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Among the simplest helical capsids are those of the well-known bacteriophages of
the family Inoviridae, such as M13 and fd - known as Ff phages. These phages are about 900
nm long and 6-7 nm in diameter and the particles contain 5 proteins. All are similar and
are known collectively as Ff phages - they require the E.coli F pilus for
The major coat protein is the product of
phage gene 8 (g8p) and there are 2,700 - 3,000 copies of this protein per particle,
together with approximately 5 copies each of four minor capsid proteins, g3p, g6p, g7p and
g9p which are located at the ends of the filamentous particle.
The primary structure of the major coat protein g8p explains many of the properties of the
particle. Mature molecules of g8p consist of approximately 50 amino acid residues (a
signal sequence of 23 amino acids is cleaved from the precursor protein during its
translocation into the outer membrane of the host bacterium), and is almost entirely
alpha-helical in structure so that the molecule forms a short rod. There are three
distinct domains within this rod:
- A negatively-charged region at the amino
terminal end which contains acidic amino acid residues and which forms the outer,
hydrophilic surface of the virus particle
- A basic, positively-charged region at the
carboxyl terminal end which lines the inside of the protein cylinder adjacent to the
negatively-charged DNA genome
- A hydrophobic region which is responsible
for interactions between the g8p subunits which allow the formation of and stabilize the
Ff phage particles are held together by the
hydrophobic interactions between the coat protein subunits and this is demonstrated by the
fact that the particles fall apart in the presence of chloroform, even though they do not
contain any lipid component. The g8p subunits in successive turns of the helix interlock
with the subunits in the turn below, are tilted at an angle of approximately 20° to the
long axis of the particle and have been described as overlapping one another 'like the
scales of a fish'. The value of µ (protein subunits per complete helix turn) is
4.5 and p (axial rise per subunit) = 1.5nm.
Since the phage DNA is packaged inside the
core of the helical particle, the length of the particle is dependent on the length of the
genome. In all Ff phage preparations the following forms occur:
- Polyphage: containing more than one
genome length of DNA
- Miniphage: deleted forms containing
0.2-0.5 phage genome lengths of DNA
- Maxiphage: genetically defective
forms but containing more than one phage genome length of DNA
This plastic property of these filamentous
particles has been exploited by molecular biologists to develop the M13 genome as a
cloning vector - insertion of foreign DNA into the non-essential inter-genic region
results in recombinant phage particles which are longer than the wild-type filaments.
Unlike most viruses, there is no sharp cut-off genome-length at which the genome can no
longer be packaged into the particle.
However, as M13 genome size increases, the efficiency of replication declines such that
while recombinant phage genomes 1-10% longer than the wild-type do not appear to be
significantly disadvantaged, those 10-50% longer than the wild-type replicate
significantly more slowly and above 50% increase over the normal genome length it becomes
progressively more difficult to isolate recombinant phage. This property has been
exploited in M13 cloning vectors.
||NS membrane protein; few
copies/cell; interacts with host fip gene product (thioredoxin); required for assembly
||Site- & strand-specific
endonuclease/topoisomerase; ~103 copies/cell; required for replication of RF
||N-terminal fragment of gene 2;
~500 molecules/cell; required for replication of RF
||5 copies at one end of
particle; required for correct morphogenesis of unit-length particles; N-terminal domain
binds to F pilus of host cell (receptor)
||NS membrane protein; few
copies/cell; required for assembly
||Major structural protein
during replication; ~105 copies/cell; controls expression of g2p; binds to DNA;
replaced by g8p during assembly; controls switch from RF replication to progeny (+)stand
||~5 copies at same end of
particle to g3p; involved in attachment & morphogenesis
||~5 copies at opposite end of
particle to g3p/g6p; involved in assembly
||Major coat protein: ~2700-3000
Ff phages are 'male-specific', i.e. they
require the F pilus on the surface of E. coli for infection. The first event in
infection is an interaction between g3p, located at one end of the filament together with
g6p, and the end of the F pilus.
This interaction causes a conformational changes in g8p: Initially, its structure changes
from 100% alpha-helix to 85% alpha-helix and this causes the filament to shorten. The end
of the particle attached to the F pilus flares open, exposing the phage DNA.
Subsequently, a second conformational change in the g8p subunits reduces its alpha-helical
content from 85% to 50%, causing the phage particle to form a hollow spheroid about 40nm
in diameter and expelling the phage DNA, thus initiating the infection of the host cell.
g8p is stripped off and ends up in the inner cell membrane, where it may possibly be
stored and reused to produce new particles.
Infecting (+)strand DNA is converted in d/s
'RF' form by host cell enzymes, which together with g2p build up a pool of RF DNA in the
cell. Virus proteins are synthesized from this pool of DNA.
Later in the infection, the concentration of g5p in the cell builds up. This protein binds
to newly formed (+)strands, causing a switch from RF to (+)strand replication (coated with
Assembly occurs at the inner membrane of
the cell. DNA is extruded into the periplasmic space and the g5p coat is replaced by g8p.
g5p is recycled to form further particles (efficient!).
These are temperate (i.e. non-lytic) phages
- establish permanent infections without lysogeny. ~300 particles/cell/generation are
produced; titres in infected cultures up to 5x1012/ml (~150mg/l) - good