Activation of the SARS-CoV-2 spike protein by 𝘤𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 secreted proteases

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Mjokane, Nozethu
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University of the Free State
The thesis is not structured in a classical way. As such, it is composed of a literature review section (Chapter 1) and two research chapters (Chapters 2 and 3). A general discussion section (Chapter 4) and addendums are also included. As some chapters are in a publication format, repetition of essential information could not be avoided. Chapter 1 reviews the emergence of SARS-CoV-2 and its impact. In particular, it considers the co-infection of this virus with respiratory fungal pathogens, which are major independent risk factors that complicate COVID-19 by causing a more severe infection resulting in higher mortality than that of either infection on its own. These fungal pathogens secreted furin-like proteases to further their virulence during host invasion. In this context, the thesis argues that it is foreseeable that the virus could also access these fungal furin-like proteases and pervert them in order to activate its latent spike protein. Therefore, this set up a number of questions, which are addressed in the thesis concerning the possible activation of the viral latent spike protein by fungal furin-like proteases. In Chapter 2, it was sought to characterise 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 (𝘊.) neoformans proteases and assess if they could theoretically bind to the SARS-CoV-2 spike protein. To be specific, previous papers reporting on cryptococcal serine proteases were perused, and this made it possible to select a number of proteases, namely cryptococcal serine carboxypeptidase (CNBF4600), cryptococcal cerevisin (CNBJ2870) and cryptococcal peptidase (CNBA1340), cryptococcal peptidase (CNAG_00150) and cryptococcal cerevisin (CNAG_04625). By designing specific primers, it was possible to show that these serine proteases were expressed in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 H99, the prototypical cryptococcal strain used in this thesis. Therefore, the expressed gene products were expected to be secreted into the culture media. This was important for the work that follows in Chapter 3. Through using the computational programme, High Ambiguity Driven protein-protein DOCKing (HADDOCK), it was possible to show that some of the selected cryptococcal serine proteases could interact with the coronavirus spike protein and yield a binding affinity greater than and comparable to furin. However, as HADDOCK is a computational programme, the predicted binding affinities might not correlate with the experimental binding affinities in solution, more so since the used 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 proteases structures were predicted and not solved. To account for this, Chapter 3 sought to provide enzymatic evidence using the collected culture media – in the form of supernatant. To do this, a mimetic fluorogenic peptide of the SARS-CoV-2 spike protein was designed and modified to have intra-molecular fluorescence quenching capability using 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. The assay was performed using the cryptococcal supernatant. For reference, recombinant furin was included as this is the serine protease present in humans that catalyses the activation of the spike protein. Here, it was determined that cryptococcal serine proteases present in the supernatant could cleave the mimetic spike protein at S1/S2 site with biochemical efficiency comparable to furin. To test the veracity of these data, SARS-CoV-2 pseudovirion containing a full-length spike protein was used. It was possible to show that the pseudovirion could be transduced into HEK-293T cells in the presence of the cryptococcal supernatant. Chapter 4 takes into account the obtained results and provides a summary of the major observations. Of note, the thesis theorises that yeast kexin proteases are responsible for the observed activity. This is because there is a functional homology between yeast kexin proteases and furin (both are convertases); thus, it is reasonable that the supernatant (which contains yeast kexin proteases) could activate the latent SARS-CoV-2 spike protein. The thesis further proves that other respiratory fungal pathogens have yeast kexin proteases that activate the spike protein. This evidence is documented in Addendum no. 1. All things considered, the findings point to the regulation of protease activity as a viable approach to control the activation of the spike protein by either mammalian protease or fungal proteases. To this end, protease inhibitors could be used to control unwanted proteolysis. Addendum no. 2 attempted to show this. Here, it was possible to show that the South African-based medicinal plant Artemisia tea infusion extract and its active compound artemisinin could control the activation of the mimetic SARS-CoV-2 spike protein by furin but not the supernatant. The latter highlights the need to purify the supernatant and isolate yeast kexin proteases. The idea of exploring the control of unwanted proteolysis is also an interventional measure considered by Pfizer, the pharmaceutical company. This American multinational pharmaceutical and biotechnology corporation successfully piloted Paxlovid to control SARS-CoV-2. This drug contains an anti-protease (PF-07321332) that inhibits the protease (SARS-CoV-2 3CLp) responsible for viral replication.
Thesis (Ph.D.(Microbiology and Biochemistry))--University of the Free State, 2023