RNASEK is required for internalization of diverse acid-dependent viruses Brent A. Hacketta, Ari Yasunagaa, Debasis Pandaa, Michael A. Tartella, Kaycie C. Hopkinsa, Scott E. Hensleyb, and Sara Cherrya,1 aDepartment of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and bVaccine Center, Wistar Institute, Philadelphia, PA 19104 Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved May 15, 2015 (received for review December 16, 2014) Viruses must gain entry into cells to establish infection. In general, The molecular mechanisms involved in these uptake mecha- viruses enter either at the plasma membrane or from intracellular nisms are complex and rely on key molecules and organelles that endosomal compartments. Viruses that use endosomal pathways are essential for cellular viability, as these uptake mechanisms are dependent on the cellular factors that control this process; bring nutrients and other metabolites into the cytosol for cellular however, these genes have proven to be essential for endogenous growth and survival. Indeed, these processes and proteins in- cargo uptake, and thus are of limited value for therapeutic inter- volved are highly conserved from yeast to humans (14, 15). vention. The identification of genes that are selectively required Depending on the virus entry requirements, some viruses fuse for viral uptake would make appealing drug targets, as their in- within early endosomal vesicles, whereas others traffic to more hibition would block an early step in the life cycle of diverse viruses. acidic compartments or macropinosomes for entry. Because At this time, we lack pan-antiviral therapeutics, in part because many viruses are dependent on these endosomal trafficking of our lack of knowledge of such cellular factors. RNAi screening pathways for entry, much effort has been made in identifying the has begun to reveal previously unknown genes that play roles in specific cellular genes required for viral entry (16). Therapeutics viral infection. We identified dRNASEK in two genome-wide RNAi targeting entry are appealing because it is the first step in the screens performed in Drosophila cells against West Nile and Rift infection cycle, and many viruses use common pathways; thus, Valley Fever viruses. Here we found that ribonuclease kappa inhibition may be broadly antiviral, rather than active against (RNASEK) is essential for the infection of human cells by divergent only a specific virus. Furthermore, many viruses have high mu- MICROBIOLOGY and unrelated positive- and negative-strand-enveloped viruses from tation rates and rapidly evolve resistance to therapeutics tar- the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae geting virally encoded genes. Conversely, therapeutics against families that all enter cells from endosomal compartments. In con- host encoded targets would likely be more difficult for the virus trast, RNASEK was dispensable for viruses, including parainfluenza to evade. virus 5 and Coxsackie B virus, that enter at the plasma membrane. Recent advances in functional genomic technologies have fa- RNASEK is dispensable for attachment but is required for uptake cilitated the use of unbiased genome-wide RNAi screens to of these acid-dependent viruses. Furthermore, this requirement ap- identify cellular genes required for viral infection (17). Such pears specific, as general endocytic uptake of transferrin is unaf- approaches allow for the discovery of otherwise unknown genes fected in RNASEK-depleted cells. Therefore, RNASEK is a potential that play essential roles in infection. We recently performed such ’ host cell Achilles heel for viral infection. screens in insect cells against two disparate insect-borne human pathogens: the flavivirus West Nile virus (WNV) and the arbovirus | entry | endocytosis | clathrin-mediated endocytosis | bunyavirus RVFV (18, 19). These are both arthropod-borne macropinocytosis human pathogens for which there are no vaccines or therapeu- tics. Furthermore, these viruses are quite divergent: WNV is a iral pathogens are quite diverse in their replication strate- flavivirus that is a globally important cause of encephalitis (20), Vgies; however, all viruses must enter cells to initiate their replication cycles. The first step involves binding of virus parti- Significance cles to the cell surface. Such interactions can involve attachment factors, which have low affinity but concentrate viruses on the Many viruses, including those of global concern, are dependent surface of cells, and receptors that intricately interact with viral on internalization for their entry. We found that ribonuclease envelope glycoproteins, which, in addition to binding, promote kappa (RNASEK) is required for infection of every virus we other aspects of infection such as internalization. Although a tested that enters cells through an acid-dependent pathway, plethora of receptors and pathways can be used, most viruses including dengue, West Nile, Sindbis, Rift Valley Fever, and in- take advantage of the cellular endocytic machinery and pene- fluenza viruses. Mechanistically, we found that RNASEK has no trate from within the cytosol (reviewed in refs. 1–3). Clathrin- effect on virus binding to cells but, rather, is required for their mediated endocytosis, macropinocytosis, and caveolin-mediated uptake. RNASEK was required for diverse viruses that are de- endocytosis are the best-studied forms of uptake used by viruses. pendent on clathrin-mediated endocytosis for entry, but we Clathrin-mediated endocytosis is the most common mechanism found that RNASEK was dispensable for general endocytic used by small viruses, as clathrin-coated vesicles have a diameter – uptake. Therefore, RNASEK appears to play a unique role in of 60 200 nm and can be enlarged to fit even larger particles (4, viral uptake and may be a therapeutically viable target to 5). This pathway is constitutive on most cells, and some viruses inhibit major human viral pathogens. use preexisting clathrin-coated pits for entry (e.g., dengue virus), whereas others induce the formation of these structures (e.g., Author contributions: B.A.H. and S.C. designed research; B.A.H., A.Y., D.P., M.A.T., and influenza virus) (6, 7). Macropinocytosis is an actin-dependent K.C.H. performed research; S.E.H. contributed new reagents/analytic tools; B.A.H. and S.C. endocytic process for the nonselective uptake of nutrients in analyzed data; and B.A.H. and S.C. wrote the paper. response to receptor engagement. It is the predominant pathway The authors declare no conflict of interest. for many larger viruses, including vaccinia virus, but is also used This article is a PNAS Direct Submission. by others, including influenza virus under some conditions (8– 1To whom correspondence should be addressed. Email: [email protected]. 10). It also remains unclear which pathways are used by some This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. viruses, including Rift Valley Fever virus (RVFV) (11–13). 1073/pnas.1424098112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1424098112 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 and RVFV is a bunyavirus that causes significant morbidity and RNASEK Promotes Flavivirus Infection in Human Cells. RNASEK is a mortality in livestock and humans in Africa (21). In our screens, highly conserved protein with 51% homology between Drosophila there were only three genes that promoted infection by both viruses: and humans (25). Therefore, we tested whether these flaviviruses dRAB5, dSTX7, and dRNASEK (CG40127). The functions of require hRNASEK for infection in human cells. We obtained two RAB5 and STX7 have been described, but little is known about independent siRNAs against hRNASEK and found that they ef- ribonuclease kappa (RNASEK). Both RAB5 and STX7 are in- ficiently deplete hRNASEK in human osteosarcoma cells (U2OS), volved in endosomal transport and have roles in viral entry (22, 23). as measured by RT-qPCR (Fig. 1D). We challenged these cells Both WNV and RVFV are enveloped RNA viruses that require an with WNV (KUN) or DENV2 (NGC) and monitored infection by acidic compartment for entry (12, 13, 24). RNASEK is a single- RT-qPCR or automated microscopy. We found that depletion of copy, 137-aa protein conserved from insects to humans (25–27) hRNASEK significantly attenuates infection of these flaviviruses in with an unknown function. We set out to determine the role of human cells (Fig. 1 E and F and Fig. S2 A and B). Again, we ob- RNASEK in viral infection and found that RNASEK is required served no change in cell number or growth properties of hRNA- for internalization of a diverse panel of viruses of medical concern. SEK-depleted cells (Fig. S2C). We also tested a different human cell line, HEK293T cells, and found that depletion of hRNASEK Results is efficient in these cells and attenuates WNV (KUN) infection RNASEK Promotes Flaviviral Infection in Insect Cells. Because we had (Fig. S3A). Our initial studies monitored infection of WNV at a identified dRNASEK as promoting infection in our genome- late point postinfection, so we next tested whether depletion of wide RNAi screens, we set out to verify this outside of a hRNASEK affects infection at early points. We depleted screening format with independent RNAi reagents. First, we hRNASEK in U2OS cells and monitored infection by WNV verified that we efficiently depleted dRNASEK, as measured by (KUN) 8 hours post infection (hpi), and