Showing we still CARE – how we have moved the world closer to pandemic preparedness

As we approach the five-year anniversary of the coronavirus pandemic, the CARE consortium draws to a close, handing the baton to other initiatives who continue the cause to get the world to a position of pandemic preparedness.

Following our series of work package infographics which were published throughout 2024, we conclude the series with a final infographic which brings together a summary of CARE: the objectives it set out with when it was rapidly mobilized in April 2020, a selection of some of its many achievements during the past five years against the shifting context of coronavirus variants, and most importantly the outcomes it has realised.

There are a number of useful resources: scientific publications, public deliverables and datasets which can be accessed on the CARE website and also reached via the infographic.

The infographic is also available here

Published in IJBM: SARS-CoV-2 Mpro oligomerization as a potential target for therapy

The CARE partner Jagiellonian University (JU) explored the mechanisms of dimerization (formation of dimers) of SARS-CoV-2 main protease (Mpro) to evaluate the impact of blocking this dimerization on the virus replication. Indeed the enzyme Mpro is critical in the virus’s replication cycle, facilitating the maturation of polyproteins into functional units. Currently available COVID-19 treatments target the Mpro.

Knowing that dimerization is indispensable for Mpro activity, JU decided to evaluate the impact of mutating two amino acids located in two different locations of the Mpro that are involved in dimerization. For this purpose, they used a range of techniques to evaluate the state (monomer or dimer) of different variants of Mpro, including biochemistry studies, biophysical analyses, together with structural, molecular dynamics, and quantum mechanical analysis.

Among the two amino acids studied (Arginine in position 4 – Arg4 – and Arginine in position 298 – Arg298), the results show that Arg4 is not the main driver for dimerization and high enzyme activity, while mutation of Arg298 completely abolishes the dimer formation and results in significant activity loss. Mutation of both arginines results in detrimental effects.

This research deepens our knowledge of the potential of inhibition of Mpro’s enzymatic activity by preventing dimerization, offering complementary approaches to limit SARS-CoV-2 virulence. This is a new perspective for the development of targeted therapies, to hit the most critical parts of the enzyme machinery. Moreover, given that the Mpro is highly conserved between different types of coronaviruses, these findings may help develop an antiviral drug effective against more than SARS-CoV-2 including potential new coronaviruses.

To learn more, click here:  SARS-CoV-2 Mpro oligomerization as a potential target for therapy

Published in NAR: formation of the SARS-CoV-2 methylation complex

The CARE partner Jagiellonian University (JU) deciphered the interaction between three viral proteins that are necessary to viral replication. To be able to replicate, coronaviruses modified their single-stranded RNA genome by adding a methylated cap to mimic the host cell mRNAs and thus highjack the host enzymes that will then replicate the virus RNA.

Three viral non-structural proteins – nsp14, nsp10 and nsp16 – have previously been shown to be involved in the implementation of the methylation cap, with nsp14 and nsp16 performing the methylation while nsp10 acts as a co-factor to both. However, structural information available suggested that the interaction between the three proteins would be impossible considering their conformation. To solve this mystery, JU applied orthogonal methodologies, i.e., used different techniques to evaluate the interactions between the three proteins.

The results shows that nsp14, nsp16 and nsp10 are able to form a trimer complex, built around nsp10. This trimer brings together two consecutive activities required for RNA cap formation, the one led by nsp14 and the one led by nsp16, likely contributing to the RNA capping mechanism.

These findings contribute to the current understanding of the intricate interplay among nsp14, nsp10 and nsp16 during the replication process, particularly in the context of RNA cap methylation in the coronaviral genome. This research may have implications for the development of novel targeted therapeutic strategies for the control of coronaviral infections.

To learn more, click here: Despite the odds: formation of the SARS-CoV-2 methylation complex

Published in Virology: a new cell line permissive to human coronavirus 229E

The CARE partner Jagiellonian University (JU) developed a new cell line that can be infected by the human coronavirus 229E (HCoV-229E), a member of the alphacoronavirus family. HCoV-229E is one of the coronaviruses that poorly propagate in cell lines and require extensive cell culture adaptation leading to changes in the virus phenotype. New cell lines are thus necessary to study in vitro HCoV-229E more easily. 

To create this new cell line, JU co-transduced CD13 and transmembrane serine protease 2 (TMPRSS2) in A549 cell line (lung-derived cell line) using lentiviral vectors. HCoV-229E can use several pathways to enter a permissive cell, one using CD13, an aminopeptidase N while another one is using TMPRSS2 to proteolytically prime the viral spike, leading to membrane fusion. Cells overexpressing CD13 and TMPRSS2 (A549++ cells) and infected by HCoV-229 showed viral RNA replication and a significant rise on virus titer, confirming their ability to produce infectious HCoV-229E progeny.  

Moreover, JU evaluated the possible entry pathways of HCoV-229E in A549++ cells by inhibiting the TMPRSS2-mediated membrane fusion entry pathway and/or the endocytic pathway. The results indicate that although HCoV-229E prefers TMPRSS2-mediated entry, it can also readily use the endocytic pathway in the absence of TMPRSS2. 

JU has thus developed a robust and physiologically relevant cell line model that is permissive to HCoV-229E clinical isolate replication. The data provide further insight into the potential of lentiviral transduction in developing permissive cell models for viral infection studies. Moreover, this line constitutes a uniform platform for studies on multiple members of the coronaviridae family. 

To learn more, click here: An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E 

Published in American Chemical Society Pharmacology & Translational Science: Effective, but Safe? Physiologically Based Pharmacokinetic (PBPK)-Modeling-Based Dosing Study of Molnupiravir for Risk Assessment in Pediatric Subpopulations

Recognising that treatment options for vulnerable paediatric populations with COVID-19 are limited to intravenous remdisivir, the CARE team at Helmholtz Centre for Infection Research (HZI, Germany) wanted to validate the assumption that oral molnupiravir should not be prescribed to under 18-year-olds, due to the potential for cartilage and bone toxicity, which would put normal paediatric development at risk.

Through the application of physiologically based pharmacokinetic (PBPK) modelling, the HZI team was able to show that to match the efficacy of oral molnupiravir seen in adults, a proportionately much higher dose would be needed in paediatric populations, substantially increasing the toxicity risk.

To learn more, click here: Effective, but Safe? Physiologically Based Pharmacokinetic (PBPK)-Modeling-Based Dosing Study of Molnupiravir for Risk Assessment in Pediatric Subpopulations