Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells

Abstract

It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells. Mammalian cells express various receptors to sense invading pathogens. Specialized receptors that survey the intracellular milieu are the NOD-like receptors (NLRs), the RIG-I-like receptors (RLRs), and numerous receptors that detect cytosolic DNA (1). Recently, a ubiquitously expressed DNA sensor, cyclic GMP-AMP synthase (cGAS), has been identified (2, 3). Upon binding of cytosolic DNA, like herring testis DNA and poly(dA: dT), cGAS produces cyclic GMP-AMP signaling molecules that subsequently activate stimulator of interferon genes (STING [4,5]). STING then activates a cascade that results in the synthesis and secretion of type I interferons (interferon alpha/beta [IFN-␣/ ␤]), ultimately leading to the induction of an antiviral state of the cells.Plasmid DNA transfection is often used as a transient gene delivery system to overexpress proteins (e.g., dominant negative or green fluorescent protein [GFP] fusion proteins). Unfortunately, in many cases the transfection procedure blocks virus infection. Although it is conceivable that transfected plasmid DNA activates the cGAS/STING pathway, which might lead to an antiviral state of the cells, this has not been properly investigated.To study the plasmid DNA-mediated repression of virus infection, we transfected red fluorescent protein (RFP)-encoding plasmid DNA into HeLa-R19 and BGM cells using Fugene 6 (similar experiments, with comparable results, have been performed using Lipofectamine 2000 and JetPRIME [data not shown]). Twentyfour hours posttransfection, the cells were infected with the coxsackievirus B3-enhanced GFP (CVB3-EGFP) and mengovirus-EGFP (mengo-EGFP) reporter viruses, and infection efficiency was monitored using flow cytometry analysis. We observed a marked decrease in CVB3-EGFP infection efficiency in plasmid DNA-transfected HeLa-R19 cells (from 70% to 31%) and BGM cells (from 43% to 11%) compared to mock-treated cells (Fig. 1A). A comparable decrease in infection efficiency was also observed for mengo-EGFP (Fig. 1B). It is important to note that virus infection was hampered in both RFP-positive and RFP-negative cells. This suggests that plasmid DNA transfection induces the secretion of a soluble factor that restricts virus infection, possibly cGAS/STING-induced IFN-␣/␤. To investigate this, we performed transfection/infection experiments in HEK293T and Vero-E6 cells, which are known to be deficient in cGAS and STING signaling (3, 6) and the production of IFN-␣/␤ (7), respectively. In these cells, plasmid DNA transfection...

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