Activation, Penetration, and Uncoating of M13

Griffith, Jack

Abstract

Of central interest to cell biologists are the phenomena of membrane directed assembly and disassembly of protein filaments. The filamentous phage M13, bacterial pili, numerous viruses and possibly cytoskeletal filaments in higher cells provide examples. Of these, M13 offers a powerful, yet simple system for studying membrane-localized assembly and disassembly including how the protein subunits are removed from the membrane, transit of DNA across a membrane barrier, and finally how these structures are extruded without killing the cell. Previously, the extraordinary hardiness of M13 filaments made it difficult to follow in detail the assembly and disassembly of the phage at the cell membrane. Recently we have been able to trigger highly organized rearrangements of the virus in vitro which make them more amenable for study. In particular these triggered forms now reveal interactions with membrane components that provide a new window into the M13 life cycle. In brief, M13 filaments undergo a highly ordered structural change when exposed to certain solvent-water interfaces. Upon contact with the interface at 15 degrees or less, the filaments contract 3 fold into thick rods. At 15 degrees or higher the rods contract 7 fold more into sperical shells, ejecting 2/3 of the genome from the shell. We will apply our long experience with M13, electron microscopy, and biochemistry to examine the central question of how membranes participate in the assembly and disassembly of protein structures. We will study the insertion of partially contracted M13 into liposomes and define the lipid parameters (e.g. phase transition temperature and degree of saturation) required for insertion. We will examine the orientation of the inserted protein and under what conditions DNA can transit the bilayer. We will examine the activation of M13 filaments prior to penetration and will characterize complexes we have isolated which contain partially extruded M13 filaments. The experiments proposed here will use the contracted M13 forms as tools to speed our understanding of how a simple viral capsid undergoes an ordered disassembly, how its coat protein becomes membrane soluble, and how the viral DNA transits the membrane barrier.