The Ebola virus, along with three of its close relatives, causes the Ebola hemorrhagic fever. Ever since the first Ebola outbreak occurred in 1976 in Yambuku, the virus has been considered one of the deadliest to affect humans, causing a very high fatality rate – up to 90 percent in some reported epidemics. There is no definitive treatment or vaccination for the Ebola virus, and, until recently, researchers were limited in their understanding of how the virus enters a host cell.
Now, scientists from Harvard Medical School, Albert Einstein College of Medicine and U.S. Army Medical Research Institute of Infectious Diseases have found that the cholesterol transporter protein Niemann-Pick C1 (NPC1) is essential for the Ebola virus to enter cells and begin replicating. Another important factor is the homotypic fusion and vacuole protein-sorting (HOPS) complex, which is involved in the fusion of endosomes, or membrane-bound compartments inside eukaryotic cells, to lysosomes, or organelles that contain enzymes to break down cellular waste.
The finding, which was published online in Nature in Aug., reports that cells defective for the HOPS complex or NPC1 function were resistant to infection from the Ebola and Marburg filoviruses, but remained susceptible to several other unrelated viruses.
When a cell is infected by a virus, the machinery used to produce proteins for the cell is used to produce components of a virus instead. These components come together in vesicles that have to travel to the edges of the cell and fuse with the cell membrane. The membrane fusion and escape of viruses from the vesicular compartment in the cell require the NPC1 protein, independent of its cholesterol transport function.
The study was carried out using human cell lines. Unlike human cells, which inherit one copy of each chromosome from both parents and contain two copies of an individual gene, cell lines contain only a single copy of each gene. A problem with human cells is the presence of two copies of a particular gene. Knocking out one copy of a gene still leaves the other fully functioning, and the effects of disrupting the function of that gene remain unknown.
However, Jan Carette, one of the leading authors of the paper, used a technique he relied on in a previous study. He used a line of haploid cells, or cells that contain half the number of chromosomes (examples are sperm and egg cells), isolated from a chronic myeloid leukemia patient. The haploid cells had only one copy of each chromosome, except for chromosome 8.
The researchers performed haploid genetic screens to investigate filovirus entry pathways in detail. They relied on the method of gene disruption, removing one functional gene at a time, in order to identify which cells survived as the result of a mutation that provided them protection against viruses.
In their experiments, adherent haploid cells (HAP1) were generated using several transcription factors. Carette and colleagues used a harmless virus covered in the Ebola virus's glycoprotein coat, essentially making the virus a sheep in wolf's clothing. By altering the haploid cells, they were able to identify the precise cellular genes the Ebola virus relies on to gain entry into the host cell.
Several genes involved in organelles that transport and recycle proteins were identified as important factors involved in way the Ebola virus enters cells. The NPC1 gene, in particular, was revealed to be essential for the virus to enter the cytoplasm of the cell and begin cell replication. While mutations in the NPC1 gene cause the Niemann-Pick Disease, a fatal neurological disorder diagnosed mainly in children, the same mutations afforded cells protection against Ebola virus entry.
At the U.S. Army Medical Research Institute of Infectious Diseases, scientists tested the effects of active Ebola virus on two groups of mice, a control group with two functioning copies of the NPC1 gene and another group with one copy knocked out. While the mice with two copies were quickly infected by the Ebola virus, the mice with a copy knocked out were, for the most part, protected. These results further supported the role of the NPC1 gene as a crucial point of entry for the Ebola filovirus.
Altogether, these findings reveal features of Ebola virus entry pathways that were previously little understood, offering new potential for the development of drugs and antiviral strategies targeted at these crucial points of entry. With further research in this area, researchers may eventually be able to develop effective ways to combat the deadly Ebola virus.
