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Master's Dissertation
DOI
10.11606/D.42.2017.tde-08052017-142231
Document
Author
Full name
Juliana Kaori Ogawa
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Ventura, Armando Morais (President)
Botosso, Viviane Fongaro
Strauss, Bryan Eric
Title in Portuguese
Caracterização da interação entre o metilossomo e o nucleocapsídeo do vírus respiratório sincicial humano.
Keywords in Portuguese
Interação célula-vírus
Metilossomo
Nucleoproteína
PRMT5
Vírus respiratório sincicial humano
Abstract in Portuguese
Neste projeto caracterizamos a interação da nucleoproteína viral (N) do Vírus Respiratório Sincicial Humano (HRSV) com as proteínas PRMT5 e WDR77, que constituem o metilosomo celular. Confirmamos que essa interação ocorre através de co-imunoprecipitação em células humanas, e de interação in vitro dessas proteínas purificadas. Demonstramos a co-localização dessas proteínas na célula através de microscopias de imunofluorescência e confocal. Inibindo ou aumentando a expressão de PRMT5 não observamos impacto na replicação viral. Verificamos que ocorre metilação em N tanto em resíduos de argininas como de lisinas, com anticorpos específicos para essas modificações, e por espectrometria de massas, indicando significado funcional. Com essa evidência testamos o efeito de inibidores de metilação e demetilação, em argininas e lisinas. Obtivemos efeito inibitório significativo da replicação do HRSV com um inibidor de metilação de lisina, UNC0646, indicando que a interação N-metilossomo tem potencial como alvo terapêutico contra HRSV.
Title in English
Characterization of humam respiratory syncytial virus nucleoprotein and methylosome interaction.
Keywords in English
Human respiratory syncytial virus
Methylossome
Nucleoprotein
PRMT5
Virus-cell interaction
Abstract in English
In this project we had as objective to characterize the interaction observed previously in the laboratory of viral nucleoprotein (N) with PRMT5 and WDR77 proteins that constitute the cell metilosome. We confirmed that this interaction occurs through co-imunoprecipitation in human cells and in vitro interaction of these purified proteins. We also demonstrated the co-localization of these proteins in inclusion bodies, by immunofluorescence and confocal microscopy. Inhibiting or enhancing PRMT5 expression we didnt see effect on viral replication. Our results show that methylation occurs in both arginine and lysine residues, through reactivity with antibodies specific to these modifications, and analysis by mass spectrometry. We tested the effect of arginine and lysine methylation and de-methylation inhibitors in viral replication. We obtained significant inhibitory effect on HRSV replication with a lysine methylation inhibitor, UNC0646, indicating that N-metilossome interaction has the potential to be exploited as a therapeutic target in developing antiviral drugs.
 
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Release Date
2019-05-08
Publishing Date
2017-05-08
 
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