Characterization of Zika Virus Endocytic Pathways in Human Glioblastoma Cells

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 NH 4 Cl, 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 dominantnegative 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 Rab7positive 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.

Novel Nucleoside Analogues as Effective Antiviral Agents for Zika Virus Infections

“…In particular, our early findings in the case of DENV serotype 2 suggest 5a as an inhibitor of autophagy and micropinocytosis mechanisms, which are relevant to DENV and other flaviviruses [ 26 , 27 ]. This inhibitory effect would likely affect the replication of other flaviviruses , which enter host cells via multiple pathways, including micropinocytosis, as has been demonstrated for ZIKV infections in glioblastoma cells [ 28 ].…”

“…The selected compounds were first evaluated at a fixed 10 μM concentration for their potential inhibition of ZIKV-induced cytopathic effect in a MTS based TCID 50 assay [ 37 ], using cell lines which are physiologically relevant for ZIKV infection, including HUH7 hepatoma cells [ 38 ], dbtrg cells [ 28 ], and A549 cells, which support Zika virus infection in vitro [ 39 ]. The cell lines used were chosen to reflect the pathogenesis of ZIKV in liver (HUH7) and CNS tissues (dbtrg), in addition to a cell line where the ZIKV strain originally isolated in Uganda in 1947 grows and forms plaques particularly well (A549).…”

Antiviral activity of chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine towards RNA-viruses. A review

“… Li et al (2020) showed that chlorpromazine in the non-toxic concentrations in T98G cells inhibits ZIKV cell infection in a dose-dependent manner without influencing JEV copies of viral RNA ( Li et al, 2020 ).…”

The Large Action of Chlorpromazine: Translational and Transdisciplinary Considerations in the Face of COVID-19

“…Moreover, transfection of cells with small interfering RNAs specific to clathrin heavy chain can also inhibit viral gene expression, indicating the essential role of clathrin in viral entry (PuZhang, 2008). Similarly, entry of Zika virus has also been shown to be significantly inhibited by CPZ (Li et al, 2020). CPZ slows or suppresses the replication of alphaviruses, hepatitis C virus, SARS-CoV-1, and MHV-2.…”