Abstract
Cytoplasmic viral RNA and DNA are recognized by RIG-I-like receptors and DNA sensors that include DAI, IFI16, DDX41, and cGAS. The RNA and DNA sensors evoke innate immune responses through the IPS-1 and STING adaptors. IPS-1 and STING activate TBK1 kinase. TBK1 is phosphorylated in its activation loop, leading to IRF3/7 activation and Type I interferon (IFN) production. IPS-1 and STING localize to the mitochondria and endoplasmic reticulum, respectively, whereas it is unclear where phosphorylated TBK1 is localized in response to cytoplasmic viral DNA. Here, we investigated phospho-TBK1 (p-TBK1) subcellular localization using a p-TBK1-specific antibody. Stimulation with vertebrate DNA by transfection increased p-TBK1 levels. Interestingly, stimulation-induced p-TBK1 exhibited mitochondrial localization in HeLa and HepG2 cells and colocalized with mitochondrial IPS-1 and MFN-1. Hepatitis B virus DNA stimulation or herpes simplex virus type-1 infection also induced p-TBK1 mitochondrial localization in HeLa cells, indicating that cytoplasmic viral DNA induces p-TBK1 mitochondrial localization in HeLa cells. In contrast, p-TBK1 did not show mitochondrial localization in RAW264.7, L929, or T-23 cells, and most of p-TBK1 colocalized with STING in response to cytoplasmic DNA in those mammalian cells, indicating cell type-specific localization of p-TBK1 in response to cytoplasmic viral DNA. A previous knockout study showed that mouse IPS-1 was dispensable for Type I IFN production in response to cytoplasmic DNA. However, we found that knockdown of IPS-1 markedly reduced p-TBK1 levels in HeLa cells. Taken together, our data elucidated the cell type-specific subcellular localization of p-TBK1 and a cell type-specific role of IPS-1 in TBK1 activation in response to cytoplasmic viral DNA.
Highlights
RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that play an essential role in Type I interferon (IFN) expression in response to RNA virus infection [1]
We found that exogenous expression of RIG-I CARDs, TICAM-1, IPS-1, or STING induced TBK1 phosphorylation, whereas total TBK1 levels were not affected (Figure 1A)
Total TBK1 was dispersed through the cytoplasm, whereas p-TBK1 exhibited mitochondrial localization in HeLa cells that expressed RIG-I CARDs, IPS-1, or STING (Figure 1B and 1C)
Summary
RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that play an essential role in Type I interferon (IFN) expression in response to RNA virus infection [1]. A recent study reported that RLRs localize on antiviral stress granules in response to cytoplasmic polyI:C or viral infection [2]. A recent study reported that a part of IPS-1 localizes on mitochondriaassociated membranes (MAMs), which is a distinct membrane compartment that links the endoplasmic reticulum (ER) to the mitochondria [8]. Toll-like receptor 3 (TLR3) recognizes viral dsRNA and polyI:C; TLR3 localizes to early endosomes or the cell surface and requires the adaptor TICAM-1 to induce Type I IFN expression [14,15,16]
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