Researchers uncover a new way the immune system detects coronavirus infection

By Tarun Sai LomteReviewed by Emily Henderson, B.Sc.Sep 27 2022

In a recent study posted to bioRxiv*, researchers in the United States demonstrated that the main protease (M^pro) of coronaviruses (CoVs) activates host inflammasome responses.

Effector-triggered immunity (ETI) is a defense strategy of hosts wherein innate sensors recognize pathogens, and a subset of ETI sensors in eukaryotes form inflammasomes. Inflammasomes are large immune complexes inside cells that activate pro-inflammatory caspases, mainly caspase 1 (CASP1), to trigger inflammatory signaling and pyroptotic cell death.

In humans, inflammasome-forming sensors such as caspase recruitment domain family member 8 (CARD8) sense the activity of viral proteases. CARD8 contains a disordered N-terminus and a C-terminal function-to-find domain (FIIND) and CARD. Self-cleavage of FIIND results in a bipartite sensor, where the N-terminus acts as a tripwire for viral proteases.

Study: Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome. Image Credit: Kateryna Kon / Shutterstock

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

The study and findings

In the present study, researchers evaluated the sensing of viral proteases by CARD8 inflammasome. Using a previously reported bioinformatic approach, the authors created a predictive model for the M^pro [also known as 3-chymotrypsin-like protease (3CL^pro)] of CoVs. They identified two putative M^pro cleavage sites in the N-terminus of human CARD8. The CARD8 isoform was co-expressed with M^pro of severe acute respiratory syndrome (SARS)-CoV-2.

A 34kDa CARD8 product was detected in the presence of SARS-CoV-2 M^pro, predicted to result from cleavage at Q37. The cleavage site was confirmed by mutating the residue at the site (to Q37A), which eliminated the 34kDa product after M^pro treatment. Interestingly, the Q37A mutant generated a cryptic M^pro-dependent 37kDa product, resulting from cleavage at Q61. Moreover, the Q37A Q61A mutant of CARD8 was insensitive to cleavage by SARS-CoV-2 M^pro.

Transfecting HEK293T cells with CASP1, pro-interleukin-1-beta (pro-IL-1β), and M^pro resulted in robust processing of pro-IL-1β to active IL-1. Inflammasome activation did not occur in CARD8 knocked-out (KO) cells. Further, CARD8 KO cells were complemented with wild-type CARD8 or cleavage site mutants. Wild-type CARD8 complementation rescued M^pro-induced inflammasome activation. Contrastingly, M^pro-induced inflammasome activation was reduced (with Q37A mutant) or abolished (with Q37A Q61A mutant) in the CARD8 KO cells.

Additionally, they tested whether proteases from other relevant CoVs also cleave CARD8. To this end, they cloned M^pros from other CoVs such as human CoV (HCoV)-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV, Middle-East respiratory syndrome (MERS)-CoV, and murine hepatitis virus (MHV). All tested proteases cleaved and activated CARD8 in a site-specific manner. In addition, the THP-1 cell line was infected with HCoV-229E to evaluate the consequences of CARD8 inflammasome activation.

HCoV-229E infection significantly resulted in the release of IL-1β and cell death of wild-type cells but not CARD8 KO cells. The wild-type or CARD8-KO THP-1 cell line was engineered to express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), and infected with SARS-CoV-2. Cell death and IL-1β release were evident in ACE2 and TMPRSS2-expressing wild-type cells but not in KO cells.

Next, the research team investigated whether the CARD8 inflammasome served as a M^pro sensor in Rousettus aegyptiacus (a megabat species). The megabat CARD8 lacks Q37 and Q61 and was not cleaved by the M^pro of SARS-CoV-2. A TEV^pro cleavage site was inserted into the N-terminus of megabat CARD8 to determine if functional degradation could activate it. TEV^pro cleavage of the engineered CARD8 resulted in inflammasome activation. Further investigations revealed that SARS-CoV-2 M^pro failed to activate and instead antagonized the TEV^pro-mediated activation of the bat CARD8. Similarly, all other M^pros prevented TEV^pro-mediated activation of the bat CARD8 inflammasome.

The researchers analyzed non-synonymous single-nucleotide polymorphisms (SNPs) in CARD8 within the human population. They found multiple high-frequency SNPs, including an S39P variant residing at the P2’ position within the proteolytic cleavage site that was present in more than 20% of African and Afro-American samples. The CARD8 P39 variant showed reduced sensitivity to cleavage and activation by CoV Mpros. 

Lastly, the team investigated whether the CARD8 P39 variant affected the recognition of other pathogens. The P2’ proline in the cleavage site was preferred for the enterovirus 3C protease (3C^pro) prediction model. Cleavage assays with 3C^pro from human rhinovirus (HRV), a picornavirus, revealed that CARD8 cleavage at Q37 was more pronounced for the P39 variant. Moreover, inflammasome activation by HRV 3C^pro was almost absent in HEK293T CARD8 KO cells, complemented with CARD8 S39 but robust when complemented with CARD8 P39. The team also observed that variations in CARD8 affected cleavage by 3C^pros from other picornaviruses.

Conclusions

Overall, the study demonstrated that CARD8 is a polymorphic, rapidly evolving innate immune sensor of infection by positive-sense RNA viruses. That SARS-CoV-2 infection causes CARD8 inflammasome activation in THP-1 cells underscores the role of inflammasomes in severe coronavirus disease 2019 (COVID-19), suggesting that CARD8 inflammasome in macrophages/monocytes contribute to inflammation in COVID-19 patients.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources