Utility of primary cells to examine NPC1 receptor expression in Mops condylurus, a potential Ebola virus reservoir

Abstract

The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, nonhuman primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied.In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication.The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro. Author summaryAlthough there have been Ebola virus (EBOV) outbreaks for more than 40 years, the animal natural reservoir that maintains this virus in nature has not been identified. Viruses PLOS Neglected Tropical Diseases | https://doi.org/10.and their respective reservoirs coevolve over millions of years, often without causing diseases in the reservoir itself. Upon entering a new host, infection can have devastating consequences, as in the case of EBOV. To gain entry into cells prior to replication, all ebolaviruses utilize the cellular receptor Niemann-Pick C1 (NPC1). In this study the authors focus their work on the Angolan free-tailed bat (Mops condylurus) as a potential reservoir for EBOV. Cells from various organs of this bat were isolated in culture and tested for the presence of NPC1. Most bat cell types had a lower amount of NPC1 compared to the tested human cells. These bat cells were also less efficiently infected by EBOV, indicating adaptation to EBOV. These results suggest low levels of virus replication in the respective tissues of M. condylurus and might be indicative of a virus-natural reservoir relationship.NPC1 receptor expression in bat primary cells PLOS Neglected Tropical Diseases | https://doi.org/10.

NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry

“…We compared antiviral activity following U18666A treatment and NPC1 deficiency and demonstrated that NPC1 inhibition offers a protective function against viral infection, suggesting the essential role of NPC1 in the replication of a wide range of enveloped viruses. NPC1, an essential protein involved in cholesterol delivery from the lysosomal lumen to the membrane, is highly conserved and ubiquitously expressed among mammalian cells (Bokelmann et al, 2020). A previous report demonstrated that an Ebola virus surface glycoprotein binds to luminal loop 2 of NPC1, which serves as a passive attachment site for downstream cellular infection (Miller et al, 2012).…”

Cross-Neutralisation of Novel Bombali Virus by Ebola Virus Antibodies and Convalescent Plasma Using an Optimised Pseudotype-Based Neutralisation Assay

“…Yet, it is conceivable that S142 may represent an adaptation of BOMV to M. condylurus NPC1. It has previously been suggested that each ebolavirus is adapted to a specific bat host [ 62 ], with variation occurring across the NPC1 binding domain, although a partial M. condylurus NPC1 sequence shows F504 is conserved [ 64 ].…”

Tolerance and Persistence of Ebola Virus in Primary Cells from Mops condylurus, a Potential Ebola Virus Reservoir

“…Additionally, NPC1 was shown to be a genetic determinant of filovirus susceptibility in bat cells; NPC1 polymorphisms found in specific bat species result in reduced interactions between filoviruses and NPC1, influencing the cellular susceptibility of bats to filovirus infection, replication, and virulence [34]. In culture, M. condylurus cells typically have low NPC1 expression levels compared to cells from highly symptomatic hosts, such as humans [35]. Infection of M. condylurus cells with EBOV revealed a potential correlation between NPC1 receptor expression level and virus replication rate [35].…”

“…In culture, M. condylurus cells typically have low NPC1 expression levels compared to cells from highly symptomatic hosts, such as humans [35]. Infection of M. condylurus cells with EBOV revealed a potential correlation between NPC1 receptor expression level and virus replication rate [35].…”