Search

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. • Some bat cell lines show differential susceptibilities to Ebola and Marburg viruses • Distinctive amino acid sequences exist in the filovirus receptor of some bats • Receptor heterogeneity in bat species controls their susceptibility to filoviruses • Receptor preference is important for differential filovirus tropism to bat cells Differential susceptibilities of bats to filoviruses have been suggested. Takadate et al. compare structures of the filovirus receptor among a variety of bat cell lines and discover a molecular mechanism determining their susceptibility to Ebola and Marburg viruses, providing information for understanding the ecology of filoviruses. [ABSTRACT FROM AUTHOR]

2-Hyroxypropyl-beta-cyclodextrin (HPβCD) is being used to treat Niemann-Pick C1, a fatal neurodegenerative disease caused by abnormal cholesterol metabolism. HPβCD slows disease progression, but unfortunately causes severe, rapid onset hearing loss by destroying the outer hair cells (OHC). HPβCD-induced damage is believed to be related to the expression of prestin in OHCs. Because prestin is postnatally upregulated from the cochlear base toward the apex, we hypothesized that HPβCD ototoxicity would spread from the high-frequency base toward the low-frequency apex of the cochlea. Consistent with this hypothesis, cochlear hearing impairments and OHC loss rapidly spread from the high-frequency base toward the low-frequency apex of the cochlea when HPβCD administration shifted from postnatal day 3 (P3) to P28. HPβCD-induced histopathologies were initially confined to the OHCs, but between 4- and 6-weeks post-treatment, there was an unexpected, rapid and massive expansion of the lesion to include most inner hair cells (IHC), pillar cells (PC), peripheral auditory nerve fibers, and spiral ganglion neurons at location where OHCs were missing. The magnitude and spatial extent of HPβCD-induced OHC death was tightly correlated with the postnatal day when HPβCD was administered which coincided with the spatiotemporal upregulation of prestin in OHCs. A second, massive wave of degeneration involving IHCs, PC, auditory nerve fibers and spiral ganglion neurons abruptly emerged 4-6 weeks post-HPβCD treatment. This secondary wave of degeneration combined with the initial OHC loss results in a profound, irreversible hearing loss., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ding, Jiang, Manohar, Liu, Li, Chen and Salvi.)

Filoviruses, including marburgviruses and ebolaviruses, have a single transmembrane glycoprotein (GP) that facilitates their entry into cells. During entry, GP needs to be cleaved by host proteases to expose the receptor-binding site that binds to the endosomal receptor Niemann-Pick C1 (NPC1) protein. The crystal structure analysis of the cleaved GP (GPcl) of Ebola virus (EBOV) in complex with human NPC1 has demonstrated that NPC1 has two protruding loops (loops 1 and 2), which engage a hydrophobic pocket on the head of EBOV GPcl. However, the molecular interactions between NPC1 and the GPcl of other filoviruses remain unexplored. In the present study, we performed molecular modeling and molecular dynamics simulations of NPC1 complexed with GPcls of two ebolaviruses, EBOV and Sudan virus (SUDV), and one marburgvirus, Ravn virus (RAVV). Similar binding structures were observed in the GPcl-NPC1 complexes of EBOV and SUDV, which differed from that of RAVV. Specifically, in the RAVV GPcl-NPC1 complex, the tip of loop 2 was closer to the pocket edge comprising residues at positions 79-88 of GPcl; the root of loop 1 was predicted to interact with P116 and Q144 of GPcl. Furthermore, in the SUDV GPcl-NPC1 complex, the tip of loop 2 was slightly closer to the residue at position 141 than those in the EBOV and RAVV GPcl-NPC1 complexes. These structural differences may affect the size and/or shape of the receptor-binding pocket of GPcl. Our structural models could provide useful information for improving our understanding the differences in host preference among filoviruses as well as contributing to structure-based drug design.

The COVID-19 plague is hitting mankind. Several viruses, including SARS-CoV-1, MERS-CoV, EBOV, and SARS-CoV-2, use the endocytic machinery to enter the cell. Genomic variants in NPC1, which encodes for the endo-lysosomal Niemann-Pick type C1 protein, restricts the host-range of viruses in bats and susceptibility to infections in humans. Lack of NPC1 and its pharmacological suppression inhibits many viral infections including SARS-CoV-1 and Type I Feline Coronavirus Infection. Antiviral effects of NPC1-inhibiting drugs for COVID-19 treatment should be explored., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Lloviu virus (LLOV), a bat-derived filovirus that is phylogenetically distinct from human pathogenic filoviruses such as Ebola virus (EBOV) and Marburg virus (MARV), was discovered in Europe. However, since infectious LLOV has never been isolated, the biological properties of this virus remain poorly understood. We found that vesicular stomatitis virus (VSV) pseudotyped with the glycoprotein (GP) of LLOV (VSV-LLOV) showed higher infectivity in one bat ( Miniopterus sp.)-derived cell line than in the other bat-derived cell lines tested, which was distinct from the tropism of VSV pseudotyped with EBOV (VSV-EBOV) and MARV GPs. We then focused on the interaction between GP and Niemann-Pick C1 (NPC1) protein, one of the cellular receptors of filoviruses. We introduced the Miniopterus bat and human NPC1 genes into NPC1-knockout Vero E6 cells and their susceptibilities to the viruses were compared. The cell line expressing the bat NPC1 showed higher susceptibility to VSV-LLOV than that expressing human NPC1, whereas the opposite preference was seen for VSV-EBOV. Using a site-directed mutagenesis approach, amino acid residues involved in the differential tropism were identified in the NPC1 and GP molecules. Our results suggest that the interaction between GP and NPC1 is an important factor in the tropism of LLOV to a particular bat species.

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann-Pick C1.

Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus disease (EVD) outbreak in western Africa. One obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. Here, we investigated the role of the intracellular filovirus receptor, Niemann-Pick C1 (NPC1) as a molecular determinant of Ebola virus (EBOV) host range at the cellular level. Whereas human cells can be infected by EBOV, a cell line derived from a Russell's viper (Daboia russellii) (VH-2) is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russell's viper NPC1 ortholog bound poorly to the EBOV spike glycoprotein (GP). Analysis of panels of viper-human NPC1 chimeras and point mutants allowed us to identify a single amino acid residue in NPC1, at position 503, that bidirectionally influenced both its binding to EBOV GP and its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1's endosomal localization nor its housekeeping role in cellular cholesterol trafficking. Together with other recent work, these findings identify sequences in NPC1 that are important for viral receptor activity by virtue of their direct interaction with EBOV GP and suggest that they may influence filovirus host range in nature. Broader surveys of NPC1 orthologs from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection. IMPORTANCE Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell's viper are not susceptible to infection because EBOV cannot bind to viper NPC1. This resistance to infection can be mapped to a single amino acid residue in viper NPC1 that renders it unable to bind to EBOV GP. The newly solved structure of EBOV GP bound to NPC1 confirms our findings, revealing that this residue dips into the GP receptor-binding pocket and is therefore critical to the binding interface. Consequently, this otherwise well-conserved residue in vertebrate species influences the ability of reptilian NPC1 proteins to bind to EBOV GP, thereby affecting viral host range in reptilian cells.

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.

Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.

Background: The current Ebola virus (EBOV) outbreak has highlighted the troubling absence of available antivirals or vaccines to treat infected patients and stop the spread of EBOV. The EBOV glycoprotein (GP) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-EBOV drugs. We report the identification of 2 novel EBOV inhibitors targeting viral entry., Methods: To identify small molecule inhibitors of EBOV entry, we carried out a cell-based high-throughput screening using human immunodeficiency virus-based pseudotyped viruses expressing EBOV-GP. Two compounds were identified, and mechanism-of-action studies were performed using immunoflourescence, AlphaLISA, and enzymatic assays for cathepsin B inhibition., Results: We report the identification of 2 novel entry inhibitors. These inhibitors (1) inhibit EBOV infection (50% inhibitory concentration, approximately 0.28 and approximately 10 µmol/L) at a late stage of entry, (2) induce Niemann-Pick C phenotype, and (3) inhibit GP-Niemann-Pick C1 (NPC1) protein interaction., Conclusions: We have identified 2 novel EBOV inhibitors, MBX2254 and MBX2270, that can serve as starting points for the development of an anti-EBOV therapeutic agent. Our findings also highlight the importance of NPC1-GP interaction in EBOV entry and the attractiveness of NPC1 as an antifiloviral therapeutic target., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

A genome-wide association study (GWAS) and subsequent replication studies in diverse ethnic groups indicate that common Niemann-Pick C1 gene ( NPC1 ) polymorphisms are associated with morbid-adult obesity or diabetes independent of body weight. The objectives for this prospective cross-sectional study were to determine allele frequencies for NPC1 polymorphisms (644A>G, 1926C>G, 2572A>G, and 3797G>A) and association with metabolic disease phenotypes in an ethnically diverse New Mexican obstetric population. Allele frequencies for 1926C>G, 2572A>G, and 3797G>A were significantly different between race/ethnic groups (non-Hispanic white, Hispanic, and Native American). The results also indicated a significant pairwise linkage-disequilibrium between each of the four NPC1 polymorphisms in race/ethnic groups. Moreover, the derived and major allele for 1926C>G was associated (OR 2.11, 95% CI 1.10-3.96, P = 0.022) with increased risk for maternal prepregnancy overweight (BMI 25.0-29.9kg/m 2 ) while the ancestral and major allele for 2572A>G was associated (OR 4.68, 95% CI 1.23-17.8, P = 0.024) with increased risk for gestational diabetes in non-Hispanic whites, but not Hispanics or Native Americans. In summary, this is the first transferability study to investigate common NPC1 polymorphisms in a multiethnic population and demonstrate a differential association with increased risk for maternal prepregnancy overweight and gestational diabetes.

Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Niemann-Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1(-/-) mice (Npc1(nih)) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70days of age). Similar measurements were made in Npc2(-/-) mice at 70days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1(-/-) mice and remained so at 70days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1(-/-) mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1(-/-) mice starting as early as 28days. Similar metabolic and histologic changes were evident in the lungs of the Npc2(-/-) mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time., (© 2013.)

A high throughput screen identified adamantane dipeptide 1 as an inhibitor of Ebola virus (EboV) infection. Hit-to-lead optimization to determine the structure-activity relationship (SAR) identified the more potent EboV inhibitor 2 and a photoaffinity labeling agent 3 . These anti-viral compounds were employed to identify the target as Niemann-Pick C1 (NPC1), a host protein that binds the EboV glycoprotein and is essential for infection. These studies establish NPC1 as a promising target for anti-viral therapy.

Ebola and Marburg viruses cause outbreaks of highly lethal infection in central Africa. In the last few years, rapid progress has been made in understanding how these viruses are transmitted and spread. These studies show that the glycoprotein GP that protrudes from the virus envelope mediates membrane fusion and infection. Activation of the GP membrane fusion activity is triggered by a multi-step pathway initiated by binding to lectins expressed on the cell surface. After uptake of lectin-bound particles by macropinocytosis, virus-containing vesicles are transported to late endosomes and lysosomes containing the protease cathepsin B and the membrane protein Niemann-Pick C1 (NPC1), which are essential for infection. Recent studies indicate that cathepsin B cleaves GP, removing heavily glycosylated sequences and exposing the domain in GP that is a ligand for NPC1. Although more studies are needed, current evidence strongly suggests that binding of protease-cleaved GP to NPC1 is the signal that activates virus membrane fusion and infection. Importantly, small molecules that target NPC1 and interfere with GP binding and ebolavirus infection have been identified. A major goal is to develop these inhibitors into anti-viral drugs.