Cytoplasmic‐translocated Ku70 senses intracellular DNA and mediates interferon‐lambda1 induction

Abstract

We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN-k1) rather than IFN-alpha or IFN-beta, through a STING-dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN-k1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA-triggered IFN-k1-inducible cells. Additionally, infection by Herpes simplex virus type-1 (HSV-1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN-k1 induction in a strain-dependent manner: the translocation and IFN-k1 induction were detected upon infection by HSV-1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN-k1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70's cytoplasmic accumulation, and therefore increases DNA-mediated IFN-k1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization-dependent manner.

The Role of Ku70 as a Cytosolic DNA Sensor in Innate Immunity and Beyond

“…All this happened before IFN-l1 induction. Consequently, it is reasonable to speculate that the translocation of Ku70 happened first and that IFN-l1 induction occurred later since we further confirmed that recombinant IFN-l1 does not induce the cytoplasmic translocation of Ku70 (Sui et al, 2021). Like Ku70, IFI16, another DNA sensor protein, has been reported to recognize and sense DNA not only in the cytoplasm but also in the nucleus of the cells, and its sensing capabilities depend on the distribution of IFI16 (Li et al, 2012;Dell'oste et al, 2014;Ansari et al, 2015).…”

“…Ku70 cytoplasmic translocation and IFN-l1 induction only in HEK cells infected with the HSV-1 McKrae strain. Those results further emphasized that the cytoplasmic translocation of Ku70 is a required step for Ku70-mediated IFN-l1 induction (Sui et al, 2021). A quantification analysis with Western blot using cytosolic fractions was adapted to characterize the accumulation kinetics of cytoplasmic Ku70.…”

“…And it has been consistently confirmed in our previous study that Ku80 translocates from the nucleus to the cytoplasm together with Ku70. The confocal microscopy analysis indicated that Ku70 and Ku80 colocalized together in the nucleus of unstimulated cells, and then both translocate from the nucleus to the cytoplasm upon a DNA stimulation (Sui et al, 2021). However, Koik et al demonstrated that the localization of Ku80 does not entirely coincide with that of Ku70, Ku80 protein was transported to the nucleus without heterodimerization with Ku70.…”

“…Bax, a cytoplasmic protein, has been discovered to interact with Ku70, and this Ku70-Bax binding is indicated to inhibit Bax-mediated apoptosis ( Cohen et al., 2004 ). In addition, many studies have implicated that cytosolic Ku has been shown to serve as a PRR that recognizes viral DNA in human cells and then induces type I and type III interferons or proinflammatory cytokines ( Zhang et al., 2011a ; Ferguson et al., 2012 ; Li et al., 2016b ; Sui et al., 2017 ; Wang et al., 2017 ; Burleigh et al., 2020 ; Sui et al., 2021 ; Wang et al., 2021 ). This article summarized and discussed the Ku70-mediated innate immune response in detail and then highlighted potential strategies to modulate the innate immune cascades.…”

“…Ku70 is identified as a cytosolic DNA sensor that induces type III IFNs through a STING-TBK1-IRF3, IRF1, and IRF7 signaling pathway. In this signaling pathway, cytoplasmic translocation of Ku70 is an initial and essential step (black arrows) ( Zhang et al., 2011a ; Sui et al., 2017 ; Sui et al., 2021 ); Ku70 and Ku80, together with DNA-PKcs (DNA-PK), are also involved in a STING-dependent ( Ferguson et al., 2012 ) (solid arrow) and STING-independent ( Burleigh et al., 2020 ) (dashed arrow) pathway to induce type I IFNs (red arrows). Ku70 is reported to sense HTLV-1 transcription intermediate product ssDNA90 and interacts with STING to induce IFNs and inflammatory cytokines, thereby modulating HTLV-1 replication ( Wang et al., 2017 ) (brown arrows).…”