Flavivirus internalization is regulated by a size-dependent endocytic pathway Brent A. Hacketta and Sara Cherrya,1 aDepartment of Microbiology, University of Pennsylvania, Philadelphia, PA 19104 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved March 1, 2018 (received for review November 16, 2017) Flaviviruses enter host cells through the process of clathrin- was dispensable for transferrin, suggesting that RNASEK may mediated endocytosis, and the spectrum of host factors required represent a protein required for particular cargo (15, 16). How- for this process are incompletely understood. Here we found that ever, it is unknown how RNASEK facilitates the internalization of lymphocyte antigen 6 locus E (LY6E) promotes the internalization these viruses and whether there are other factors that selectively of multiple flaviviruses, including West Nile virus, Zika virus, and promote viral endocytosis. dengue virus. Perhaps surprisingly, LY6E is dispensable for the Here we report that lymphocyte antigen locus 6 E (LY6E) is internalization of the endogenous cargo transferrin, which is also required for flavivirus infection at the level of entry. LY6E is a dependent on clathrin-mediated endocytosis for uptake. Since small (101 aa) GPI-anchored protein of the Ly6/uPAR super- viruses are substantially larger than transferrin, we reasoned that family of proteins that can be IFN-inducible (17, 18). Ectopic LY6E may be required for uptake of larger cargoes and tested this expression of LY6E can promote replication of the flavivirus using transferrin-coated beads of similar size as flaviviruses. LY6E yellow fever virus (19, 20). Additionally, a genome-wide siRNA was indeed required for the internalization of transferrin-coated screen revealed that knocking down LY6E reduces susceptibility beads, suggesting that LY6E is selectively required for large cargo. to West Nile virus infection (21). However, the mechanism by Cell biological studies found that LY6E forms tubules upon viral which LY6E promotes flavivirus infection was unknown. We infection and bead internalization, and we found that tubule found that LY6E facilitates infection of flaviviruses but not formation was dependent on RNASEK, which is also required for parainfluenza virus 5 (PIV5), which fuses at the plasma mem- flavivirus internalization, but not transferrin uptake. Indeed, we brane. Further, we find that LY6E is required for the in- MICROBIOLOGY found that RNASEK is also required for the internalization of ternalization of flavivirus virions, but is dispensable for the transferrin-coated beads, suggesting it functions upstream of internalization of canonical clathrin cargo, transferrin. To di- LY6E. These LY6E tubules resembled microtubules, and we found rectly test the role of size in this process, we coated beads of that microtubule assembly was required for their formation and similar size of flaviviruses with transferrin and found that LY6E flavivirus uptake. Since microtubule end-binding proteins link is specifically required for the internalization of this large cargo. microtubules to downstream activities, we screened the three Since we had previously found that RNASEK promoted in- end-binding proteins and found that EB3 promotes virus uptake ternalization of virions, but not transferrin, we also tested the and LY6E tubularization. Taken together, these results highlight a requirement for this factor in transferrin-bead uptake. We found specialized pathway required for the uptake of large clathrin- that RNASEK is also necessary for the uptake of these large dependent endocytosis cargoes, including flaviviruses. cargoes. Morphological studies revealed that virus infection in- duces tubularization of LY6E, which is dependent on RNASEK endocytosis | flavivirus | entry | virus | LY6E and microtubules. Moreover, disruption of microtubules, or the plus-end–binding, microtubule-associated protein EB3 abrogates lathrin-mediated endocytosis is a major endocytic route by LY6E tubule formation and attenuates viral entry. Taken to- Cwhich cells sample their environment and take up nutrients. gether, these data demonstrate that large cargoes, including Thus, many viruses, including flaviviruses, hijack this endocytic viruses, are dependent on additional cellular factors for their pathway to gain access to internal compartments of host cells (1– 3). Flaviviruses are a large group of reemerging viruses trans- Significance mitted to humans by mosquito species of global significance (4). West Nile virus (WNV) is widespread in the Americas, neuro- tropic, and can cause encephalitis (5, 6). Zika virus (ZIKV) is Flaviviruses are a globally important group of human viral newly emerging in the Americas and can also be neurotropic (7– pathogens. These viruses enter cells by hijacking an endocytic pathway, clathrin-mediated endocytosis, used by cells to take 9). Dengue virus (DENV) is reemerging globally, infecting up growth factors and nutrients. While most cargo is small, 350 million people annually (10). Presently, there are no specific virions are large, and we identified host factors specifically antiviral treatments or vaccines approved to treat these viruses. required for the internalization of large cargo including these Clathrin-mediated endocytosis is a tightly orchestrated path- viruses. These studies define a set of requirements for viral way whereby cargoes bound to surface receptors are internalized internalization and which may be amenable to therapeutic using a number of well-characterized cellular factors (reviewed interventions. in ref. 11). Indeed, for those viruses that depend on clathrin- mediated endocytosis for entry, the classically known canonical Author contributions: B.A.H. and S.C. designed research; B.A.H. performed research; components are required to facilitate this process (12, 13). B.A.H. contributed new reagents/analytic tools; B.A.H. and S.C. analyzed data; and However, most of our understanding of this cellular pathway B.A.H. and S.C. wrote the paper. comes from studies on endogenous cargoes, such as transferrin, The authors declare no conflict of interest. which are an order of magnitude smaller than viral particles. This article is a PNAS Direct Submission. Studies on vesicular stomatitis virus found that actin, which is This open access article is distributed under Creative Commons Attribution-NonCommercial- dispensable for transferrin uptake, is required for internalization NoDerivatives License 4.0 (CC BY-NC-ND). of this virus (14). Therefore, it is likely that there are additional 1To whom correspondence should be addressed. Email: [email protected]. requirements for the internalization of flavivirus particles. Indeed, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RNASEK, a recently identified ∼100-aa transmembrane protein, 1073/pnas.1720032115/-/DCSupplemental. was found to be required for the internalization of flaviviruses, but www.pnas.org/cgi/doi/10.1073/pnas.1720032115 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 uptake and that elucidation of this process may aid in the increased permissivity (Fig. 1D). These data suggest that LY6E future development of new classes of broad-spectrum antiviral promotes flavivirus entry. therapies. To directly assay entry we implemented a RT-qPCR–based assay we previously developed that allows us to quantitatively Results assess viral binding and uptake (15). Briefly, we prebind virus to LY6E Promotes Flavivirus Infection. Previous studies suggested that cells at 4 °C, wash off unbound virus, and monitor the levels of LY6E promotes infection of multiple flaviviruses (20, 21). LY6E bound virus by RT-qPCR. Using this assay, we found that WNV- is ubiquitously expressed and in some studies has been shown to KUN binding to cells is not affected by RNAi against LY6E be IFN-inducible (17, 18). However, in human osteosarcoma or clathrin light chain (CLTC), which is known to be required for E cells (U2OS) we found that LY6E is basally expressed and internalization (Fig. 1 ). Next, we monitored virus internaliza- largely insensitive to IFN stimulation (Fig. S1). We next tested tion in cells prebound with virus by subsequently incubating the the role of LY6E in WNV infection. First, we transfected either cells at 37 °C for 4 h. We remove the surface-bound virus with trypsin and quantify the internalized virus. Under these condi- nontargeting control or LY6E-directed siRNAs and found that tions we found that depletion of either LY6E or the control we could deplete LY6E mRNA (Fig. 1A). We then challenged – CLTC leads to significant reduction in virus internalization these cells with WNV Kunjin, as it is closely related to circu- (Fig. 1F). lating strains of WNV in the United States (98% identical) and can be used under BSL2 conditions (22). We found that there is LY6E and RNASEK Facilitate Internalization of Large Endocytic Cargo. a significant decrease in WNV-KUN RNA at 8 hours post- Since LY6E promotes WNV internalization, which is dependent infection (hpi) as measured by RT-qPCR (Fig. 1B) and protein on clathrin-mediated endocytosis, we reasoned that LY6E may at 24 hpi as measured by immunoblot (Fig. 1C). We next took also play a role in canonical clathrin-mediated endocytosis. advantage of cells that express a WNV replicon with a GFP re- Therefore, we tested whether LY6E is required for uptake of porter in place of the structural proteins (23). We found that transferrin—a well-characterized