Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism

Abstract

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.

Keywords: Ebola virus; Marburg virus; Niemann-Pick C1; bat; filovirus; glycoprotein; host range; natural host; receptor; virus-host interaction.

Figure 1.. Susceptibility of Cell Lines to VSVs Pseudotyped with Filovirus GPs

(A) Vero E6 and bat-derived cells were infected with VSVs pseudotyped with filovirus GPs (VSV-EBOV, -SUDV, -TAFV, -BDBV, -RESTV, and -MARV). (B) Vero E6, bat cells (FBKT1 and ZFBK13-76E) expressing exogenous human NPC1, and empty vector-transduced FBKT1 and ZFBK13-76E were infected with VSVs pseudotyped with filovirus GPs. Viral infectious units (IUs) in each cell line were determined by counting the number of GFP-expressing cells as described in STAR Methods. Each experiment was conducted three times, and average and standard deviations are shown. Asterisks represent IUs under the limit of detection (20 IU/mL).

Figure 2.. Comparison of aa Sequences of the Domain C Loops of Bat NPC1 Orthologs

(A) The three-dimensional structure of domain C of human NPC1 (PDB: 5F1B) is represented as a ribbon model. GP-interacting regions, loop 1 and loop 2 (indicated in violet and sky blue, respectively), are shown in the boxed regions. Nitrogen and oxygen atoms in side chains are shown in blue and red, respectively. (B) Deduced aa sequences of the domain C loop regions of NPC1 orthologs are aligned. The aa positions including the unique aa residues observed in FBKT1 (positions 425, 426, and 427 in the loop 1 region) and ZFBK13-76E (positions 502 and 505 in the loop 2 region) are enclosed by rectangles.

Figure 3.. Effects of aa Substitutions in the NPC1-C Loops on Cell Susceptibility to Pseudotyped VSVs, EBOV, and MARV

(A) Wild-type and mutant NPC1 genes were constructed to assess the importance of the unique aa sequences (shown in boldface) in loop 1 of FBKT1 and loop 2 of ZFBK13-76E. (B) Locations of the unique aa residues of the loop regions are indicated in light green (loop 1) and orange (loop 2). Nitrogen and oxygen atoms in side chains are shown in blue and red, respectively. (C and D) Vero E6/NPC1-KO cl.19 cells transduced with exogenous NPC1 genes and control cells (NPC1 knockout and vector control) were infected with pseudotyped VSVs (C) or infectious filoviruses (D). Relative infectivity was determined as described in STAR Methods. Each experiment was conducted three times (C) or in triplicate (D), and averages and standard deviations are shown. For comparison of viral infectivity among NPC1-expressing cells, one-way analysis of variance was performed, followed by Dunnett’s test, and significant differences compared to cells expressing wild-type human NPC1 (HEK293T-NPC1) are shown with asterisks (*p < 0.05).

Figure 4.. Effects of aa Substitutions in the GP RBD on the Infectivity of Pseudotyped VSVs in FBKT1 and ZFBK13-76E Cells

(A and B) In the three-dimensional structure of the complex of EBOV GP and human NPC1-C, the GP-NPC1 interfaces are indicated in the boxed regions. The aa residues at positions 425–427 (S, G, and A) in NPC1-C loop 1 and at position 142 (S) of EBOV GP are shown in light green and pink, respectively (A). The aa residues at positions 502 and 505 (D and V) in NPC1-C loop 2 and at positions 147 and 148 (C and A) of EBOV GP are shown in orange and green, respectively (B). Oxygen atoms in side chains are shown in red (A and B). (C) Deduced aa sequences of filovirus GPs are aligned. The aa residues at positions 142 and 147/148, which are assumed to interact with the aa at positions at 425–427 in loop 1 and at 502 and 505 in loop 2 of human NPC1-C, respectively, are enclosed by rectangles. (D and E) Vero E6, FBKT1, and ZFBK13-76E cells were infected with VSVs pseudotyped with wild-type and mutant GPs of EBOV, SUDV, TAFV, BDBV, RESTV, and MARV, whose aa at positions 142 (D) or 148 (E) were substituted (EBOV numbering). Viral infectious units (IUs) in each cell line were determined by counting the number of GFP-expressing cells. Each experiment was conducted three times, and averages and standard deviations are shown. Asterisks represent IUs under the limit of detection (20 IU/mL).

Figure 5.. Interaction between GP and NPC1 and Predicted Structure of the NPC1 Loops and EBOV GP

(A) A solid phase immunosorbent assay to detect binding activity of NPC1 and GP was carried out as described in STAR Methods. Each experiment was conducted three times and averages and standard deviations of relative OD values are shown. Significant differences are shown with asterisks (*p < 0.05). (B and C) The three-dimensional co-crystal structure of domain C of human NPC1 and EBOV GP (PDB: 5F1B) was used as a template. The aa residues G426 or D502 of NPC1 and S142 or A148 of EBOV GP were substituted to E426 or F502 and Q142 or P148 by in silico mutagenesis. (B) The interface of NPC1 loop 1 and EBOV GP is shown as a ribbon model. G426/E426 of NPC1 and S142/Q142 of GP are shown in light green/purple and pink/yellow, respectively. (C) Loop 2 of NPC1 is shown as a ribbon model. GP1 (dark gray) and GP2 (light gray) are shown in a surface model. The aa residues forming a hydrophobic cavity of GP1 (i.e., V79, P80, T83, W86, G87, F88, L111, E112, I113, V141, G145, P146, C147, A152, and I170) are colored light cyan. D502/F502 of NPC1 and A148/P148 of GP are shown in orange/dark blue and green/deep red, respectively. Nitrogen and oxygen atoms in side chains are shown in blue and red, respectively (B and C). All mutagenesis procedures were performed using PyMOL (Schrödinger).