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ABSTRACT

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.

INTRODUCTION

The boundaries of all living cells and their compartments are detined by lipitl hilayers. Intracellular lransport, cell entry, and secretion require vesicular lipid structures to fuse with target membranes. Membrane fusion proteins are involved in catalyzing almost every situation of biological membrane fusion. Despite decades of intense study, the precise molecular mechanism by which fusion proteins mediate membrane fusion remains a subject of much debate (1-11).

Among the best-studied membrane fusion protein machines arc those present in enveloped viruses. Enveloped viruses have evolved highly efficient fusion proteins that allow the viral genome to penetrate targeted cells (12-18). For most enveloped viruses, environmental signals trigger these proteins to catalyze fusion of the viral membrane with the cell membrane. The decreased pH encountered along the endocytotie pathway is a common trigger for the fusion proteins of many enveloped viruses, most notably influenza (19,20). In a few cases, atomic resolution structures are available for viral fusion proteins under both neutral pH and low pH conditions (12) that have led to formulation of models of their molecular action (12,13).

Many enveloped viruses will fuse to protein free lipid hilayers (20-22). In vitro liposome fusion experiments have been extremely useful in characterizing the biochemical properties of viral fusion, including the pH and lipid species dependences (23,24). Bulk liposome measurements have also confirmed that viral fusion is often nun leaky and can fully mix both the tipids and the contents of fusing structures (25,26).

As valuable as these bulk assays have been in advancing our understanding of membrane fusion, they have limitations. Low-pH triggered viral membrane fusion occurs rapidly after acidification, with timescales measured in lens of seconds or minutes in the bulk liposome experiments(16,21). The stochastic nature of each occurrence of fusion prevents the precise synchronization of all of the individual virus-liposome fusion events within a cuvette-based bulk solution assay. These experimental challenges mask transient, intermediate stales along the fusion pathway and obscure detailed analysis of the trajectory of the nonequilibrium processes driving the dynamics. Difficulty separating the linked processes of binding and fusion in bulk assays also complicates the interpretation of experimental results.

We have developed a fluorescence assay to make detailed measurements of the early stages of individual Sindhis and influenza virus particles fusing to supported lipid bilayers under acidic conditions. Our assay detects lipid mixing of ocladecyl rhodumine (R18) between virus particles and the supported lipid bilayer. As R18 is known to “flip-flop” between the inner and outer leaflets of a labeled bilayer (27,28), this signal does not differentiate between hemifusion and full fusion. Throughout this article we will use hemifusion/fusion to indicate this ambiguity.

The supported lipid bilayer geometry is desirable for its compatibility with high-resolution optical measurements as well as its ease of integration with biotechnological instrumentation and sensors. Supported bilayers have been successfully applied in membrane fusion studies of SNARE proteins (29-31) and influenza virus (32-36). Illumination of the supported lipid bilayer by total internal refection yields a signal/noise ratio sufficient for precise measurements of individual virus particles during fusion. Continued development of this single-particle approach will allow detection of the transient, stochastic intermediate states that arc commonly averaged in liposome fusion assays.

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