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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2018.tde-13092018-105525
Document
Author
Full name
Roberta Maraninchi Silveira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Silva, Luis Lamberti Pinto da (President)
Arruda Neto, Eurico de
Figueiredo, Luiz Tadeu Moraes
Hinds, Luciana Barros de Arruda
Title in Portuguese
Localização subcelular do vírus da Zika durante a infecção em células humanas
Keywords in Portuguese
Calnexina; Célula U251; ESCRT; Flavivírus; TGN46; TSG101; ZIKV
Abstract in Portuguese
O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas.
Title in English
Subcellular localization of Zika virus during infection in human cells
Keywords in English
Calnexine; ESCRT; Flavivirus; TGN46; TSG101; U251 cell; ZIKV
Abstract in English
Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.
 
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Publishing Date
2018-10-02
 
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