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Review
. 2023 Jul-Aug;14(4):e1770.
doi: 10.1002/wrna.1770. Epub 2022 Dec 7.

Regulation of ribonucleoprotein condensates by RNase L during viral infection

James M Burke  1
Affiliations
Review

Regulation of ribonucleoprotein condensates by RNase L during viral infection

James M Burke. Wiley Interdiscip Rev RNA. 2023 Jul-Aug.

Abstract

In response to viral infection, mammalian cells activate several innate immune pathways to antagonize viral gene expression. Upon recognition of viral double-stranded RNA, protein kinase R (PKR) phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) on serine 51. This inhibits canonical translation initiation, which broadly antagonizes viral protein synthesis. It also promotes the assembly of cytoplasmic ribonucleoprotein complexes termed stress granules (SGs). SGs are widely thought to promote cell survival and antiviral signaling. However, co-activation of the OAS/RNase L antiviral pathway inhibits the assembly of SGs and promotes the assembly of an alternative ribonucleoprotein complex termed an RNase L-dependent body (RLB). The formation of RLBs has been observed in response to double-stranded RNA, dengue virus infection, or SARS-CoV-2 infection. Herein, we review the distinct biogenesis pathways and properties of SGs and RLBs, and we provide perspective on their potential functions during the antiviral response. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Turnover and Surveillance > Regulation of RNA Stability RNA Export and Localization > RNA Localization.

Keywords: RNase L; RNase L-dependent body; innate immunity; stress granules; virus.

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Conflict of interest statement

Conflict of interest: The authors declare no conflict of interests.

Figures

Figure 1.
Figure 1.. Select antiviral pathways.
Diagrams showing the key activity of the RLR-MAVS, PKR, and OAS/RNase L pathways in response to viral double-stranded RNA.
Figure 2.
Figure 2.. RNase L inhibits stress granules and promotes RNase L-dependent bodies.
(A) Immunofluorescence assay for G3BP1 (Abcam: ab56574) and UBAP2L (Abcam: ab70319), which are both SG markers, in parental (WT) or RNase L-KO A549 cells (Burke et al., 2019) (12-well format) 4 hours post-lipofection with lipofectamine 2000 (Thermo Fisher Scientific: 11668019) and 500 ng of poly(I:C) (Invivogen:tlrl-pic), a viral double-stranded RNA mimic. As we previously reported (Burke et al., 2019; Burke et al., 2020), lipofection of poly(I:C) in A549-WT cells results in small punctate foci that enrich G3BP1. These structures are dependent on RNase L catalytic activity, and thus are termed RNase L-dependent bodies. These data demonstrate that UBAP2L strongly enriches in RLBs, which was previously unknown. In contrast to WT cells, lipofection of poly(I:C) in RNase L-KO cells leads to large (canonical) stress granules that highly enrich both G3BP1 and UBAP2L. See Burke et al., 2019 for detailed methodology. (B) As previously reported (Burke et. al, 2022), immunofluorescence assay for the G3BP1 and PABPC1 (Abcam: ab21060) in A549 cells infected with dengue virus serotype 2 16681 strain. In cells infected with dengue virus (DENV+), G3BP1 and PABP form small punctate foci consistent with RLB morphology. PABPC1 also concentrates in the nucleus in dengue viruses infected cells, which is a marker of RNase L activation (Burke et al., 2019). Figure adapted from Burke et. al, 2022. (C) As previously reported (Burke et. al, 2021b), immunofluorescence assay for PABPC1 and smFISH for SARS-CoV-2 ORF1a 24 hours post-infection with SARS-CoV-2 in A549ACE2 or A549ACE2-RNase L-KO cells. Notably, RLBs form in SARS-CoV-2-infected WT cells, whereas SARS-CoV-2-infected RNase L-KO cells do not generate PABPC1-positive foci. Figure adapted from Burke et al., 2021b.
Figure 3.
Figure 3.. Distinct biogenesis pathways of SGs and RLBs
(A) Biogenesis pathway of SGs in response to viral dsRNA. (B) Biogenesis pathway of RLBs in response to viral dsRNA. (C) Live-cell imaging adapted from Burke et al., 2020. Panels show A549 cell expressing mRuby-2-PABPC1 following poly(I:C) lipofection at indicated times. RLB foci assemble at 60 minutes, disassemble by 120 minutes, but then re-assemble at 520 minutes.
Figure 4.
Figure 4.. RNA-binding proteins that localize to SG and/or RLBs.
The proteins that have been confirmed to localize specifically to either SGs or RLBs or that localize to both SGs and RLBs. The asterisks indicate proteins reported to localize to RLBs based on mass spectrometry, but have not been verified by immunofluorescence.
Figure 5.
Figure 5.. Hypothetical models for the functions of SGs, RNase L-mediated inhibition of SGs, and RLBs
(A) Stress granules have been shown to concentrate PRRs, which is modeled to promote their activation and/or activity. However, direct evidence that stress granules, as opposed to the constituents of stress granules, are responsible for this is unclear. This is because most SG-associated RNA-binding proteins, such as G3BP1, are primarily localized to the cytoplasm and can directly modulate antiviral signaling and/or viral gene expression. (B) RNase L inhibits both the assembly of SGs and promotes the disassembly of SGs. This could inhibit the pro-survival functions of SGs, promote the pro-apoptotic functions of RNase L, or could inhibit SG-mediated antiviral signaling. (C) Hypothetical model for RLB function. RLBs enrich for RNase L-cleaved RNAs and associated RBPs. The close association of RLBs with P-bodies allows for transfer of RNA fragments into P-bodies, which enrich for RNA decay machinery. This promotes rapid decay of mRNAs and releases RBPs for exchange with the cytoplasm and/or RLBs.
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