It was a peer review request that got Alessia Ruggieri and her group at University of Heidelberg to look more closely at how Dengue virus manipulates the cellular translation and stress machineries during infection.
In a previous study, Ruggieri showed that Hepatitis C virus infection induced an oscillating stress response that correlated with phases in which host cell translation is switched off and then reactivated. She visualized cycles of assembly and disassembly of huge aggregates called stress granules in which stalled mRNA transcripts get bound up. The reviewers asked whether the closely related Dengue virus elicited the same type of stress response. It turned out that Dengue virus does it differently: no stress granules were seen at all. Dengue virus is a positive single-strand RNA virus, which has a genome that is capped with a similar structure to that of cellular mRNAs. This feature makes it dependent on active host translation—or so it was thought. (image: In Dengue virus-infected human liver cells (green), global cellular protein synthesis (red) is blocked. credit: Alessia Ruggieri)
Ruggieri’s interest was piqued: inducing stress granules and the associated host translation suppression could be detrimental for Dengue’s replication. However, she recalled previous findings from Eva Harris´s lab showing that under very particular experimental conditions, in which global host translation is suppressed, Dengue virus could switch its own RNA genome translation to a process that did not require the RNA capping complex for initiation. The two observations made her wonder: why would Dengue virus do that?
In a paper published this week in mBio, her group shows that flaviviruses like Dengue and Zika viruses use an unexpected approach to hijack the cell’s machinery for their own replication.
In other viral infections, when the host cell senses the presence of viral RNA, a stress response is activated. This shuts down cellular translation in an attempt to thwart virus spread. “Interestingly, in the case of flavivirus infection, although the virus actively blocks stress granule formation, it is also switching off the host cell translation,” says Ruggieri, a group leader and virologist at Heidelberg.
Her team wanted to investigate further how the virus might be manipulating cell machinery and signaling pathways to do this. Of course, taking a closer look at translation repression brought its challenges: “This is such a complicated process regulated by hundreds of signaling pathways in the cell,” Ruggieri recalls thinking.
Their first step was to establish that Dengue virus could indeed continue its own viral protein translation under the global cellular translation suppression. Her team measured the amount of actively translated transcripts during Dengue infection. The percentage of actively translated host cellular transcripts, represented by the housekeeping gene GAPDH, significantly decreased during the course of infection. In contrast, the percentage of Dengue RNA genome being translated remained constant. They also measured at the protein level and showed that while global protein synthesis dropped off, the amount of the Dengue NS4B protein increased over time.
“The viral protein levels are not affected—which is not expected for a virus with a capped RNA genome that is supposed to be sensitive to such a strong repression of the host cell translation,” says Ruggieri. As infection progresses, Dengue virus must be using some other mechanism to ensure that its own translation proceeds. The team also showed that this host cell translation repression happens similarly in other flaviviruses as well, including Zika and West Nile viruses.
Next, the team added only the non-structural Dengue proteins to cells—a set of proteins not able to assemble new virus particles—and saw that these proteins alone were enough to trigger the host translation blockade. The team demonstrated that the two processes—blockage of both global cell translation and inhibition of stress granule formation—were completely uncoupled in flavivirus infection.
“It seems to be a different mechanism at play here. The virus itself is blocking the activation of the stress pathway and also repressing translation,” says Ruggieri. She speculates that by inhibiting stress granule formation, Dengue virus is ensuring that its RNA genome doesn’t get sequestered away in these structures.
Although her team has good evidence that Dengue virus is blocking the global cell translation at the initiation step, they have not yet found how this occurs.
“It seems to be a two-step process and a novel strategy used by flaviviruses: block the global host translation and then escape this block by switching to a different mechanism for viral translation” says Ruggieri. “It is extremely complicated, but amazing to study, too.”