Unlocking the M13 (f1 and fd) virion : investigation into the role of the pIII C-domain of F specific filamentous bacteriophage in infection : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand.
Ff filamentous bacteriophage infect male (F+) strains of Escherichia coli and are
assembled at the cell membranes, by a secretion-like, non-lethal process. The pIII
protein, located at one end of the virion-filament, is required at both the beginning and
the end of the phage life cycle. During infection, the N-terminal domains of pIII, N2 and
N1, bind to the primary and secondary host receptors, F pilus and TolA protein,
respectively. At the end of the life cycle, the pIII C-domain mediates the termination
and release of virions. Thus, both entry and release involve structural transitions of the
virus coupled to membrane transactions of the virion proteins. "Unlocking” of the
highly stable virion presumably results in membrane integration during entry, whereas a
reverse event, “locking” of the virion, occurs upon detachment from the membrane at
termination step of assembly/secretion. Recently, it was shown that the pIII C-domain
plays an active role at the step of entry. This finding implicates the C-domain of pIII in
“unlocking” of the virion, presumably resulting in the exposure of the membrane anchor
at the very C-terminus of pIII (Bennett & Rakonjac, 2006).
To further this work, this thesis has mapped the portion of the pIII C-domain required
for infection, by constructing a set of nested deletions of the C-domain fused to the
receptor binding domains N1 and N2, and then determined the infectivity of phage
carrying the mutant proteins. This mapped the portion of the C-domain required for
phage infection is different to that required for termination of assembly. The different
requirement for entry and release suggests that the two processes are carried out by
distinct mechanisms and/or depend on different sets of accessory proteins.
In addition, a system was designed for the efficient production and purification of very
short virions, the length of which is 1/20 that of the wild-type f1. These short virions,
called microphage, are the first step towards the structural analyses of the phage termini
cap structures, of which one contains pIII in the “locked” conformation.