Antibody binding captures high energy state of an antigen: The case of Nsp1 SARS-CoV-2 as revealed by hydrogen-deuterium exchange mass spectrometry

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1

Oropouche orthobunyavirus (OROV

Monoclonal antibodies binding data for SARS-CoV-2 proteins

ELISA EC50 The raw dataset of EC50 for the selected IgGs that are specific for SARS2-CoV-2 proteins. Data points and analyzed set were included in the raw data in excel format. The data were fit to standard four-parameter logistic equations using GraphPad Prism (GraphPad Software, La Jolla, CA). BLI (Bio-Layer Interferometry) The raw data are in excel format which includes data points and fitting points for selected IgGs that are specific for SARS2-CoV-2 proteins. The binding responses were analyzed by subtracting data from reference, and data were globally fitted with a 1:1 binding model using ForteBio’s Data Analysis software 9.0

Data Figure2_EC50_Monoclonal antibodies binding data for SARS-CoV-2 proteins

Data for figure 2. Data were processed. Data points and the fitting points were included in the raw data in excel format. The data were fit to standard four-parameter logistic equations using GraphPad Prism (GraphPad Software, La Jolla, CA)

Biochemical and HDX Mass Spectral Characterization of the SARS-CoV‑2 Nsp1 Protein

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions. To better define how this multifunctional protein can facilitate distinct functions, we characterize SARS-CoV-2 Nsp1 by using a combination of biophysical techniques, including light scattering, circular dichroism, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and temperature-dependent HDX-MS. Our results reveal that the SARS-CoV-2 Nsp1 N- and C-terminus are unstructured in solution, and in the absence of other proteins, the C-terminus has an increased propensity to adopt a helical conformation. In addition, our data indicate that a short helix exists near the C-terminus and adjoins the region that binds the ribosome. Together, these findings provide insights into the dynamic nature of Nsp1 that impacts its functions during infection. Furthermore, our results will inform efforts to understand SARS-CoV-2 infection and antiviral development