados para ensaios de validação em que células C33 foram co-transfectadas com o plasmídeo repórter, além de quantidades crescentes dos plasmídeos de expressão de cada FT. Assim, verificou-se que SNAI2 e E2F1 reprimiram significativamente a atividade da LCR da variante B1, enquanto, HOXA13 ativou significativamente apenas a LCR dessa variante. Adicionalmente, ELF5 apresentou efeito transrepressor sobre a LCR de ambas as variantes testadas. Ensaios de co-transfecção de FTs para avaliar o impacto sinergético destes FTs sobre a LCR revelaram que quando HOXA13 é co-expresso com SNAI2 o efeito ativador e repressor destas proteínas, respectivamente, é anulado. Ensaios de imunoprocipitação de cromatina (ChIP) mostraram a ligação in vitro de HOXA13 e SOX7 à LCR de ambas as variantes. Estes resultados indicam um papel de HOXA13 na ativação diferencial da transcrição da variante pertencente a sublinhagem B1, em comparação com a atividade transcricional da variante B3. A identificação de FTs celulares que interagem diferencialmente com a LCR de diferentes variantes do HPV-6 poderia explicar, pelo menos em parte, as diferenças no risco de desenvolver VGs atribuídos a essas variantes e futuramente servir como um potencial alvo terapêutico
Title in English
Characterization of HPV-6 trancriptional activity
Keywords in English
Genome Viral
Human papillomavirus 6
Papillomaviridae
Transcription factors
Transcription genetic
Warts
Abstract in English
Genital warts (GW), recurrent respiratory papillomatosis and some rare cases of cancer are associated with HPV-6 infection. Variants of the HPV-6 B1 sublineage are associated with an increased risk of developing GWs in men compared to infections with variants of the B3 sublineage what could be at least in part associated to the higher transcriptional activity of the B1 variant compared to the B3 variant. Viral transcription is regulated by cellular and viral proteins that bind to LCR and little has been described about the regulation of HPV-6 transcription. Therefore, the objectives of the present study were to identify transcription factors (TFs) that affect HPV-6 transcription; identify TFs that act distinctly in the transcription of variants B1 and B3; determine which TFs bind to the LCR; and determine the impact of TFs synergistically on HPV-6 transcription. Using the GFC-Transfection Array (Origene) technology, more than 700 FTs expression plasmids were transformed into E. coli bacteria and the plasmidial DNA isolated by mini-preparation. Then, C33 cells were co-transfected with the TFs expression plasmids and recombinant reporter plasmids LCR-Luc. We observed that 77 TFs activated or suppressed the LCR of at least one of the tested variants. Among these, 9 TFs showed potential binding sites within the LCR of both variants, according to in silico analysis, and were selected for validation tests in which C33 cells were co-transfected with the reporter plasmid, in addition to increasing amounts of the expression plasmids of each TF. Thus, we verified that SNAI2 and E2F1 significantly repressed the LCR activity of the B1 variant, while HOXA13 activated significantly solely the LCR of this variant. Additionally, ELF5 showed a transrepressor effect upon the LCR of both variants tested. TFs co-transfection assays to assess the synergistic impact of these TFs upon the LCR revealed that when HOXA13 is co-expressed with SNAI2, the activation and repression effect of these proteins, respectively, is abolished. Chromatin immunoprocipitation (ChIP) assays showed the in vivo binding of HOXA13 and SOX7 to the LCR of both variants. These results indicate a role for HOXA13 in the differential transcriptional activation of the variant belonging to the B1 sublineage, The identification of cellular TFs that differentially interact with the LCR of different HPV-6 variants could explain, at least in part, the differences in the risk of developing GWs attributed to these variants and in the future serve as a potential therapeutic target
 
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Publishing Date
2020-10-27
 
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