Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway

Abstract

The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of -(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection. Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

Chikungunya virus antagonizes cGAS-STING mediated type-I interferon responses by degrading cGAS

“…Infectious virus release was quantified from the supernatant of infected cells and revealed a sharp increase in CHIKV replication from both the cGAS and STING null cells when compared to their WT counterpart ( Fig 3D ). Taken together, these data support previous reports that the cGAS-STING pathway restricts the replication of CHIKV[ 21 , 23 , 25 ] and provides direct evidence that the pathway poses a significant restriction upon viral replication.…”

“…cGAS and STING have been previously implicated in restricting the replication of alphaviruses[ 21 , 23 , 25 ]. In MEF and RAW 264.7 cells deficient for cGAS or STING, we observed that the individual proteins in this DNA-sensing pathway serve to functionally restrict CHIKV replication and infectious particle release.…”

“…Successful replication and subsequent release of viral progeny is largely dependent upon the ability of a virus to evade or inhibit both detection and activation of cellular antiviral responses. CHIKV is no exception, the type-I interferon (IFN) response has been demonstrated to be key in controlling CHIKV infection[ 21 – 26 ] and primary sensing of the virus is via the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I)-mitochondrial antiviral signaling protein (MAVS) axis[ 24 , 26 ]. Alternatively to pathogen associated molecular patterns (PAMPs), cell intrinsic molecules released as a result of cellular stress, termed danger associated molecular patterns (DAMPs), are also potent inducers of innate immune responses[ 27 , 28 ] which lead to the induction of type-I IFNs.…”

“…Upon cGAMP binding, STING dimerizes, translocates to the Trans-Golgi-Network (TGN), where it associates with Tank Binding Kinase 1 (TBK1), which ultimately results in phosphorylation of Interferon Regulatory Factor 3 (IRF3) and induction of type-I IFN transcription[ 41 , 42 , 49 , 50 ]. Interestingly, activation of the cGAS-STING pathway with chemical activators or via overexpression has been shown to restrict alphaviruses, including CHIKV[ 21 , 23 , 25 ]; however, no studies have identified direct antagonism of the cGAS-STING pathway by CHIKV. Given the importance of type-I IFN in restricting CHIKV, as well as recent work highlighting the relevance of the cGAS-STING pathway in restricting RNA viruses, we asked whether CHIKV was able to antagonize the cGAS-STING pathway.…”

High Throughput Screening of FDA-Approved Drug Library Reveals the Compounds that Promote IRF3-Mediated Pro-Apoptotic Pathway Inhibit Virus Replication

“…Using cells and mice expressing a RIPA active nt-IRF3 mutant, we demonstrated the relative contribution of RIPA to the antiviral response of IRF3 [7]. Several high throughput screens have revealed small molecules that regulate the transcriptional activity of IRF3 [39][40][41][42][43]. However, because RIPA is newly identified, there are no known regulators of this pathway.…”