Dissecting the Interaction of Human SUMOylation E3 ligases with the Influenza A Virus M1 Protein using qFRET Technology

Introduction:: Influenza viruses remain a significant public health issue, causing hospitalizations and mortalities during each annual flu season in the United States. The influenza A virus caused notable pandemics within the last few centuries. The severity of this crisis highlights the urgent need to investigate the influenza virus life cycle and discover novel therapeutic strategies. This urgency is further underscored by the inefficacy of current antiviral drugs for influenza, which are effective only in the early stages of infection and are susceptible to drug resistance development. Our research group has discovered that the influenza virus relies on the human SUMOylation pathway to modify its M1 capsid protein, which is essential for virality in human hosts. Human SUMOylation is mediated by three essential enzymes: the E1 and E2 conjugating enzymes and E3 ligases. The E1 and E2 enzymes have been thoroughly studied, while the E3 ligases are still poorly understood. Using quantitative Förster Resonance Energy Transfer (qFRET), we determined the interaction affinities of the PIAS family of SUMOylation E3 ligases with the essential influenza M1 protein. Our goal here is to discover and characterize specific E3 ligases that mediate the SUMOyaltion of influenza A viral M1 (IAV M1) protein, thereby providing insight into a potential therapeutic target for novel drug design and development.

Materials and Methods:: The potential contact affinity of the IAV M1 protein with the SUMOylation E3 ligase candidates (PIAS1, PIAS2, PIAS3, and PIAS4) was determined by a specific FRET-based Kd assay technique. First, a full-length encoding, optimized SUMOylation E3 ligase and substrate IAV M1 genes were each cloned into the pET28 plasmid (NovagenTM). These genes include the qFRET fluorescence protein partners (CyPet and YPet), fused to E3 PIAS1-4 and IAV M1 genes, respectively. Next, multiple E. coli strains were transformed with these genes, and the strains with the highest expression levels were selected. The selected strains expressed the fusion fluorescent proteins, which were then purified through a series of washes using a Ni-His affinity column. Once we obtained the target proteins, the CyPet E3 (0.1μM) ligase was mixed with a series of increasing concentrations of YPet IAV M1 protein in order to determine the dissociation constant, Kd, by utilizing our unique qFRET method. This method quantifies the strength of the E3 ligase affinity with the M1 protein based on the strength of the energy transfer from the CyPet donor to the YPet acceptor.

Results, Conclusions, and Discussions:: We used our qFRET approach to determine the SUMOylation E3 ligase candidates’ affinity for the IAV M1 protein. Varying the YPet-IAV M1 concentrations from 0μM to 30μM, the CyPet-PIAS1-4 were each held at a constant concentration of 0.1μM. Using our established formula, we determine the Kd for the interaction between: IAV M1 and PIAS1, which had a Kd value of 1.2μM; IAV M1 and PIAS2, which had a Kd value of 15.3μM; IAV M1 and PIAS3, which had a Kd value of 7.3μM; IAV M1 and PIAS4, which had a Kd value of 6.2μM. The affinity of IAV M1 with PIAS1 was higher than that of IAV M1 with PIAS1-3. These results were significant when compared to the results of testing PIAS1-4 with a blank, negative control.

We evaluated more than a dozen strains using our unique technique before attempting to express different SUMOylation E3 ligases at high levels. Once evaluated, we used our qFRET technique to evaluate the affinity of CyPet-tagged SUMOylation E3 ligases, PIAS1-4, with the YPet-tagged IAV M1 protein. The ideal CyPet-PIAS1-4 concentration was established by a titration procedure, in which the FRET signal was monitored as YPet-IAV M1 concentration was increased and CyPet-PIAS1-4 concentrations were fixed. The interaction affinity of each E3 ligase with IAV M1 protein was measured and the results demonstrate the viability of SUMOylation E2 ligases (PIAS1-4), which exhibit varying degrees of affinity for the IAV M1 protein. The need for anti-influenza medications has arisen as a result of the influenza virus's persistent impact on the global population in recent years. To combat the influenza virus, developing new medication is necessary. Numerous studies have tested human host factors and pathways for their importance in IAV infection and replication; SUMOylation is a motif that appears frequently across genome-wide searches. Our current understanding of IAV-M1's significant affinity for SUMOylation E3 suggests that inhibiting SUMOylation may present promising new opportunities for the development of antiviral medicines. A significant opportunity exists for the development of a novel class of inhibitors to treat illnesses garnered by influenza viruses and other SUMOylation-exploiting viruses.


Acknowledgements (Optional): : We are grateful to all of Liao's group’s members for their close, collaborative work.

References (Optional): : [1] Dang et al. Molecules, 2021 Nov., 26(21): 6339.

[2] Liu et al. UC Riverside Undergraduate Research Journal Submit, 2022 16(1): 9-17

[3] Song et al. Mol. Biosyst., 201 2, 8, 1723