ABSTRACT
Several endemic corona viruses (eCoVs) have been reported to be the most common etiologic agents for the seasonal common cold and also cause pneumonia. These eCoVs share extensive sequence homology with SARS-CoV-2, and immune responses to eCoVs can cross-react with SARS-CoV-2 antigens. Based on such cross-reactivity of antigens among eCoVs, the IgG antibodies against the spike protein (SP) of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated from pig serum using magnetic beads immobilized with SARS-CoV SP and a protein-A column. The selectivity of the isolated antibodies was tested using different types of antigens, such as SARS-CoV-2 nucleoprotein (NP), influenza A virus (Beijing type), influenza B virus (Tokio and Florida types), human hepatitis B virus surface antigen (HBsAg), and bovine serum albumin (BSA). From the selectivity test, the anti-SP antibodies isolated from pig serum had sufficient selectivity to other kinds of viral antigens, and the apparent binding constant of the isolated antibodies was approximately 1.5 × 10-8 M from the surface plasmon resonance (SPR) measurements. Finally, the isolated anti-SP antibodies were applied to the immunoassay of SP using competitive immunoassay configuration. The feasibility of the detection as well as the quantitative analysis of the SARS-CoV viral culture fluid was determined using four viral culture samples, namely, SARS-CoV, SARS-CoV-2, MERS-CoV, and CoV-229E.
ABSTRACT
Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and the consensus around the molecular origins behind binding preferences of SARS-CoV-2 for binding with the angiotensin-converting enzyme 2 (ACE2) host receptor is yet to be established. In this work, we performed a comprehensive computational investigation in which sequence analysis and modeling of coevolutionary networks are combined with atomistic molecular simulations and comparative binding free energy analysis of the SARS-CoV and SARS-CoV-2 spike protein receptor binding domains with the ACE2 host receptor. Different from other computational studies, we systematically examine the molecular and energetic determinants of the binding mechanisms between SARS-CoV-2 and ACE2 proteins through the lens of coevolution, conformational dynamics, and allosteric interactions that conspire to drive binding interactions and signal transmission. Conformational dynamics analysis revealed the important differences in mobility of the binding interfaces for the SARS-CoV-2 spike protein that are not confined to several binding hotspots, but instead are broadly distributed across many interface residues. Through coevolutionary network analysis and dynamics-based alanine scanning, we established linkages between the binding energy hotspots and potential regulators and carriers of signal communication in the virus-host receptor complexes. The results of this study detailed a binding mechanism in which the energetics of the SARS-CoV-2 association with ACE2 may be determined by cumulative changes of a number of residues distributed across the entire binding interface. The central findings of this study are consistent with structural and biochemical data and highlight drug discovery challenges of inhibiting large and adaptive protein-protein interfaces responsible for virus entry and infection transmission.