Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus

Abstract

Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive pro-inflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic RIG-I-like receptors (RLRs), and the viral RNA sensor PKR are known to initiate a pro-inflammatory response upon recognition of viral genomes. 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 YFV infection. 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. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granules disruption did not affect the cytokine response to YFV infection, as assessed by siRNA-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings 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 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. IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of pro-inflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR-, but not RIG-I-dependent manner. Whilst stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for pro-inflammatory mediators' production upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions on disease management.

Roles of antiviral sensing and type I interferon signaling in the restriction of SARS-CoV-2 replication

“…We further investigated the importance of type I IFN signaling in the restriction SARS-CoV-2 growth by profiling transcriptional responses in human liver carcinoma cell lines, Huh7 and Huh7.5, at 0 and 48 HPI (Figure 3). Previous studies with hepatitis C, dengue, and yellow fever viruses have shown that enhanced viral permissivity in Huh7.5 cells compared with Huh7 cells is due to defective RIG-1-mediated signaling (both cell lines lack IFNA10 and TRIM56) (Beauclair et al, 2020;Chatel-Chaix et al, 2016;Chen et al, 2021;Sumpter et al, 2005). In line with these previous studies, Huh7.5 cells supported greater viral growth and release compared with Huh7 cells (Figures 1A-1C).…”

Stress granules: regulators or by‐products?

“…These results support a model where SGs act as a platform for recognition of viral RNAs and activation of innate immune signaling. Other studies, however, observed that disruption of SGs (by drug treatments, mutating eIF2α, or depleting PKR or SG components) did not reduce interferon production [51–53]. Therefore, a growing body of evidence implicates SGs in innate immune signaling, but their role might be restricted to specific viruses and conditions, depending on the properties and localization pattern of the viral genome [23].…”

A yellow fever virus NS4B inhibitor not only suppresses viral replication, but also enhances the virus activation of RIG-I-like receptor-mediated innate immune response

“…Both RIG-I and MDA5 are involved in responses to dengue virus serotype-2 (DENV-2) infection in mouse embryonic fibroblasts [ 36 ] and Zika virus infection in human trophoblasts [ 37 ]. Concerning the role of RLRs in YFV infection, it was reported that replication of YFV vaccine strain YF-17D in human pDCs and pDC-like cell lines, and YFV reference strain Asibi in human hepatoma cells stimulated type I IFN production through activation of RIG-I [ 38 , 39 ].…”