Introduction
A large number of people in our world are suffering from viral diseases. The majority of these viruses do not have a well-understood life cycle and mechanism of pathogenesis, leading to poor understanding and treatment of the diseases caused by them. Several studies carried out in the recent past have identified a few unique pathways and proteins which are involved in the pathogenic response of different viral species, e.g., poxviruses and rhinoviruses (RVs). cGAS-STING plays a vital role in the virulence of many viruses, and its role varies in response to individual pathogens. For studying Poxviruses, the ectromelia virus (ECTV), causing mousepox, was used to study the impact of the cGAS-STING pathway on its virulence due to its similarities with OPV (Cheng et al. 2018). A few research articles are reviewed that discuss the role of chemical mediators in the pathogenesis and life cycle of a few viral diseases. In 2008, a protein called stimulator of interferon genes (STING) was found as the primary stimulator of type-1 interferon (IFN) in response to foreign DNA. A ligand protein for STING called cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthesized by “cyclic-GMP-AMP synthase” (cGAS) was discovered in 2013 (Wan et al. 2020). RVs are a huge source of upper and lower respiratory tract infections around the globe. A unique genotype of RVs, RV-C, was studied in relation to STING due to the poor understanding of its mechanism, and relatively severe disease caused by it (Gagliardi et al. 2022).
Materials and Methods
In the research carried out on poxviruses, the WT strain of ECTV was obtained and inserted into Vero cells through various plasmids. After that, transfection was done, and luciferase activity was calculated. RNA samples were isolated, and interference was run with artificially manufactured siRNA. Macrophage samples were obtained from the peritoneal cells of infected mice kept under controlled conditions at specified intervals for ELISA assay and western blot analysis. A PCR was done to calculate DNA copies, and histologic analysis was performed. (Cheng et al. 2018).The
cGAS pathway gets activated after identifying B-type foreign dsDNA in the cytoplasm of the cell. This activation produces cGAMP and causes the digestion of the cGAS-DNA complex. cGAMP can cross cell borders in several ways, causing dimerization of STING, leading to increased formation of type-1 IFN. After ubiquitination, cGAS-STING is lysed through autophagosomes. Factors affecting the regulation have been tabulated. This pathway can interact with other DNA-sensing pathways of the cell, having a positive or negative impact on apoptosis. Increased IFN production contributes to cell ageing. cGAS-STING pathway plays a vital role in defence against certain viral (HIV, herpes), bacterial (mycobacterium) and protozoal (T. gondii) diseases. On the other hand, disorders of the cGAS-STING pathway can cause autoimmune diseases like SLE. It can also contribute to certain inflammatory diseases like non-alcoholic steatohepatitis
(NASH) and scoliosis. Certain neurodegenerative disorders, like Parkinson’s disease (PD) are complicated by the apoptotic cascade that this pathway triggers.IFN-1 plays an essential role in
regressing tumour cells and limiting their abnormal growth. However, certain research demonstrated STING to act as a double-edged sword by increasing chronic inflammation and contributing to the development of certain tumours. This interaction of cGAS-STING with malignant cells is complicated and depends upon several variable factors involved. The practical ability to modulate the cGAS-STING pathway for the treatment of certain diseases was put to the test. It can help by boosting the immunological status on the one hand and by controlling abnormal inflammation on the other hand. The effect of certain anti-tumour drugs, like CD47 blockers, was reduced in mice in the absence of STING. A similar decrease in the effectiveness of certain anti-cancerous drugs was seen when they were used without a STING agonist. A few STING agonists like dimethylxanthenone-4-acetic acid (DMXAA), 10-carboxymethyl-9 (10H) acridone (CMA) and amidobenzimidazole have been tested for practical application. Certain drugs like aspirin and suramine have been found to have an inhibitory effect on cGAS. (Wan et al. 2020).
Human airway epithelial (HAE) cells were taken from four subjects and were placed in in vitro cultures of the human upper airway tract. Different variants of RV were obtained. STING was removed using a variant of CRISPR. Variants of RV were incubated at different temperatures for a fixed period, and then they were quantified through a qPCR. Before inoculating RV cells into modified HAE, they were rinsed with 100μl of phosphate-buffered saline (PBS) to make them optimal for use. Cultures were obtained after the addition of buffers from various chambers and stored for Western blot (WB) analysis at specified temperatures. After numerous rounds of incubation and washing of the apical surface of the culture in a controlled way, fluorescent antibodies were applied along with stains. All the required imaging was taken with a Zeiss Axio Observer 3 Inverted fluorescence microscope, and the same was done for all assays. The calculation of fluorescent levels was done by the Corrected Total Cell Fluorescence (CTCF).
A method and colocalization study were conducted using the specified method. The method used in this study was based on the issued guidelines of Transmission Electron Microscopy (TEM). Immunoblotting assays were carried out with primary and secondary antibodies. In order to determine the amount of lactate dehydrogenase produced as a response to RV infection, samples were taken and studied with the Cytotoxicity Assay kit. The Transepithelial Electrical Resistance (TEER) assay was also measured. Junction Analyzer Program (JAnaP) was applied to measure the properties of the junctional region. The clearance of mucociliary transport was assessed via transport of 2μm red-fluorescent polystyrene microspheres. (Gagliardi et al. 2022).
Results
The results of the first research showed that ECTV infection was able to produce IFN-1 in certain cells while failing to do so in others. An increase in the production of IFN-β only in the co-presence of STING and cGAS pointed towards the fact that they are essential for its production. It was determined that the cGAS-STING pathway was essential for the making of Tbk1 and Irf3 during ECTV infection. It was observed in knocked-out macrophages, infected by ECTV, that either the levels of IFN were reduced or completely depleted. This signified the driving value of cGas–Sting–Tbk1–Irf3 Pathway in response to viral infections. In vitro experimentation demonstrated the significance of cGAS and STING to limit the replication of the ECTV virus. In conclusion, this pathway was also found to be essential for developing resistance against mousepox in vivo. Even in the presence of TLR9, the absence of cGAS and STING led to an inadequate resistance among mice. (Cheng et al. 2018).
In the second article, it was concluded that the cGAS-STING pathway performs a binary role in providing immunity. In some cases, it can enhance the immunological response while dampening it at other occasions. It’s pro-inflammatory can enhance the adaptive immunity response transiently and cause cell death in the long term, if needed to protect against harmful inflammatory reactions. It can provide the groundwork for understanding the inflammatory processes involved in neurological diseases. It can have practical application in curative medicine if appropriate drugs are developed to enhance the desirable effects of this pathway and mask the undesirable ones. (Wan et al. 2020).
In the third research, replication of RV-C15 was found to be associated with cells lined by cilia. It also shows a variation in the levels of CDHR3. The shaping of replicative complexes inside the RV was noted, and infection caused the formation of a number of accumulations filled with pockets. The Golgi apparatus serves as the raw material for the formation of complexes during infection, causing a surge in PIP4 levels. Endoplasmic reticulum was found to be the host site for the multiplication of the genome for certain viruses like RV-C15. This virus showed the ability to generate incomplete autophagy because it was unable to inhibit autolysosome production. An increase in the generation of STING was beneficial for the multiplication of the viral genome. RV-C15 was determined to play a pivotal role in changing the permeability of the membrane and the arrangement of proteins present in the junctional zone. (Gagliardi et al. 2022).
Conclusion
All these experiments were carried out on non-human subjects, which greatly limits the validity of the results obtained by these trials. There is a great contradiction in the responses produced by the cGAS-STING pathway to the same genus of pathogens. This response also varies with the type of cells involved, the type of overall reaction, and interaction with other pathways. All these factors leave many question marks on the practical application of this pathway. There is still a lot of work to be done in completely understanding the mechanism of this pathway in humans. Human testing can only be possible after and only if the safety of its application is confirmed. Moreover, a thorough review needs to be done about the cost-benefit relationship of all these projects. These experiments would have had a better impact if different species were tested upon and the results could be cross-referenced for the possibility of human application. To conclude, understanding this pathway is a work in progress with bright prospects provided that the trials are systematically organized towards its practical application shortly.
Referecnes
- Cheng et al. (2018) ‘The cGAS–sting Signalling Pathway is required for the innate immune response against Ectromelia Virus’ Frontiers in Immunology. Available at: https://doi.org/10.3389/fimmu.2018.01297
- Gagliardi et al. (2022) ‘Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium’ Plos Pathogens. Available at: https://doi.org/10.1371/journal.ppat.1010159
- Wan et al. (2020) ‘Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response’ Frontiers in Immunology. Available at: https://doi.org/10.3389/fimmu.2020.00615