Characterization of Zika Virus Endocytic Pathways in Human Glioblastoma Cells.

Mei Li,Di Zhang,Yalan Liu,Fengfeng Ni,Hanzhong Wang,Tao Du,Qinxue Hu,Mudan Zhang,Chuntian Li,Ming Fu,Zifeng Zheng,George E Griffin

doi:10.3389/fmicb.2020.00242

Abstract

Zika virus (ZIKV) infections can cause microcephaly and neurological disorders. However, the early infection events of ZIKV in neural cells remain to be characterized. Here, by using a combination of pharmacological and molecular approaches and the human glioblastoma cell T98G as a model, we first observed that ZIKV infection was inhibited by chloroquine and NH4Cl, indicating a requirement of low intracellular pH. We further showed that dynamin is required as the ZIKV entry was affected by the specific inhibitor dynasore, small interfering RNA (siRNA) knockdown of dynamin, or by expressing the dominant-negative K44A mutant. Moreover, the ZIKV entry was significantly inhibited by chlorpromazine, pitstop2, or siRNA knockdown of clathrin heavy chain, indicating an involvement of clathrin-mediated endocytosis. In addition, genistein treatment, siRNA knockdown of caveolin-1, or overexpression of a dominant-negative caveolin mutant impacted the ZIKV entry, with ZIKV particles being observed to colocalize with caveolin-1, implying that caveola endocytosis can also be involved. Furthermore, we found that the endocytosis of ZIKV is dependent on membrane cholesterol, microtubules, and actin cytoskeleton. Importantly, ZIKV infection was inhibited by silencing of Rab5 and Rab7, while confocal microscopy showed that ZIKV particles localized in Rab5- and Rab7-postive endosomes. These results indicated that, after internalization, ZIKV likely moves to Rab5-positive early endosome and Rab7-positive late endosomes before delivering its RNA into the cytoplasm. Taken together, our study, for the first time, described the early infection events of ZIKV in human glioblastoma cell T98G.

Highlights

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flavivirus genus, which includes other pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and yellow fever virus (YFV)
Using a combination of pharmacological and molecular approaches, we have demonstrated that both clathrin-dependent and clathrinindependent pathways can be involved in the ZIKV infection of glioblastoma T98G cells
To determine whether ZIKV infection before trafficking to the acidic vesicles was sensitive to the perturbation of lipid rafts, we evaluated the effects of MβCD, a drug selectively extracting cholesterol from the plasma membrane (Kilsdonk et al, 1995; Acosta et al, 2009), and filipin, a compound that binds selectively to cholesterol-rich microdomains on virus infection (Bolard, 1986)

Summary

Introduction

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flavivirus genus, which includes other pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and yellow fever virus (YFV). ZIKV was originally isolated from a sentinel monkey in the Zika Forest of Uganda (Dick et al, 1952), and the first human infections were reported in 1977. Characterization of ZIKV Endocytic Pathways in Central Java, Indonesia (Olson et al, 1981). The outbreak of ZIKV in French Polynesia and in Brazil, which expanded rapidly throughout South and Central America, raised a global health emergency (Avšic Županc and Petrovec, 2016; Bharucha and Breuer, 2016; Plourde and Bloch, 2016). Reports have revealed the capability of ZIKV to cross the human placental barrier and, to infect the developing central nervous system (CNS) (Calvet et al, 2016). ZIKV infection in unborn fetuses showed cerebral calcifications, microcephaly, and other congenital malformations (Brasil et al, 2016; Rasmussen et al, 2016). Neurological manifestation is known as Guillain–Barre syndrome, with symptoms of neuropathy and paralysis (Acosta et al, 2009; Oehler et al, 2014; Cao-Lormeau et al, 2016)

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