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    • Using our infection assays we also

    2022-11-18

    Using our infection assays, we also explored whether low-pH compartments were required for the entry of ZIKVs in mammalian cells. Lysosomotropic agents such as ammonium chloride, chloroquine and Bafilomycin A1, strongly inhibited the ability of ZIKVs to enter mammalian cells. Our results are in agreement with the findings of others suggesting that ammonium chloride and chloroquine blocks ZIKVs infection (Delvecchio et al., 2016; Li et al., 2017; Rausch et al., 2017). These experiments suggested that ZIKVs infection require a low pH compartment and that ZIKVs are endocytosed before the viral membrane fuses with the cellular membrane. Our findings are in agreement with the notion that the entry of other flaviviruses such as dengue, Tick-borne encephalitis and West Nile viruses demonstrating a requirement for low pH during entry (Gollins and Porterfield, 1986, Randolph and Stollar, 1990, Smit et al., 2011, Vorovitch et al., 1991). Our results suggested that ZIKV entry requires low pH for fusion and involves endocytosis, which has been shown to be important for cell entry of flaviviruses (Pierson and Kielian, 2013). To determine the kinetics of entry of ZIKV into mammalian cells, we prebound viruses to the surface of protoporphyrin ix receptor and stopped infection by adding a lyssomotropic agent at the indicated times. This allowed us to calculate a t1/2 of 2–4 h for ZIKVs to reach the fusion compartment from the cell surface, illustrating the time required by the particle to reach the fusion compartment. Cortese and colleagues have shown that the virus requires a total time of 3 h to reach the fusion compartment in the human hepatic cell line Huh7 (Cortese et al., 2017). One possibility is that the differences may be due to that we are comparing different cell lines. After contacting the cell surface, viruses that require low pH for infection, undergo receptor-mediated endocytosis. Here we tested whether clathrin mediated endocytosis is important for ZIKVs entry. Interestingly, the use of chlorpromazine, which is a cationic amphiphilic drug that disrupts clathrin-mediated uptake by relocating clathrin and adaptor protein 2 complexes from the cell surface (Pho et al., 2000, Wang et al., 1993), completely blocks the infection by ZIKVs. These experiments suggested that clathrin-mediated endocytosis is involved in the ability of ZIKVs to enter mammalian cells, which is in agreement with similar findings for ZIKVs and other flaviviruses such as dengue (Meertens et al., 2017, Pierson and Kielian, 2013van der Schaar et al., 2008). In summary our work suggested that ZIKVs require AXL, low pH and endocytosis for infection of mammalian cells.
    Materials and methods
    Acknowledgements
    Introduction The receptor tyrosine kinase Axl, a 140 kDa protein, is a member of the TAM (Tyro3, Axl, and Mer) family.1, 2 Axl is ubiquitously expressed in various cancers, including breast cancer, and overexpression of Axl has been reported to be correlated with increased invasiveness in preclinical models.4, 5 Recent studies have shown that Axl plays an important role in several chemotherapy-resistant cancers.6, 7 Therefore, Axl has been considered as a new potential therapeutic target for cancer treatments. Axl is activated through several mechanisms, including binding of its ligand, growth arrest specific 6 (Gas6), extracellular domain mediated dimerization, and cross-talk with human epidermal growth factor receptor 2 (HER-2).8, 9, 10 Axl activation has been associated with several signal transduction pathways, including PI3K, MAP kinases, STAT, and NF-κB.11, 12, 13 Axl has also been shown to be a downstream effector of epithelial-to-mesenchymal transition (EMT). EMT is characterized by the down-regulation of E-cadherin in adherence junctions, down-regulation of occludin and claudin in tight junctions, up-regulation of mesenchymal proteins vimentin, N-cadherin, and fibronectin and activation of transcription factors Snail, Slug, Zeb1, Zeb2, and Twist.14, 15, 16 Among these factors, vimentin is regarded as a major canonical marker of EMT. Our previous study demonstrated that vimentin expression is significantly associated with poor prognosis in triple-negative breast cancer (TNBC). Vimentin has also been shown to be required for induction of Axl expression. The association between Axl and vimentin expression has been observed in cell lines; however, the association between Axl and vimentin expression, as well as the impact on prognosis in breast cancer tissue, has not been fully determined.