ABSTRACT
Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PAN) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PAN, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed Km (150 ± 11 nM) and kcat [(1.4 ± 0.2) x 10-3s-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC50 values of 10-20 nM, demonstrating the utility of this system for future high throughput screening.
Subject(s)
Endonucleases/antagonists & inhibitors , Endonucleases/metabolism , Enzyme Inhibitors/pharmacology , Influenza A virus/drug effects , Influenza A virus/enzymology , RNA-Dependent RNA Polymerase/metabolism , Endonucleases/chemistry , Enzyme Activation/drug effects , Inhibitory Concentration 50 , RNA, Messenger/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to a 96-well plate format, we have shown that low-pH or high-salt concentrations decrease the thermal stability of mAb1, whereas some excipients, such as sucrose, polysorbate 80, and sodium phosphate, increase its stability. The basal fluorescence of SYPRO Orange was enhanced by the presence of detergents, limiting the use of this approach to diluted detergent solutions. Throughput of DSF can be increased further with the use of a 384-well plate. DSF thermograms are in good agreement with the melting profiles obtained by differential scanning calorimetry (DSC). The Tms determined by DSF and DSC were well correlated, with the former being on average lower by 3 °C.
