Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV (“SARS-1”), SARS-CoV-2 (“SARS-2”) and MERS-CoV (“MERS”). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2’ cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion. In this study, we want to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.

Targeted Delivery of siRNA through Nanocomplex using Specific Fusion Peptides for Breast Cancer Treatment

Breast cancer is one of the serious diseases and has the second-highest mortality in women worldwide. RNA interference has been developed as a promising way of specific cancer treatment by silencing oncogenes efficiently. However, small RNAs exhibits difficulties in specific cellular uptake and instability. Therefore, we designed novel fusion peptides (RS and RT) for an efficient, stable, and specific delivery of small RNAs. Both RS and RT peptides could form self-assembled nanocomplexes via electrostatic attraction. RS nanocomplexes exhibited prolonged stability, enhanced cellular uptake, and target gene silencing by siRNAs to MDA-MB-231 breast cancer cells. Moreover, RS nanocomplexes successfully inhibited breast cancer cell growth via specific and efficient siRNA delivery. Furthermore, in vitro and in vivo safety tests showed negligible cytotoxicity and neither tissue damage nor significant inflammatory cytokine release. Therefore, the RS nanocomplexes could be expected to become a promising siRNA delivery platform for the treatment of breast cancer or other cancers.

Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides

To rationalize the antiviral actions of plant alkaloids, the ability of 20 compounds to inhibit calcium-mediated fusion of lipid vesicles composed of phosphatidylglycerol and cholesterol was investigated using the calcein release assay and dynamic light scattering. Piperine, tabersonine, hordenine, lupinine, quinine, and 3-isobutyl-1-methylxanthine demonstrated the most potent effects (inhibition index greater than 50%). The introduction of phosphatidylcholine into the phosphatidylglycerol/cholesterol mixture led to significant changes in quinine, hordenine, and 3-isobutyl-1-methylxanthine efficiency. Comparison of the fusion inhibitory ability of the tested alkaloids, and the results of the measurements of alkaloid-induced alterations in the physical properties of model membranes indicated a potent relationship between a decrease in the cooperativity of the phase transition of lipids and the ability of alkaloids to prevent calcium-mediated vesicle fusion. In order to use this knowledge to combat the novel coronavirus pandemic, the ability of the most effective compounds to suppress membrane fusion induced by fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 fusion peptides was studied using the calcein release assay and confocal fluorescence microscopy. Piperine was shown to inhibit vesicle fusion mediated by both coronavirus peptides. Moreover, piperine was shown to significantly reduce the titer of SARS-CoV2 progeny in vitro in Vero cells when used in non-toxic concentrations.

Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering

Coronavirus disease-2019 (COVID-19), a lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of the SARS-CoV-2 Spike protein, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the protein to the lipid leaflet in contact with the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Sterically confined rearrangements of SARS-CoV-2 Spike protein control cell invasion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale (200–300 Å) conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. While infection relies on this transition between the prefusion and postfusion conformations, there has yet to be a biophysical characterization reported for this rearrangement. That is, structures are available for the endpoints, though the intermediate conformational processes have not been described. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. With the current lack of data on the pre-to-post transition, the precise role of glycans during cell invasion has also remained unclear. To provide an initial mechanistic description of the pre-to-post rearrangement, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans can induce a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. In contrast, in the absence of glycans, the viral particle would likely fail to enter the host. This analysis reveals how the glycosylation state can regulate infectivity, while providing a much-needed structural framework for studying the dynamics of this pervasive pathogen.