RIG-I and PKR, but not stress granules, mediate the pro-inflammatory response to Yellow fever virus

Yellow fever virus (YFV) is a single-stranded RNA primarily targeting the liver. Severe YF cases are responsible for viral hemorrhagic fever, plausibly precipitated by excessive pro-inflammatory cytokine response, resulting in up to 50% fatality. Pathogen recognition receptors (PRRs), such as the ubiquitously expressed cytoplasmic RIG-I-like receptors (RLRs), as well as the viral RNA sensor PKR are known to initiate a pro-inflammatory response upon recognition of viral genomes and replication intermediates. The PRRs responsible for cytokine production in cells infected with YFV strains are not known. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon infection with the Asibi strain. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR, and are so far recognized as hubs orchestrating RNA virus sensing. Using PKR-deficient hepatoma cells, we found that PKR contributes to both stress granule formation and cytokine induction upon Asibi infection. However, we showed that stress granules disruption did not affect the cytokine response to Asibi infection, as assessed by siRNA-knockdown-mediated inhibition of stress granule assembly and an automated determination of granule size and number. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Collectively, our data strongly suggest that both RIG-I and PKR mediate pro-inflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the early cytokine response from the stress granules formation, our model challenges the current view by which stress granules are required for the mounting of the acute antiviral response.