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Previews mechanism that presumably prevents dimerization and subsequent downstream activation. In addition, this study could have far-reaching consequences because in an age of antibiotic resistance, alternative targets of bacterial pathogens are being sought to treat or prevent disease (Dickey et al., 2017). Not only does this study uncover lipoyl-E2-PDH as a candidate factor to target, but by boosting support for the outside moonlighting hypothesis in general, Grayczyk et al. (2017) may refocus attention to target a cohort of ‘‘cytoplasmic’’ proteins that mischievously moonlight outside of cells.
ACKNOWLEDGMENTS This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (1ZIA-AI000904-17) and the Postdoctoral Research Associate Program of the National Institute of General Medical Sciences (1FI2GM11999101) in the U.S. National Institutes of Health. REFERENCES
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Breaking the Barrier: Host Cell Invasion by Lujo Virus Stefan Kunz1 and Juan Carlos de la Torre2,* 1Institute
of Microbiology, Lausanne University Hospital, Lausanne, Switzerland of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.chom.2017.10.014 2Department
Lujo virus (LUJV) is an arenavirus that emerged in 2008 associated with a cluster of human cases of severe hemorrhagic fever. In this issue of Cell Host & Microbe, Raaben et al. (2017) identify neuropilin (NRP)-2 as cell surface receptor and the tetraspannin protein CD63 as intracellular entry factor for LUJV. Viral hemorrhagic fevers (VHFs) are severe human diseases associated with high mortality. Human VHFs are zoonotic infections caused by a range of emerging RNA viruses of the filo-, arena-, flavi-, and bunyavirus family. Upon breaking the species barrier, these viruses are capable of igniting chains of human-to-human transmission, which could result in explosive outbreaks, as illustrated by the devastating Ebola virus (EBOV) epidemic in Western Africa in 2013–2016. Host cell attachment and entry represent the first and necessary steps for a successful virus infection. To execute these early steps, viruses critically depend on host cell factors, including receptors, co-receptors, and other entry factors. Dissecting the complex interaction of a virus with cellular factors implicated in host cell en-
try can help to predict the risk of zoonotic transmission, define tissue tropism, and assess disease potential. Haploid genetic screens have emerged as a powerful approach to identify host cell factors involved in productive infection of infectious agents, including major HF viruses, such as EBOV (Carette et al., 2011) and Lassa virus (LASV) (Jae et al., 2014). These studies have identified host cell factors required for productive infection and uncovered mechanisms of virus cell entry that shaped concepts in virus-host cell interactions. Arenaviruses are enveloped viruses with a bi-segmented negative strand RNA genome that include several agents of severe VHF. In nature, arenaviruses are carried by persistent infection of rodent hosts and human infection occurs
mainly via reservoir-to-human transmission, but inter-human transmission can also occur with case fatality rates >50%. Lujo virus (LUJV), named after the cities of Lusaka and Johannesburg, was identified in 2008 as an arenavirus associated with a cluster of fatal human VHFs in Southern Africa (Briese et al., 2009). Sequence comparison revealed that this virus was a distant relative of the Old World arenaviruses LASV and lymphocytic choriomeningitis virus (LCMV) (Briese et al., 2009). The index case was a young female living in Lusaka, Zambia, who acquired the virus by an unknown route and developed signs and symptoms of a severe viral infection. Emergency airlift evacuation to Johannesburg initiated a chain of inter-human transmission including three
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Previews healthcare workers and a janitor (Briese et al., 2009). The index patient and three of the contacts died within 10–12 days after disease onset, representing a case-fatality rate of 80%. The clinical disease associated with LUJV VHF resembled severe Lassa fever (Sewlall et al., 2014), with early unspecific flu-like symptoms, followed by gastrointestinal involvement and hallmarks of severe infection involving facial and neck swelling, rash, and rapid deterioration toward shock associated with multi-organ system failure. Similar to Lassa fever, hemorrhages were not a salient feature of fatal LUJV VHF, but in contrast to Lassa fever, LUJV VHF had a more abrupt onset and was characterized by stronger coagulopathy, involving disseminated intravascular coagulation (DIC), which is rarely seen in LASV infection (Sewlall et al., 2014). Infectious LUJV was isolated from the blood of the second case and a reverse genetic system established, opening the avenue of structure-function studies. Experimental infection of LUJV in non-human primates (NHPs), the ‘‘gold standard’’ animal model for experimental pathology of LASV, did not result in fatal disease, suggesting important speciesspecific differences between LUJV and LASV disease potential. Initial studies on cell entry of LUJV revealed that the virus was independent of dystroglycan (DG) and human transferrin receptor 1 (TFRC), cellular receptors used by pathogenic Old World and New World arenaviruses, respectively (Tani et al., 2014), which was consistent with the highly divergent sequence of LUJV envelope glycoprotein (GP)-1 implicated in arenavirus-host cell binding (Briese et al., 2009). In their elegant study, Raaben et al. (2017) generated a recombinant vesicular stomatitis virus expressing LUJV GP (rVSV-LUJVGP) and used it in a genome-wide haploid screen for LUJV cell entry factors. The rationale for this approach was that arenavirus cell attachment and entry are mediated exclusively by the viral surface GP complex, and thereby infection with rVSV-LUJVGP recreates LUJV GP-mediated cell entry, whereas the VSV core allows cytopathic effects to be used as a readout. After confirming that entry of LUJV into haploid human (HAP)-1 cells was independent of DG and TfR1, the authors infected >100
million HAP-1 cells bearing retroviral insertion mutations with rVSV-LUJVGP, selected cells resistant to VSV-induced killing, and mapped sites of retroviral insertion by parallel deep sequencing. Similar to a previous haploid screen (Jae et al., 2014), hits included a prominent gene cluster involved in heparan sulfate biosynthesis, which serves as an attachment factor for many viruses. Other prominent hits included neuropilin (NRP)-2, a cell surface receptor for semaphorins, and the tetraspannin protein CD63, which, based on their subcellular location and known biological function, were of particular interest as candidate entry factors for LUJV. The authors used a variety of assays to demonstrate a role for NRP2 as a cell surface receptor for LUJV. Virus binding to NRP2 is of high affinity and involves the N-terminal domain of NRP2. Interestingly, NRP2 is highly expressed on microvascular endothelial cells, and the consequent high susceptibility of the microvasculature to LUJV may explain, at least in part, the extent of coagulopathy observed in LUJV clinical disease (Sewlall et al., 2014). Likewise, expression of NRP2 on alveolar macrophages may facilitate aerosol transmission of LUJV, a proposed route of transmission (Sewlall et al., 2014). NRP2 and its homolog NRP1 serve as receptors for semaphorins and can interact with vascular endothelial growth factor (VEGF). Notably, NRP1 was recently identified as an entry factor for the g-herpesvirus Epstein-Barr virus (EBV) into nasopharyngeal epithelial cells, where it promotes viral uptake via macropinocytosis (Wang et al., 2015). Intriguingly, while NPR1 promoted EBV entry involving receptor tyrosine kinase activation, NRP2 impaired EBV infection. NRP1 is also involved in cell entry of human T cell lymphotropic virus (HTLV)-1, where it acts in concert with heparan sulfate (Lambert et al., 2009). Specifically, the surface protein of HTLV-1 mimics VEGF and interacts with NRP1 and heparan sulfate, which act as viral receptors in a cooperative manner. It will be of interest to see if heparan sulfate contributes also to the role of NRP2 in LUJV cell entry. In contrast to the cell surface receptor NRP2, CD63 localizes to the multivesicular body and the late endosome. Overexpression of a CD63 variant that localized to the cell surface facilitated LUJV GP-
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mediated fusion induced by low pH in a cell-based assay. This finding, together with the observed co-localization of CD63 with incoming virus at the level of late endosomes, suggested a role of CD63 in viral fusion. The identification of CD63 as a candidate intracellular entry factor for LUJV draws interesting parallels to cell entry of LASV, another HF arenavirus. Engagement of cell surface receptors by LASV is followed by the internalization of the virus-receptor complex via endocytosis and delivery to acidified late endosomes, where viral fusion occurs under low pH (<5.5). Haploid screens identified the late endosomal resident protein LAMP1 as a crucial entry factor for cell entry of LASV, and functional studies revealed a modus operandi that involves a late endosomal ‘‘receptor switch’’ (Jae et al., 2014). Upon delivery to late endosomes, the low luminal pH induces dissociation of LASV from its surface receptor, followed by engagement of the late endosomal entry factor LAMP-1. Structural studies revealed the existence of a stable low pH conformation of LASV GP1 displaying a triad of histidine residues that form a binding site for LAMP-1. Engagement of the late endosomal receptor LAMP1 requires protonation of critical residues within the histidine triad of LASV GP1, which orchestrates the ‘‘receptor switch’’ and assures optimal spatial conditions for fusion triggering (Cohen-Dvashi et al., 2016). Among Old World arenaviruses, the use of LAMP-1 is a unique feature of the highly pathogenic LASV (Israeli et al., 2017), suggesting a possible link to pathogenesis. Interestingly, exposure to low pH within the lumen of late endosomes resulted in dissociation of LUJV from NRP2, supporting a similar ‘‘receptor switch’’ scenario in the late endosome (Figure 1). In contrast to LAMP1, which binds LASV GP1 with high affinity under low pH conditions (Jae et al., 2014), no direct interaction of LUJV GP1 and CD63 was detected so far. This may be explained by either a transient interaction of the virus with CD63 at the late endosome or the involvement of another, yet unknown component involved in LUJV fusion triggering. The findings reported by Raaben et al. have uncovered that two HF arenaviruses, LASV and LUJV, use distinct
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ACKNOWLEDGMENTS
LUJV LUJV
This research was supported by Swiss National Science Foundation grants 310030_149746 and 310030_170108 to S.K. and NIH/NIAID grants AI047140 and AI077719 to J.C.T.
GP2 GP1 Endocytosis Acidification
a1
NRP2
a2
a1 a2
REFERENCES
Dissociation
NRP2 NRP2
pH < 5.5
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Late endosome
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Binding
CD63
CD63
CD63
Fusion triggering
Figure 1. LUJV Cell Entry Interaction at the cell surface between LUJV GP1 and NRP2 mediates LUJV endocytosis. The acidic environment of the late endosome promotes dissociation of the trimeric GP1 from NRP2, which allows the late endosome resident protein CD63 to trigger fusion between the viral membrane and the limiting membrane of the late endosome.
host cell factors to execute a similar cell entry strategy. Differences in the natural reservoir between LASV, Mastomys natalensis, and LUJV, currently unknown, may have contributed to selection of different host cell factors for cell entry. LUJV illustrates how zoonotic transmissions of novel highly pathogenic viruses are likely to be future recurrent events. The advent of powerful next-generation sequencing greatly facilitates the rapid and accurate identification of viruses involved in these events. Because cell en-
try is a major barrier for reservoir-tohuman and inter-human transmission, haploid screens for cell entry factors exemplified by the present study will be invaluable to assess the risk of zoonotic emergence and the capacity to ignite human-to-human transmission. Cellular factors required for viral entry represent important host susceptibility factors and attractive targets for the development of anti-viral drugs aimed at blocking the pathogen before it can take control of the host cell.
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