Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike protein (S) is the main determinant of viral entry. Proteolytic cleavage of S exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca²⁺) play an important role for fusogenic activity via a Ca²⁺ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FP share a high sequence homology and here, we investigated whether Ca²⁺ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate infection of pseudo-particles (PPs) on host cells in modulating Ca²⁺ environments. Our results demonstrate that intracellular Ca²⁺ enhances MERS-CoV WT PPs infection by approximately two-fold and that E891 is a crucial residue for Ca²⁺ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca²⁺ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca²⁺ ratio, as opposed to an 1:2 SARS-CoV FP to Ca²⁺ ratio, suggesting significant differences in FP Ca²⁺ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity. Significance MERS-CoV is a major emerging infectious disease with zoonotic potential and a reservoir in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data has found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This can guide therapeutic solutions to either block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Lipid bilayers are widely employed as a model system to investigate interactions between cells and their environment. Supported lipid bilayers (SLB) with integrated transmembrane proteins are emerging as a preferred platform for sensing applications. Challenges lie in the generation of SLB on surfaces which allow transduction of signals for characterization of lipid bilayer and incorporated transmembrane proteins. For the first time, the formation of SLBs is shown on films of the conducting polymer, poly(3,4‐ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), using traditional methods for characterizing lipid bilayer quality and function (QCM‐D, FRAP) combined with impedance spectroscopy. Further, partial formation of SLBs on PEDOT:PSS based organic electrochemical transistors (OECTs) is successfully demonstrated, as well as the ability to integrate and sense the ion pore α‐hemolysin, confirming the sensitivity of the OECT as a transducer of biological membrane function. This work represents a highly promising first step toward the use of such OECTs for functional readout of transmembrane proteins in their native environment.