Bacteriophage T7

• T7 structure

  • Phage particles outside the cell are called virions
  • They are like ‘spores’ and are energetically inert
  • Genome – 40,000 bp
• finding a host

  • Phage have no way of generating ATP, and no motility, so entirely dependent on diffusion and random chance to meet a suitable host
  • More bacteria = more phage More phage = more predation More predation = less bacteria
  • Lokta-Volterra Dynamics
  • Shape of these curves is a function of how many phages get made per infection, host density etc.
  • When phage have killed almost all the target pathogen, the probability of phages finding a new uninfected host heads towards zero, making subsequent infections very unlikely.
  • In a patient with no working immune system to finish off the stragglers, the few remaining cells can repopulate the area once the phages have been cleared from the system
  • Some phages decorate their capsids to slow their diffusion upon release
• adsorption

  • Viral particles have tail fibres that interact with specific receptors on a cell surface. This could be the lipopolysaccharide layer (LPS), or a transporter such as the OprM efflux pump.
  • BUT – their interaction with a cell is random, they can’t guide themselves onto the surface
  • Upon landing on a cell, phages T7 and T4 walk to find their desired protein target
• Host Defenses – Mutation or inhibition of receptor binding proteins

  • Turning off an efflux pump or capsule makes host susceptible to antibiotics
  • Turning off LPS can make hosts less ‘sticky’ and immunogenic
  • Known as phage-antibiotic synergy

  
• injection of T4

  • Viruses need to get their DNA into the cell for replication
  • T4 encodes a long, contractile tail with a spike on the end, which it drives through the double cell wall into the cytoplasm
  • As the DNA starts to enter the cell, water enters the capsid and ‘flushes’ the DNA out
• injection of T7

  • The short, stubby tail of T7 doesn’t allow it to span the periplasm
  • It starts to drill and builds a tube behind it for the DNA to go through
  • It has to package the drill and tube proteins in the capsid, because it has no way of synthesizing proteins until it is in the cell
  • Once it breaks through, these proteins ratchet the first 850 bp at 70 bp/s into the cell, and then gp16 clamps it in place
  • The first part of the genome encodes a promoter recognized by the host RNA polymerase
  • Host RNA polymerase binds and starts to read the genome, overcoming the gp16 clamp and pulling it into the cell at 40-50 bp/s until the first 7kbp is in the cell, and then hits a terminator and falls off
  • T7’s own RNA polymerase is transcribed from that 7kb fragment and then pulls the rest of the genome into the cell at 250 bp/s. Almost all subsequent genes in the genome have a promotor specific to T7 RNAP
• hijacking

  • Need to stop the cell wasting resources that could be used for viruses, and from mounting a counterattack
  • Gene 0.7 encodes serine kinase that inactivates host transcription
  • Gp2 binds to host RNA polymerase and stops it recognizing host promoters
  • As a final insult, genes gp3 and gp6 conspire to degrade the host DNA to prevent it from doing anything at all.
  • This also releases nucleotides that may be used by T7 to replicate its own genome
• assembly

  • Forms a long concatemer of 100-200 genomes, one after the other
  • Before the virion (viral particle) is mature, the structure of the particle is built into a procapsid
  • Each T7 virion comprises 415 copies of major capsid protein (gp10) that self-assemble around scaffold protein (gp9)
  • does not require ATP, is purely driven by protein-protein interactions
  • Once the procapsid is built, the terminase loads the DNA into the capsid from the concatemer through the portal protein
  • Packaging continues until either for a specific length (for T7) or until the capsid is full.
  • The concatemer is then cleaved and prepared for the next procapsid
  • As the capsid is loaded, the pressure increases and the terminase motor has to work harder.
  • Near completion, it costs 2 ATP for every basepair loaded in. The DNA is so dense in the capsid it approaches a liquid crystal state
  • Terminases are the most powerful motor proteins in nature
• what are jumbophages

  phages >250kbp in length. Appear to protect their new phages from attack by cellular defenses by constructing a nucleus-like shell in their bacterial host
• release

  • Gram negative bacteria have TWO walls that need to be broken down before viruses can escape
  • Holins and lysins are expressed simultaneously to phage virion proteins.
  • Lysins can degrade the outer membrane, but not the cytoplasmic membrane
  • Inactive holins accumulate in the cytoplasmic membrane
  • When all the phage progeny are mature and packaged, the holins are triggered and they form channels in the membrane
  • In T7, every infected cell releases releases ~200 new viral progeny. This is known as the burst size
  • These go on to infect other cells in a chain reaction