Efficient production of fluorophore-labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase.

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti-inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti-chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion-tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N-terminal fusion partner is carried out with lab-produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab-produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP-fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti-inflammatory therapeutic, showing a binding constant for vCCI:vMIP-fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP-fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 µM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.

The Effect of Select SARS-CoV-2 N-Linked Glycan and Variant of Concern Spike Protein Mutations on C-Type Lectin-Receptor-Mediated Infection.

The SARS-CoV-2 virion has shown remarkable resilience, capable of mutating to escape immune detection and re-establishing infectious capabilities despite new vaccine rollouts. Therefore, there is a critical need to identify relatively immutable epitopes on the SARS-CoV-2 virion that are resistant to future mutations the virus may accumulate. While hACE2 has been identified as the receptor that mediates SARS-CoV-2 susceptibility, it is only modestly expressed in lung tissue. C-type lectin receptors like DC-SIGN can act as attachment sites to enhance SARS-CoV-2 infection of cells with moderate or low hACE2 expression. We developed an easy-to-implement assay system that allows for the testing of SARS-CoV-2 trans-infection. Using our assay, we assessed how SARS-CoV-2 Spike S1-domain glycans and spike proteins from different strains affected the ability of pseudotyped lentivirions to undergo DC-SIGN-mediated trans-infection. Through our experiments with seven glycan point mutants, two glycan cluster mutants and four strains of SARS-CoV-2 spike, we found that glycans N17 and N122 appear to have significant roles in maintaining COVID-19's infectious capabilities. We further found that the virus cannot retain infectivity upon the loss of multiple glycosylation sites, and that Omicron BA.2 pseudovirions may have an increased ability to bind to other non-lectin receptor proteins on the surface of cells. Taken together, our work opens the door to the development of new therapeutics that can target overlooked epitopes of the SARS-CoV-2 virion to prevent C-type lectin-receptor-mediated trans-infection in lung tissue.

The Antiviral Activity of the Lectin Griffithsin against SARS-CoV-2 Is Enhanced by the Presence of Structural Proteins.

Although COVID-19 transmission has been reduced by the advent of vaccinations and a variety of rapid monitoring techniques, the SARS-CoV-2 virus itself has shown a remarkable ability to mutate and persist. With this long track record of immune escape, researchers are still exploring prophylactic treatments to curtail future SARS-CoV-2 variants. Specifically, much focus has been placed on the antiviral lectin Griffithsin in preventing spike protein-mediated infection via the hACE2 receptor (direct infection). However, an oft-overlooked aspect of SARS-CoV-2 infection is viral capture by attachment receptors such as DC-SIGN, which is thought to facilitate the initial stages of COVID-19 infection in the lung tissue (called trans-infection). In addition, while immune escape is dictated by mutations in the spike protein, coronaviral virions also incorporate M, N, and E structural proteins within the particle. In this paper, we explored how several structural facets of both the SARS-CoV-2 virion and the antiviral lectin Griffithsin can affect and attenuate the infectivity of SARS-CoV-2 pseudovirus. We found that Griffithsin was a better inhibitor of hACE2-mediated direct infection when the coronaviral M protein is present compared to when it is absent (possibly providing an explanation regarding why Griffithsin shows better inhibition against authentic SARS-CoV-2 as opposed to pseudotyped viruses, which generally do not contain M) and that Griffithsin was not an effective inhibitor of DC-SIGN-mediated trans-infection. Furthermore, we found that DC-SIGN appeared to mediate trans-infection exclusively via binding to the SARS-CoV-2 spike protein, with no significant effect observed when other viral proteins (M, N, and/or E) were present. These results provide etiological data that may help to direct the development of novel antiviral treatments, either by leveraging Griffithsin binding to the M protein as a novel strategy to prevent SARS-CoV-2 infection or by narrowing efforts to inhibit trans-infection to focus on DC-SIGN binding to SARS-CoV-2 spike protein.

Sustained Delivery of the Antiviral Protein Griffithsin and Its Adhesion to a Biological Surface by a Silk Fibroin Scaffold.

The protein Griffithsin (Grft) is a lectin that tightly binds to high-mannose glycosylation sites on viral surfaces. This property allows Grft to potently inhibit many viruses, including HIV-1. The major route of HIV infection is through sexual activity, so an important tool for reducing the risk of infection would be a film that could be inserted vaginally or rectally to inhibit transmission of the virus. We have previously shown that silk fibroin can encapsulate, stabilize, and release various antiviral proteins, including Grft. However, for broad utility as a prevention method, it would be useful for an insertable film to adhere to the mucosal surface so that it remains for several days or weeks to provide longer-term protection from infection. We show here that silk fibroin can be formulated with adhesive properties using the nontoxic polymer hydroxypropyl methylcellulose (HPMC) and glycerol, and that the resulting silk scaffold can both adhere to biological surfaces and release Grft over the course of at least one week. This work advances the possible use of silk fibroin as an anti-viral insertable device to prevent infection by sexually transmitted viruses, including HIV-1.

SARS-CoV-2 pseudotyped virus persists on the surface of multiple produce but can be inactivated with gaseous ozone.

Due to the immense societal and economic impact that the COVID-19 pandemic has caused, limiting the spread of SARS-CoV-2 is one of the most important priorities at this time. The global interconnectedness of the food industry makes it one of the biggest concerns for SARS-CoV-2 outbreaks. Although fomites are currently considered a low-risk route of transmission for SARS-CoV-2, new variants of the virus can potentially alter the transmission dynamics. In this study, we compared the survival rate of pseudotyped SARS-CoV-2 on plastic with some commonly used food samples (i.e., apple, strawberry, grapes, tomato, cucumber, lettuce, parsley, Brazil nut, almond, cashew, and hazelnut). The porosity level and the chemical composition of different food products affect the virus's stability and infectivity. Our results showed that tomato, cucumber, and apple offer a higher survival rate for the pseudotyped viruses. Next, we explored the effectiveness of ozone in deactivating the SARS-CoV-2 pseudotyped virus on the surface of tomato, cucumber, and apple. We found that the virus was effectively inactivated after being exposed to 15 ppm of ozone for 1 h under ambient conditions. SEM imaging revealed that while ozone exposure altered the wax layer on the surface of produce, it did not seem to damage the cells and their biological structures. The results of our study indicate that ozonated air can likely provide a convenient method of effectively disinfecting bulk food shipments that may harbour the SARS-CoV-2 virus.