Leaked genomic and mitochondrial DNA contribute to the host response to noroviruses in a STING-dependent manner

Abstract

The cGAS-STING pathway is central to the IFN response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here we show that the cGAS-STING pathway also contributes to the IFN response against noroviruses, positive-sense single-stranded RNA viruses that are now one of the most common causes of infectious gastroenteritis world-wide. We show a significant reduction in IFN-β induction and a corresponding increase in viral replication in norovirus-infected cells following STING inhibition, knockdown or deletion. Upstream of STING, we show that cells lacking either cGAS or IFI16 also have severely impaired IFN responses. Further, we demonstrate that immunostimulatory host genome-derived DNA, and to a lesser extent mitochondrial DNA, accumulate in the cytosol of norovirus-infected cells. And lastly, overexpression of the viral NS4 protein was sufficient to drive the accumulation of cytosolic DNA. Together, our data elucidate a role for cGAS, IFI16 and STING in the restriction of noroviruses, and demonstrate for the first time the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus.

TRIM7 Restricts Coxsackievirus and Norovirus Infection by Detecting the C-Terminal Glutamine Generated by 3C Protease Processing

“…To generate the relevant lentiviruses, the untagged pSMPP-TRIM7 constructs were co-transfected into HEK293T cells with pMDG2 (a gift from D. Trono [EPFL; Addgene, plasmid 12259]) and pCRV-GagPol (a gift from S. Neil, Kings College London) plasmids using FuGENE6 (Promega) reagent and incubated for 3 days. Supernatants were harvested by filtration and used for transduction of HeLa cells expressing the mouse CD300lf [ 28 ] as previously described [ 20 ]. Briefly, 2.5 × 10 5 cells were infected with the appropriate lentivirus in a 6-well plate in DMEM supplemented with 3% FBS, 20 mM HEPES and 4 μg/mL Polybrene, and spinoculated at 1000× g and 25 °C for 45 min.…”

Crosstalk between RNA viruses and DNA sensors: Role of the cGAS‐STING signalling pathway

“…[76][77][78] Similarly, researchers revealed that plasmid DNA transfection activates the cGAS-STING pathway, preparing the cells to be in an antiviral state that compromises the infectivity of coxsackievirus B3 (CVB3). 79 The cGAS-STING pathway also robustly evokes IFN response and effectively inhibits infection of several other RNA viruses, including chikungunya virus (CHIKV), 80,81 encephalomyocarditis virus (EMCV), 68,71 hepatitis C virus (HCV), 82 influenza A virus (IAV), 71,83 measles virus (MeV), 84,85 murine norovirus (MNV), 86,87 Nipah virus (NiV), 84 and Zika virus (ZIKV). 88 Surprisingly, in addition to antiviral role, STING has been discovered to be proviral in rare cases of RNA viral infections.…”

“…71 A number of studies have reported that RNA viral infection can induce mtDNA leakage, consequently prompting the activation of the cGAS-STING axis. For example, infection with CHIKV, 81 DENV, 64,65 IAV, 71 MeV, 85 and MNV 86 EMCV infection, respectively, in a mitochondrial antiviral signalling (MAVS)-dependent manner. 71 (Figure 2).…”

“…A number of studies have reported that RNA viral infection can induce mtDNA leakage, consequently prompting the activation of the cGAS‐STING axis. For example, infection with CHIKV, 81 DENV, 64,65 IAV, 71 MeV, 85 and MNV 86 has been shown to cause mitochondrial damage and cytosolic release of mtDNA, which can be sensed by cGAS and leads to STING activation and downstream IFN‐I induction. The M2 of IAV and 2B of EMCV, both of which possess viroporin activity, were identified as key viral proteins responsible for cytosolic mtDNA release observed during IAV and EMCV infection, respectively, in a mitochondrial antiviral signalling (MAVS)‐dependent manner 71 .…”

TRIM7 restricts Coxsackievirus and norovirus infection by detecting the C-terminal glutamine generated by 3C protease processing

“…To generate TRIM7 lentiviruses pSMPP-hTRIM7 constructs were co-transfected into HEK293T cells with pMDG2 and pCRV-GagPol plasmids using FuGENE6 (Promega) reagent and incubated for 3 days. Supernatants were harvested by filtration and used for transduction of HeLa cells expressing the mouse CD300lf (Jahun et al, 2021) as previously described (Fan et al, 2021). Briefly, 2.5E5 cells were infected with the appropriate lentivirus in a 6-well plate in DMEM supplemented with 3% FBS, 20 mM HEPES, and 4 µg/ml Polybrene, and spinoculated at 1000 × g and 25°C for 45 min.…”