Activation of cGAS-STING suppresses coxsackievirus replication via interferon-dependent signaling.

Coxsackievirus B3 (CVB3) is a non-enveloped, single-stranded, positive RNA virus known for its role in provoking inflammatory diseases that affect the heart, pancreas, and brain, leading to conditions such as myocarditis, pancreatitis, and meningitis. Currently, there are no FDA-approved drugs treating CVB3 infection; therefore, identifying potential molecular targets for antiviral drug development is imperative. In this study, we examined the possibility of activating the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway, a cytosolic DNA-sensing pathway that triggers a type-I interferon (IFN) response, in inhibiting CVB3 infection. We found that activation of the cGAS-STING pathway through the application of cGAS (poly dA:dT and herring testes DNA) or STING agonists (2'3'-cGAMP and diamidobenzimidazole), or the overexpression of STING, significantly suppresses CVB3 replication. Conversely, gene-silencing of STING enhances viral replication. Mechanistically, we demonstrated that cGAS-STING activation combats CVB3 infection by inducing IFN response. Notably, we discovered that knockdown of IFN-α/ß receptor, a key membrane receptor in type-I IFN signaling, or inhibition of the downstream JAK1/2 signaling with ruxolitinib, mitigates the effects of STING activation, resulting in increased viral protein production. Furthermore, we investigated the interplay between CVB3 and the cGAS-STING pathway. We showed that CVB3 does not trigger cGAS-STING activation; instead, it antagonizes STING and the downstream TBK1 activation induced by cGAMP. In summary, our results provide insights into the interaction of an RNA virus and the DNA-sensing pathway, highlighting the potential for agonist activation of the cGAS-STING pathway in the development of anti-CVB3 drugs.

A combination of genetically engineered oncolytic virus and melittin-CpG for cancer viro-chemo-immunotherapy.

BACKGROUND: Immunotherapy has emerged as an efficient therapeutic approach for cancer management. However, stimulation of host immune system against cancer cells often fails to achieve promising clinical outcomes mainly owing to the immunosuppressive characteristics of the tumor microenvironment (TME). Combination therapeutics that can trigger sustained immunogenic cell death (ICD) have provided new opportunities for cancer treatment. METHODS: In this study, we designed and applied an ICD inducer regimen, including a genetically engineered oncolytic virus (miRNA-modified coxsackieviruses B3, miR-CVB3), a pore-forming lytic peptide (melittin, found in bee venom), and a synthetic toll-like receptor 9 ligand (CpG oligodeoxynucleotides), for breast cancer and melanoma treatment. We compared the anti-tumor efficacy of miR-CVB3 and CpG-melittin (CpGMel) alone and in combination (miR-CVB3 + CpGMel) and investigated possible mechanisms involved. RESULTS: We demonstrated that miR-CVB3 + CpGMel had no major impact on viral growth, while enhancing the cellular uptake of CpGMel in vitro. We further showed that combination therapy led to significant increases in tumor cell death and release of damage-associated molecular patterns compared with individual treatment. In vivo studies in 4T1 tumor-bearing Balb/c mice revealed that both primary and distant tumors were significantly suppressed, and the survival rate was significantly prolonged after administration of miR-CVB3 + CpGMel compared with single treatment. This anti-tumor effect was accompanied by increased ICD and immune cell infiltration into the TME. Safety analysis showed no significant pathological abnormalities in Balb/c mice. Furthermore, the developed therapeutic regimen also demonstrated a great anti-tumor activity in B16F10 melanoma tumor-bearing C57BL/6 J mice. CONCLUSIONS: Overall, our findings indicate that although single treatment using miR-CVB3 or CpGMel can efficiently delay tumor growth, combining oncolytic virus-based therapy can generate even stronger anti-tumor immunity, leading to a greater reduction in tumor size.

Viral proteases activate the CARD8 inflammasome in the human cardiovascular system.

Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.

Coxsackievirus Protease 2A Targets Host Protease ATG4A to Impair Autophagy.

Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to usurp the cellular recycling process of autophagy for pro-viral functions, the precise manner by which this is accomplished remains to be elucidated. In the current manuscript, we sought to address the mechanism by which EVs subvert the autophagy pathway using Coxsackievirus B3 (CVB3) as a model. We showed that CVB3 infection selectively degrades the autophagy cysteine protease ATG4A but not other isoforms. Exogenous expression of an N-terminally Flag-labeled ATG4A demonstrated the emergence of a 43-kDa cleavage fragment following CVB3 infection. Furthermore, bioinformatics analysis coupled with site-directed mutagenesis and in vitro cleavage assays revealed that CVB3 protease 2A cleaves ATG4A before glycine 374. Using a combination of genetic silencing and overexpression studies, we demonstrated a novel pro-viral function for the autophagy protease ATG4A. Additionally, cleavage of ATG4A was associated with a loss of autophagy function of the truncated cleavage fragment. Collectively, our study identified ATG4A as a novel substrate of CVB3 protease, leading to disrupted host cellular function and sheds further light on viral mechanisms of autophagy dysregulation.

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

Despite only comprising half of all known viral species, RNA viruses are disproportionately responsible for many of the worst epidemics in human history, including outbreaks of influenza, poliomyelitis, Ebola, and most recently, the coronavirus disease-2019 (COVID-19) pandemic. The propensity for RNA viruses to replicate in cytosolic compartments has led to an evolutionary arms race and the emergence of cytosolic sensors to recognise and initiate the host innate immune response. Although significant progress has been made in identifying and characterising cytosolic RNA sensors as anti-viral innate immune factors, the potential role for cytosolic DNA sensors in RNA viral infection is only recently being appreciated. Among these, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has attracted increasing attention. The cGAS-STING signalling pathway has emerged as a key innate immune signalling axis that is implicated in diverse human diseases from infectious diseases to neurodegeneration and cancer. Here we review the existing literature on RNA viruses and their reciprocal interactions with the cGAS-STING pathway and share insights into RNA virus diversity by touching on the similarities and differences of RNA viral strategies.