(34g) Differential Requirement and Kinetics for Viral Membrane Fusion Driven by Class I and Class II Fusion Proteins

Fusion of two lipid bilayers is a prerequisite step for infection by viruses surrounded by a lipid envelope during their entry into target cells. Membrane fusion is triggered by viral fusion proteins that currently defined into two groups, class I and II fusion proteins, based on their important structural features. However, the fusion mechanism containing multiple transient intermediates catalyzed by the two different class fusion proteins has not been well determined. Understanding of the different fusion kinetics and dynamics driven by these two class proteins can provide a crucial understanding of the underlying mechanisms of the virus infection and reveal certain previously uncovered fusion details. One obstacle to compare the fusion reaction mediated by different viruses in living cells is their different receptor binding, which may subsequently lead to different intracellular viral trafficking involved in the fusion process. To this end, we designed a synthetic virus platform that consists of lentiviral particles co-enveloped with a surface antibody as the binding protein along with a fusion protein derived either from influenza virus (HAmu, class I fusogen) or from Sindbis virus (SINmu, class II fusogen) onto the lentiviral surface, which allows for better understating of differential fusion mechanisms driven by class I and class II fusion proteins. In this report, the single virus tracking study of the early internalization indicates that both HAmu- and SINmu-lentiviruses enter cells through clathrin-dependent endocytosis. However, different requirements of endosomal trafficking for the membrane fusion of these two lentiviruses were observed. The direct visualization of single viral fusion events clearly showed that hemifusion mediated by class II fusion protein (SINmu) upon exposure to acidic pH occurs faster than that mediated by class I fusion protein (HAmu). Real-time monitoring of sequential fusion processes with a dual labeling of outer and inner leaflets of viral membrane also suggested that formation of fusion pore was remarkable delayed after hemifusion mediated by class II fusion protein (SINmu) as compared with that mediated by class I fusion protein (HAmu). By this approach, we have demonstrated that the combination of this versatile platform and single virus tracking can be a powerful tool for understanding molecular details of fusion mediated by various fusion proteins.