Severe coronavirus disease 2019 (COVID-19) is associated with the development of acute respiratory distress syndrome (ARDS), characterized by exacerbated inflammation in the lungs and significantly upregulated production of cytokines. The increased expression of genes associated with interferon production is often observed in those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, though serum levels are frequently unexpectedly low. Intranasal, though not intravenous, administration of type I interferon has also been shown to be beneficial to mice infected with SARS-CoV-2, suggesting that the way in which the cells of the airway and alveolar detect and respond to the virus by producing interferon may have been negatively impacted following infection.
The early cellular response to viral challenge is largely mediated by proteins that detect foreign nucleic acid sequences in the cytosol. Retinoic acid-inducible gene-I-like receptors are a family of pattern recognition receptor proteins that have been implicated in the sensing of coronaviruses. Two proteins within this family that detect specific types of RNA structure are: retinoic acid-inducible 60 gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5). Once bound with viral RNA, these proteins trigger a signaling cascade that results in the production of cytokines such as interferon. In a paper recently uploaded to the preprint server bioRxiv*, researchers explored the effect of SARS-CoV-2 infection on the function of RIG-I and MDA5, and ultimately on interferon production.
The role of pattern recognition receptors in SARS-CoV-2 sensing
The group screened several cell lines for their ability to host SARS-CoV-2 and detectably produce virus nucleocapsid RNA within 24 hours, finding that Calu-3, HEK293, and Huh7 cells, could host the virus, but only Calu-3 cells induced a detectable cytokine response. Calu-3 cells are sourced from human lung tissue and may therefore be better evolved to provide an immune response against coronaviruses. Upregulation of type I and III interferons was observed upon infection, alongside other proinflammatory cytokines such as interleukin-6, and the major immune signaling pathways were activated, including RIG-I and MDA5.
Short hairpin RNA (shRNA) is a synthetic molecule used to silence gene expression by RNA interference. shRNA was used to knockdown mitochondrial antiviral-signaling protein (MAVS), with which both RIG-I and MDA5 must interact following binding with viral RNA. Knockdown cells experienced a higher load of SARS-CoV-2 and exhibited depleted interferon levels, though the levels of mRNA coding for some other cytokines (interleukin-6 and tumor necrosis factor) were not altered.
Cells with knocked out MDA5 or interferon regulatory factor 3 (IRF3), which is activated downstream of MAVS, showed the lowest concentration of interferon coding mRNAs in response to SARS-CoV-2 infection, while also maintaining IL-6 and TNF levels. Knocking out RIG-I or stimulator of interferon genes (STING), a cystolic DNA sensor, had little influence on SARS-CoV-2 infection, suggesting that the cystolic GMP-AMP synthase-STING pathway is not involved in SARS-CoV-2 sensing.
Interferon suppression by SARS-CoV-2
The group demonstrated that the RNA sensor MDA5 and its downstream partners MAVS and IRF3 are involved in the type I and III interferon response to SARS-CoV-2, while RIG-I is largely uninvolved, as has been supported by several other studies. However, the authors highlight several reports of opposing results, which include indications that RIG-I is involved in sensing additionally or in lieu of MDA5. A diverse range of other pattern recognition receptors are involved in the immune response, and the reason for the observed variation is not yet entirely understood. The STING system, for example, has been shown to be activated following cell damage by other infections such as Dengue virus. Though the response to coronavirus is not yet known, it could become active at time points later than observed in this study.
Importantly, the group noticed that infected cells demonstrated lowered MxA protein production compared with uninfected bystanders, a virus inhibitor expressed in response to interferon, IL-6, and other cytokines. This suggests that SARS-CoV-2 suppresses an upstream elicitor of this protein, indicated to be interferon, by encoding a viral protein that interferes with the system, as has been suggested by this and other work. Open reading frame 3a of SARS-Cov-1 is known to target and inhibit the interferon alpha/beta receptor, a widespread membrane receptor of type I interferon, and the group suggests that SARS-CoV-2 likely produces additional proteins that block interferon signaling.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.