Author response: Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature. https://doi.org/10.7554/eLife.11785.001 eLife digest Ebola virus and other filoviruses can cause devastating diseases in humans and other apes. Numerous small outbreaks of Ebola virus disease have occurred in Africa over the past 40 years. However, in 2013–2015, the largest outbreak on record took place in three Western African nations with no previous history of the disease. Human outbreaks of Ebola virus disease likely begin when a person encounters an infected wild animal. Though it remains unclear precisely which animals harbor Ebola virus between outbreaks, and how they transmit the virus to humans or other primates, recent work showed that some filoviruses do infect specific types of bats in nature. Ng, Ndungo, Kaczmarek et al. sought to identify the genes that influence whether or not a type of bat is susceptible to infection by Ebola virus and other filoviruses. Several filoviruses, including Ebola virus, were tested to see if they could infect cells that had been collected from four types of African fruit bats. These bats are all found in areas where outbreaks have occurred in the past. The tests revealed that a small change in the sequence of the NPC1 gene in some bat cells greatly reduced their susceptibility to Ebola virus. NPC1 encodes a protein that mammals need in order to move cholesterol within their cells. In humans, the loss of the protein encoded by NPC1 causes a rare but very severe disease called Niemann-Pick type C disease. This protein also turns out to be a receptor that the filoviruses must bind to before they can infect the cells. Further analysis then revealed that NPC1 has evolved rapidly in bats, with changes concentrated in the parts of the receptor that interact with Ebola virus. Ng, Ndungo, Kaczmarek et al. went on to discover some changes in the genome sequence of Ebola virus that could compensate for the changes in the bat’s NPC1 gene. These findings hint at one way that a filovirus could evolve to better infect a host with receptors that were less than optimal. Following on from this work, the next challenges will be to expand the investigation to include additional types of bats, other types of mammals, and other host genes that could influence filovirus infection and disease. Further studies could also examine the other side of the arms race – that is, the evolution of viral genes in bats. However, such studies would be complicated by the lack of viral sequences that have been collected from bats, because to date most have been isolated from humans and other primates instead. https://doi.org/10.7554/eLife.11785.002 Introduction Ebola virus (EBOV) and some of its relatives in the family Filoviridae (filoviruses) cause sporadic outbreaks of a highly lethal disease. These outbreaks are thought to be initiated by viral spillover from an animal reservoir to a highly susceptible accidental host, such as a human or nonhuman primate (Feldmann and Geisbert, 2011; Leroy et al., 2005; Towner et al., 2009). Recent work suggests that some filoviruses infect bats in nature, and that these viruses may be distributed more widely than previously recognized. Very short RNA fragments corresponding to portions of ebolavirus genomes were detected in several frugivorous bats of the family Pteropodidae (‘Old World fruit bats’) in both Africa and Asia (Leroy et al., 2005; Jayme et al., 2015), and longer filovirus RNA fragments and near-complete RNA genomes were isolated from insectivorous Schreibers's long-fingered bats in Asia and Europe, respectively (Negredo et al., 2011; He et al., 2015). However, despite considerable efforts, infectious ebolaviruses have never been recovered from bats. By contrast, Marburg (MARV) and Ravn (RAVV) viruses were found to circulate in Egyptian rousettes (Rousettus aegyptiacus), indicating that these bats are susceptible to MARV/RAVV and encounter them frequently in nature. Egyptian rousettes have been proposed as natural hosts for these viruses (Amman et al., 2012; Towner et al., 2009). This progress notwithstanding, many key questions remain. For example, the biological factors that influence filovirus host range and interspecies transmission are still poorly understood, as are the virus-host relationships that determine which species of bats are susceptible to infection by EBOV and other filoviruses. Viral entry receptors are key determinants of tissue tropism and host range (Radoshitzky et al., 2008; Sheahan et al., 2008; Hueffer et al., 2003; Demogines et al., 2013). Niemann-Pick C1 (NPC1), a highly conserved endo/lysosomal protein involved in cellular cholesterol trafficking, was recently identified to be an essential entry receptor for all known filoviruses (Côté et al., 2011; Carette et al., 2011; Miller et al., 2012; Ng et al., 2014). In this study, we uncover a pattern of virus and host species specificity in the filovirus susceptibility of bat cells, which can be explained by changes in the affinity of the essential interaction between NPC1 and the filovirus entry glycoprotein, GP. Crucially, genetic analyses reveal that NPC1 is under positive selection in bats, with a strong signature of selection at precisely the same residue that influences the filovirus-receptor interaction. Our findings suggest that amino acid sequence changes in NPC1 at these positively-selected sites represent host adaptations to resist filovirus infection, and reveal one pathway by which a filovirus could escape from receptor control. In sum, our results support the hypothesis that bats and filoviruses have been engaged in a long-term co-evolutionary relationship, one facet of which is a molecular arms race between the viral glycoprotein and its entry receptor, NPC1. Results African straw-colored fruit bat cells are selectively refractory to EBOV infection We first explored the possibility that there exist virus- and/or bat species-dependent differences in the cellular host range of filoviruses. Kidney fibroblast cell lines derived from three African pteropodids whose ranges overlap the locations of known African filovirus disease outbreaks (Figure 1A,B) were exposed to authentic EBOV and MARV (Figure 1C). We observed a large EBOV infection defect in African straw-colored fruit bat (Eidolon helvum) cells but not in cells from Büttikofer’s epauletted fruit bats (Epomops buettikoferi) and Egyptian rousettes. By contrast, cells from bats of all three species were similarly susceptible to infection by MARV (Figure 1C). Thus, cells from African straw-colored fruit bats appear to be selectively refractory to EBOV infection. Figure 1 with 1 supplement see all Download asset Open asset African straw-colored fruit bat cells are refractory to EBOV entry and infection. (A) Ranges of African pteropodids Rousettus aegyptiacus (pink), Hypsignathus monstrosus (blue), Epomops buettikoferi (green) and Eidolon helvum (yellow) (source: IUCN Redlist). (B) Locations of known filovirus outbreaks. (C) Infection of pteropodid kidney fibroblast cell lines with authentic filoviruses. Means ± standard deviations (n ≥ 3) from two biological replicates are shown. (D) Infections with recombinant vesicular stomatitis viruses (rVSVs) bearing filovirus glycoproteins. BDBV, Bundibugyo virus; TAFV, Taı¨ Forest virus; SUDV, Sudan virus; RESTV, Reston virus; LLOV, Lloviu virus. Means ± SD (n = 3–4) from two biological replicates are shown. In panels C and D, the infectivity of each virus was normalized to that obtained in Vero grivet monkey cells. Means for infection of the different cell lines by each virus were compared by one-way ANOVA (p-value indicated above each group of bars). Tukey’s post hoc test was used to compare infection means on Hypsignathus monstrosus vs Eidolon helvum cells (*p < 0.05; ****p < 0.0001; ns, no statistical significance). https://doi.org/10.7554/eLife.11785.003 An NPC1-dependent block to cell entry accounts for the EBOV infection deficit in African straw-colored fruit bat cells The viral spike glycoprotein, GP1,2 (herein termed GP) mediates all steps of filovirus entry into the cytoplasm of host cells (Lee et al., 2008; Miller and Chandran, 2012). Vesicular stomatitis viruses bearing filovirus GP proteins (VSV pseudotypes) provide a highly validated surrogate system to recapitulate filovirus entry under biosafety level 2 containment (Takada et al., 1997; Jangra et al., 2015). To assess whether the EBOV infection defect in the African straw-colored fruit bat cells occurs at the viral entry step, we exposed an expanded panel of kidney fibroblast cell lines from four African pteropodids to VSV pseudotypes bearing GP spikes (VSV-GP) from seven filoviruses, including two non-African viruses, Reston virus (RESTV) and Lloviu virus (LLOV) (Figure 1D). As observed with authentic EBOV, VSV-EBOV GP infection was substantially reduced in the African straw-colored fruit bat cells; however, this virus could efficiently infect cells derived from the other pteropodids, including those of a proposed EBOV host, the hammer-headed fruit bat (Hypsignathus monstrosus) (Leroy et al., 2005). Strikingly, only VSVs bearing EBOV GP, and to a lesser degree, those bearing BDBV and TAFV GP, were deficient at infecting African straw-colored fruit bat fibroblasts. Similar strong but EBOV-specific reductions in infection were measured in two kidney and lung cell lines derived from additional African straw-colored fruit bats (Figure 1—figure supplement 1). Therefore, reduced infection of these bat cells by EBOV reflects a virus- and host species-specific restriction at the cell entry step. We surmised that the filovirus receptor, NPC1, might explain the selective resistance of the African straw-colored fruit bat cells to EBOV entry and infection. Accordingly, we engineered these cells to stably express human NPC1 (HsNPC1) (Figure 2—figure supplements 1,2), and then exposed them to EBOV (Figure 2A). Provision of HsNPC1 substantially enhanced authentic EBOV infection in the African straw-colored fruit bat cells. By contrast, we found no evidence that either MARV infection in these cells, or EBOV/MARV infection in permissive Büttikofer’s epauletted fruit bat cells was limited by receptor availability (Figure 2A). Finally, similar results were obtained with VSVs bearing filovirus glycoproteins (Figure 2B). Taken together, these findings indicate that EBOV infection is reduced in African straw-colored fruit bat cells because of a specific molecular incompatibility between the EBOV glycoprotein and the filovirus entry receptor. Figure 2 with 2 supplements see all Download asset Open asset The NPC1-dependent entry and infection block in African straw-colored fruit bat cells is selective for EBOV. (A) Infection of pteropodid kidney fibroblast cell lines stably expressing human NPC1 (HsNPC1) with authentic filoviruses. (B) Infection of pteropodid kidney fibroblast cell lines with recombinant VSV (rVSVs) bearing filovirus glycoproteins. IU/ml, infectious units per ml. Means ± SD (n = 3) from a representative experiment are shown in each panel. Means for infection of cell lines lacking or ectopically expressing HsNPC1 were compared by unpaired two-tailed Student’s t-test with Welch’s correction (***p< 0.001; ****p< 0.0001; ns, no statistical significance). https://doi.org/10.7554/eLife.11785.005 NPC1-dependent cell entry is reduced, but not completely eliminated, in African straw-colored fruit bat cells Although EBOV entry and infection in African straw-colored fruit bat cells was consistently reduced to 0.1–1% relative to that in cells from the other pteropodids, we noted that infection was not completely blocked. To determine if EBOV could inefficiently infect these bat cells via an NPC1-independent mechanism, we used CRISPR/Cas9 genome engineering to derive an African straw-colored fruit bat cell line fully deficient in NPC1. We identified a single cell clone (Eidolon helvum NPC1–#1 [EhNPC1–#1]) in which all NPC1 alleles bore insertions or deletions (indels) at the expected site (Figure 3A). These indels were predicted to frameshift the NPC1 open reading frame at amino acid position 81 (Homo sapiens HsNPC1 numbering), generating truncated polypeptides of 82, 83, and 109 residues that lacked the majority of the 1278-amino acid NPC1 sequence. EhNPC1–#1 cells were deficient in clearance of lysosomal cholesterol, a well-established cellular function of NPC1 (Carstea et al., 1997), but could be rescued by ectopic HsNPC1 expression, confirming that NPC1 had indeed been disrupted in these cells (Figure 3B). Figure 3 with 1 supplement see all Download asset Open asset The incompatibility between EBOV GP and Eidolon helvum NPC1 reduces, but does not eliminate, EBOV entry into African straw-colored fruit bat cells. (A) CRISPR/Cas9 genome engineering was used to knock out the NPC1 gene in African straw-colored fruit bat kidney fibroblasts. WT NPC1 gene sequence aligned with the sequences of all three alleles in the knockout (NPC1–#1) cell clone. The gRNA target sequence is marked in red, and the protospacer adjacent motif (PAM) sequence of the gRNA target site is underlined. (B) The capacity of WT and NPC1–#1 cells, and NPC1–#1 cells stably expressing HsNPC1, to clear lysosomal cholesterol was determined by staining with filipin III complex from Streptomyces filipensis, as described (Carette et al., 2011). Red arrowheads indicate lysosomes with accumulated cholesterol. (C) Infection of African straw-colored fruit bat cell lines and Vero African grivet monkey cells (control) by VSVs bearing EBOV or MARV GP. Infected (eGFP-positive) cells were visualized by fluorescence microscopy. Representative fields are shown. Scale bars, 20 µm. https://doi.org/10.7554/eLife.11785.008 We next exposed wild-type (WT) and EhNPC1–#1 fibroblasts to VSVs bearing EBOV or MARV GP. No detectable infection was obtained with either virus in NPC1-deficient cells, indicating that filovirus entry into these cells is absolutely dependent on the E. helvum NPC1 ortholog (Figure 3C). Moreover, EBOV GP-dependent infection in EhNPC1–#1 cells reconstituted with HsNPC1 was dramatically enhanced over that observed in WT cells, whereas MARV GP-dependent infection was rescued by HsNPC1 expression to a level resembling that in WT cells (Figure 3C). Therefore, the low levels of EBOV infection in African straw-colored fruit bat cells likely arise from the weak, but non-zero, activity of EhNPC1 as an EBOV entry receptor. EhNPC1 is poorly recognized by EBOV GP Filovirus GPs must directly engage the second luminal domain of NPC1, domain C, during cell entry (Krishnan et al., 2012; Miller et al., 2012). Accordingly, we postulated that the African straw-colored fruit bat NPC1 ortholog is poorly recognized by EBOV GP. To test that hypothesis, we generated and sequenced NPC1 cDNAs from all four pteropodid cell lines. Alignment of their domain C amino acid sequences with that of HsNPC1 revealed a high degree of conservation (>90%), with identical arrangements of cysteine residues and similar predicted secondary structures suggestive of a similar overall fold (Figure 3—figure supplement 1). To examine GP-NPC1 binding, we engineered and expressed soluble forms of the four pteropodid NPC1 domain Cs, as described for HsNPC1 (Figure 4—figure supplement 1) (Miller et al., 2012). A cleaved form of EBOV GP could capture HsNPC1 domain C in an ELISA, as shown previously (Miller et al., 2012). EBOV GP bound with similar avidity to NPC1 domain Cs derived from Egyptian rousettes (RaNPC1), hammer-headed fruit bats (HmNPC1) and Büttikofer’s epauletted fruit bats (EbNPC1), but poorly or not at all to that of African straw-colored fruit bats (EhNPC1) (Figure 4A). Like the infection defect in African straw-colored fruit bat cells, this receptor binding defect was selective for EBOV GP, since GPs derived from MARV and the European filovirus, LLOV (Ng et al., 2014), bound equivalently to all four pteropodid domain Cs (Figure 4A). These findings strongly suggest that African straw-colored fruit bat cells are poorly susceptible to EBOV infection because EBOV GP poorly recognizes their ortholog of the filovirus receptor, NPC1. Figure 4 with 2 supplements see all Download asset Open asset African straw-colored fruit bat NPC1 binds poorly to EBOV GP because of a single amino acid change relative to NPC1 from permissive African pteropodids. (A) Binding of filovirus GP proteins to soluble NPC1 domain C proteins derived from African pteropodids measured by an ELISA. RaNPC1, Egyptian rousette; EbNPC1, Büttikofer’s epauletted fruit bat; HmNPC1, Hammer-headed bat; EhNPC1, African straw-colored fruit bat. (B) A chimera between RaNPC1 and EhNPC1 domain Cs fully rescues EBOV GP-EhNPC1 binding. (C) A single amino acid change in EhNPC1 domain C, F502D, renders it fully competent to recognize EBOV GP. Means ± SD (n = 3) from a representative experiment are shown. https://doi.org/10.7554/eLife.11785.010 The restriction in EhNPC1-EBOV GP binding can be mapped to a single amino acid change in EhNPC1 To define the molecular basis of the defect in interaction between EBOV and EhNPC1, we generated a panel of NPC1 domain C chimeras comprising sequences from permissive RaNPC1 and nonpermissive EhNPC1, and tested them in the GP-binding ELISA. A single chimera, EhNPC1 domain C containing four EhNPC1→RaNPC1 amino acid residue changes, regained the capacity to efficiently recognize EBOV GP (Figure 4B). Further dissection revealed that only a single amino acid change, F502D, in a central region of NPC1 domain C was needed to effect this complete restoration in GP-NPC1 binding (Figure 4C). The presence of residue F or D at position 502 for all four pteropodid NPC1 proteins tested was fully concordant with EBOV GP-NPC1 binding and NPC1 receptor function, and was also true for HsNPC1 (D, permissive; F, restrictive) (Figures 1,2, Figure 3—figure supplement 1). Finally, analysis of NPC1 sequences derived from nine additional wild-caught African straw-colored fruit bats confirmed that the presence of residue F at position 502 is a conserved feature of EhNPC1 (Figure 4—figure supplement 2). We conclude that a species-specific defect in virus-receptor interaction, caused by a single amino acid residue change in EhNPC1 relative to other, permissive African pteropodid NPC1 orthologs, reduces EBOV infection in African straw-colored fruit bat cells. Moreover, because residues in the NPC1-binding site are conserved among all available EBOV GP sequences (Supplementary file 1), this restriction is almost certain to be encountered by all known EBOV variants and their isolates, including those detected in EBOV disease patients during the recent epidemics in Western and Middle Africa (Park et al., 2015; Gire et al., 2014; Tong et al., 2015; Carroll et al., 2015; Kugelman et al., 2015). NPC1 has evolved under positive selection in bats Previous work has led to the hypothesis that bats in equatorial Africa and elsewhere harbor filoviruses (reviewed in [Wahl-Jensen et al., 2013]). These results, together with our findings for virus- and host species-specific differences in cellular susceptibility to filovirus infection, hinted at the possibility of a deeper co-evolutionary relationship between filoviruses and bats. One hallmark of such a relationship between a virus and its host is the evolution, under selective pressure to resist infection, of host genes encoding proviral and antiviral factors. To evaluate whether the NPC1 gene has evolved under positive selection in bats, we combined the NPC1 sequences obtained in this study with those of bats from six other species (two non-African pteropodids, two phyllostomids, and two vespertilionids) compiled through assembly of publicly available RNAseq data (Figure 5, Figure 5—figure supplement 1, Supplementary files 2–4). We then analyzed this NPC1 multiple alignment (Supplementary file for for relative to of or selection in of four we found strong evidence for positive selection in bat NPC1 (Figure 5—figure supplement 1). Figure with 1 supplement see all Download asset Open asset NPC1 is under positive selection in bats. (A) species in the analysis of NPC1. relationships are indicated at the of the sequence (B) identified with are on a of NPC1. in were identified in at one of the four analyses and the site in was identified in all four analyses (Figure 5—figure supplement 1). (C) The that each in domain C has to 502 is indicated = and two of sites with are of the specific NPC1 with on the of this selective were but only one position was identified by all four tests (Figure Figure 5—figure supplement 1). Strikingly, this is the same position in NPC1 at which a mutation in EhNPC1 reduces receptor binding to EBOV GP and viral infection, a that could a selective (Figure identified in only some of the tests for or at may still have For example, additional were identified in two of domain C that may form a of the for EBOV GP (Figure Our that signatures of sequence evolution to in NPC1 that are for virus binding C and position to that at these sites can bats from infection or severe disease caused by filoviruses and/or other A single mutation at residue in EBOV GP viral entry by its interaction with EhNPC1 arms between hosts and are thought to be by of genetic and and 2011; and 2012; Demogines et al., 2013). In this we postulated that mutation of residue 502 in EhNPC1 could be by viral To identify such compensatory viral changes, we a panel of in the NPC1-binding site of EBOV GP by for enhanced to EhNPC1 domain While no single bound to EhNPC1 as as it to the other pteropodid or to HsNPC1, EhNPC1 binding (Figure Infection by VSV bearing EBOV was substantially enhanced in African straw-colored fruit bat cells, with this binding affinity for EhNPC1 (Figure of the of EBOV GP (Lee et al., revealed that is at the of the NPC1-binding where it forms of a (Figure The mutation likely a more NPC1-binding site that can the at the GP-NPC1 binding (Figure Figure Download asset Open asset A sequence in the NPC1-binding site of filovirus GP influences GP-EhNPC1 binding and filovirus (A) Binding of EBOV GP and to soluble NPC1 domain C proteins derived from African pteropodids measured by an ELISA. RaNPC1, Egyptian rousette; EbNPC1, Büttikofer’s epauletted fruit bat; HmNPC1, Hammer-headed fruit bat; EhNPC1, African straw-colored fruit bat. (B) Infection of African straw-colored fruit bat cells with VSV pseudotypes bearing EBOV GP or Means ± SD (n = 3–4) from a representative experiment are shown in each panel. Means for VSV-EBOV GP WT vs infection were compared by unpaired two-tailed Student’s t-test with Welch’s correction < (C) of a single et al., key residues in the NPC1-binding site (yellow) and residue (D) of sequences from a panel of filoviruses. on other residues from on Infection of African pteropodid cells with VSV pseudotypes bearing GP or Means ± SD (n = from two biological replicates are shown. Means for GP WT vs infection on each cell line were compared by unpaired two-tailed Student’s t-test with Welch’s correction (*p < < ****p < sequence at residue in GP to virus- and bat species-specific of cellular susceptibility to filoviruses Although no known EBOV the we observed that LLOV and Sudan virus GP (Figure both GP proteins could viral entry into African straw-colored fruit bat cells (Figure and bind to EhNPC1 (Figure data not shown for we postulated that amino acid changes at position in the GP site influence the capacity of filovirus glycoproteins to EhNPC1 for viral Accordingly, we exposed pteropodid kidney fibroblasts to VSV pseudotypes bearing or (Figure with our hypothesis, the mutation substantially reduced GP-dependent infection in African straw-colored fruit bat cells. this virus also infected Egyptian cells less than to the of sequence that selectively binding to RaNPC1 (Figure These findings provide evidence that GP residue can influence cellular susceptibility to infection by NPC1 in a that on the sequences of both We that sequence at residue and other in the site of filovirus glycoproteins has been by selective pressure to NPC1 with potential for viral host range and virulence. Discussion The Ebola virus disease in Western Africa the need to uncover the biological and factors that the evolution, and of filoviruses. While a to this will the of multiple levels of biological from genes to to previous work has shown that studies of molecular between viruses and their host cells can to this The essential between viruses and their entry receptors provide A in receptor binding from the to the ortholog of the receptor the of a virus, and a disease in et al., 2014). of of with their receptor have to the of from bats to humans, and its of as hosts et al., 2012; et al., et al., In this study, we that between filoviruses and their entry receptor NPC1 can influence the cellular susceptibility of bats to infection. This is in of previous findings that filoviruses could efficiently infect a range of mammalian cells, including some derived from bats 2008; et al., 2011). this work and the results of infection studies in and bats have led to the hypothesis that between viral and those of the host