Subcellular localization of Zika virus during infection in human cells

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.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

PARP1 Characterization as a Potential Biomarker for <i>BCR::ABL1</i> p190+ Acute Lymphoblastic Leukemia

Detection of t(9;22), and consequent BCR::ABL1 fusion, is still a marker of worse prognosis for acute lymphoblastic leukemia (ALL), with resistance to tyrosine-kinase inhibitor therapy being a major obstacle in the clinical practice for this subset of patients. In this study, we investigated the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL, the most common isoform to afflict ALL patients, and demonstrated the use of experimental PARP inhibitor (PARPi), AZD2461, as a therapeutic option with cytotoxic capabilities similar to that of imatinib, the current gold standard in medical care. We characterized cytostatic profiles, induced cell death, and biomarker expression modulation utilizing cell models, also providing a comprehensive genome-wide analysis through an aCGH of the model used, and further validated PARP1 differential expression in samples of ALL p190+ patients from local healthcare institutions, as well as in larger cohorts of online and readily available datasets. Overall, we demonstrate the effectiveness of PARPi in the treatment of BCR::ABL1 p190+ ALL cell models and that PARP1 is differentially expressed in patient samples. We hope our findings help expand the characterization of molecular profiles in ALL settings and guide future investigations into novel biomarker detection and pharmacological choices in clinical practice