A novel SUMOylation site in the influenza a virus NS1 protein identified with a highly sensitive FRET assay.

Nonstructural protein 1 (NS1) of the influenza A virus is a major contributor to the virulence of the seasonal influenza A viruses, in part because it interferes with host viral defense mechanisms. SUMOylation regulates NS1 activity, and several residues of NS1 have been identified with traditional biochemical approaches as acceptor sites for SUMOylation. In this study, we developed a novel FRET assay to assess SUMOylation. Using this assay, we demonstrated that the lysine residue K131 in the effector domain of NS1 is a previously unidentified SUMO acceptor site. A recombinant H1N1 influenza A virus (A/PR/8/34) expressing a K131 SUMOylation-deficient NS1 had a significantly lower growth rate than the wild-type virus. These results suggest that NS1 SUMOylation at K131 is required for the rapid replication of H1N1 influenza viruses. The interaction between the NS1 protein and the host SUMOylation components may serve as a novel target for the development of anti-influenza A drugs.

Protein-Protein Affinity Determination by Quantitative FRET Quenching.

The molecular dissociation constant, Kd, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution.