br Materials and Methods br Author Contributions

Materials and Methods
Author Contributions
Conflicts of Interest
Acknowledgments This study was funded by the NIH Common Fund (UH2TR000943) through the Office of Strategic Coordination/Office of the NIH Director, an NCI/NIH grant (1R21CA199050), the MD Anderson Cancer Center support grant (P30CA016672), the American Cancer Society Research Professor Award, and The Center for RNA Interference and Non-Coding RNA. This work was partially supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC; 13345 to V.d.F.) and the Texas Center for Cancer Nanomedicine (U54CA151668 to D.G.G.) through a pilot grant awarded to D.E.V. and G.L.-B.
Introduction The recent outbreak of Zika Virus (ZIKV) in Brazil has raised important public health issues, particularly due to possible associations with neurological disorders including microcephaly and Guillian-Barré syndrome. ZIKV is a mosquito-transmitted flavivirus closely related to Dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Although ZIKV is transmitted by mosquitos, recent reports indicate the potential for male-to-female sexual transmission of the virus (Abushouk et al., 2016, Russell et al., 2017, Tang et al., 2016). Similar to other flaviviruses, ZIKV contains a positive single-stranded genomic RNA encoding a polyprotein that is processed into three structural proteins [capsid (C), precursor of membrane (prM) and envelope (E)] and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (da Fonseca et al., 2017). ZIKV is a membrane enveloped virus that requires fusion of the viral membrane with host cell membranes in order to cause infection. Entry of ZIKV to target rosmarinic acid receptor is coordinated by the E protein arrayed on the surface of the virion. Once the virion components have reached the cytoplasm, the NS proteins form a replication complex that synthesizes a negative-sense RNA, which subsequently serves as a template for the positive-sense RNA. The newly synthesized RNA is encapsidated, transported by the host secretory pathway, and released from the infected cell by exocytosis. The entry of ZIKV into mammalian cells is poorly understood, although evidence suggests that the membrane protein AXL contributes to this process by serving an attachment factor at the cell surface (Hamel et al., 2015, Liu et al., 2016, Meertens et al., 2017, Savidis et al., 2016). However, entry of ZIKV has been observed in human and mouse cells that do not express AXL (Miner et al., 2016, Rausch et al., 2017, Wells et al., 2016), suggesting that there may be additional factors that can perform the required functions for ZIKV entry on at least some types of cells. A recent study suggested that the interaction of the ZIKV virion with AXL is mediated by the AXL ligand, Gas6 (Meertens et al., 2017), which has also been shown to be involved in the entry of other viruses (Meertens et al., 2012, Morizono and Chen, 2014). In this scenario, Gas6 interacts with both the surface-exposed phosphatidylserine on the ZIKV virion and AXL on the surface of the cell, bridging the interaction of the ZIKV virion with AXL. These findings open the possibility that other proteins with the ability to bind to phosphatidyl-serine are contributing to the entry of ZIKV into different cell types, including those that do not express AXL.
Results
Discussion Using fibroblasts that were deficient in AXL expression, we demonstrated involvement of this protein in the ability of ZIKV to infect human cells. Our findings using HT1080 fibroblasts with deletion of AXL were consistent with other reports showing that knock outs of AXL expression in microgial cells (CHME3), glioblastoma cells (U87), or epithelial cells (HeLa) prevented the infection by ZIKVs (Chan et al., 2016, Coelho et al., 2017, Hastings et al., 2017, Meertens et al., 2017, Retallack et al., 2016, Savidis et al., 2016, Vicenti et al., 2018). Interestingly, with the exception of Hastings and colleagues whom rescue ZIKA infection of HeLa AXL KO cells by expressing the mouse AXL protein, none of these reports have rescued the infection by re-introducing the human AXL protein in KO cells. To remediate this, we rescued ZIKA infection in HT1080 cells knockout for the expression of AXL by expressing the human AXL protein(Fig. 3D). Altogether, these experiments suggested an essential role for AXL during ZIKV infection. By contrast, the literature also shows examples in which ablation of AXL expression does not affect ZIKV infection. For example, ablation of AXL in human neuroprogenitor cells (Wells et al., 2016), human placental cells (Rausch et al., 2017), or in mice (Govero et al., 2016, Miner et al., 2016), did not affect ZIKV infection. Overall these experiments suggested that ZIKVs might be using different attachment factors to enter cells. One possibility is that ZIKVs utilize different attachment factors such as AXL and a separate receptor that is involved in the fusion. It is important to point out that AXL binds to the Gas6 protein, which interacts with the phosphatidylserine in the viral membrane (Meertens et al., 2017), suggesting that AXL is an attachment factor. This raises the question whether the envelope protein of ZIKV, which contains the fusion machinery, interacts or not with a cellular protein that might be the receptor. Although in the case of flaviviruses is believed that the envelope does not require to interact with a cellular receptor for fusion (Stiasny and Heinz, 2006), the aforementioned controversy regarding ZIKV entry might suggest the existence of other attachment factors or receptors that may interact with the envelope protein. Future experiments in our laboratory will pursue whether the ZIKV envelope protein interacts with other membrane receptors.