Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2

Functional regulation via conformational dynamics is well known in structured proteins, but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 ( hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L- hIGFBP2) retains its intrinsic disorder upon binding IGF-1 but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

Stephan Oroszlan and the Proteolytic Processing of Retroviral Proteins: Following A Pro

Steve Oroszlan determined the sequences at the ends of virion proteins for a number of different retroviruses. This work led to the insight that the amino-terminal amino acid of the mature viral CA protein is always proline. In this remembrance, we review Steve’s work that led to this insight and show how that insight was a necessary precursor to the work we have done in the subsequent years exploring the cleavage rate determinants of viral protease processing sites and the multiple roles the amino-terminal proline of CA plays after protease cleavage liberates it from its position in a protease processing site.

Running With Scissors: Evolutionary Conflicts Between Viral Proteases and the Host Immune System

Many pathogens encode proteases that serve to antagonize the host immune system. In particular, viruses with a positive-sense single-stranded RNA genome [(+)ssRNA], including picornaviruses, flaviviruses, and coronaviruses, encode proteases that are not only required for processing viral polyproteins into functional units but also manipulate crucial host cellular processes through their proteolytic activity. Because these proteases must cleave numerous polyprotein sites as well as diverse host targets, evolution of these viral proteases is expected to be highly constrained. However, despite this strong evolutionary constraint, mounting evidence suggests that viral proteases such as picornavirus 3C, flavivirus NS3, and coronavirus 3CL, are engaged in molecular ‘arms races’ with their targeted host factors, resulting in host- and virus-specific determinants of protease cleavage. In cases where protease-mediated cleavage results in host immune inactivation, recurrent host gene evolution can result in avoidance of cleavage by viral proteases. In other cases, such as recently described examples in NLRP1 and CARD8, hosts have evolved ‘tripwire’ sequences that mimic protease cleavage sites and activate an immune response upon cleavage. In both cases, host evolution may be responsible for driving viral protease evolution, helping explain why viral proteases and polyprotein sites are divergent among related viruses despite such strong evolutionary constraint. Importantly, these evolutionary conflicts result in diverse protease-host interactions even within closely related host and viral species, thereby contributing to host range, zoonotic potential, and pathogenicity of viral infection. Such examples highlight the importance of examining viral protease-host interactions through an evolutionary lens.

Ribonuclease zymogen induces cytotoxicity upon HIV-1 infection

Background Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. Methods We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. Results The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. Conclusions These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.

Peptide Location Fingerprinting Reveals Tissue Region-Specific Differences in Protein Structures in an Ageing Human Organ

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and -resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I α2, collagen II α1 and collagen V α1, were located within their triple-helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance. Several proteins (collagen V α1, collagen II α1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young samples, suggesting that some ageing mechanisms may act locally within tissues. This study not only reveals possible age-associated differences in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF as a proteomic tool to aid in biomarker discovery.

A genetic trap in yeast for inhibitors of SARS-CoV-2 main protease

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the E. coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor down to 10 μM by a robust growth increase. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates, and is rapidly adaptable for new variants of the protease with deviant site specificities.

Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.