Stimulation of Ebola virus production from persistent infection through activation of the Ras/MAPK pathway — PNAS :
[Via Proceedings of the National Academy of Sciences]
Abstract
Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV “jumps” from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2α phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the “hit-and-run” nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission.
Ebola is a particularly nasty virus that has been described as incredibly efficent in turning an infected human into virus. The resulting damage to the victim results in high rates of death.
The natural host for the virus has not been definitively identified, even after 30 years of investigation. This paper details some interesting biology that may shed some light on this.
First, they screened a lot of mammalian cell lines to see if any were capable of maintaining a low level of infection and surviving. They “found that the mouse lines NIH 3T3 and RAW264.7 (mouse fibroblast and macrophage line, respectively), as well as a Mexican free-tailed bat line (Tb1.Lu, a bat lung fibroblast), have this capacity.”
These infected cells could be grown under the same conditions as normal cells and virus could be recovered even 10 months after infection. So now they had a lab source of cells to investigate conditions affecting viral production.
They found that stimulating the cells with chemicals that typically induce a strong immune response, such as PMA or LPS, increased virus production over two orders of magnitude. Interestingly, the increase in production was not at the level of transcription or replication but at the level of viral protein production.
They were able to show that the cellular pathways that affect virus production are ones that deal with interferon I production, in particular the Ras/MAPK pathway. Murine cell lines that have had this pathway inactivated by mutations become much more easily infected (60-80%) than wild type cell lines (<5%).
Finally they infected mice with Ebola virus. Normally there is very little viral production at all in mice. But, if they injected the mice with PMA or other activators of the Ras/MAPK pathways, they saw about 3 orders of magnitude increase in virus production.
The upshot that they can now investigate what biological processes are involved in containing an Ebola infection inside a cell. They already have hits at a specific protein involved in binding RNA. The work also hints at how to find the natural reservoir.
It may be that virus has not been detected because in most cases the amount of virus in a persistent infection is too low to detect. But under certain conditions that activate the Ras/MapK pathway, much higher levels of Ebola could be detected.
So, investigators could stimulate the pathway in proposed reservoir species and look to see if there is now virus production. It would also explain why virus production in the wild would be transient and perhaps only found in a few members of the species at a time.
The identification of some cellular pathways involved in Ebola virus production also provides hope that better methods of treatment may be identified. Mice are generally poor hosts for Ebola because of interferon I inactivation. Finding ways to effectively mimic this in human cells could have a palliative effect.
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