1. Inoviridae – M13 Microviridae – Ф X174 LECTURE 8: Viro102: Bacteriophages & Phage Therapy 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB)

2. Bacteriophage Families CystoviridaeSiphoviridae LeviviridaeMyoviridae RudiviridaeInoviridae FuselloviridaePodoviridae TectiviridaeMicroviridae LipothrixviridaeCorticoviridae Plasmaviridae

3. Inoviridae  Group II (ss circular DNA)  Helical / Filamentous bacteriophages  Inoviridae viruses are non-enveloped bacteriophages that infect E. coli by a slow-release life cycle. filamentous phages can extrude individual progeny through cell envelope (host cell does not lyse or die right away)  Two genus:  Inovirus (infect E.Coli)  Plectovirus (Infect Mycoplasma)

4.  Filamentous phage reproduce without killing the host  Some even take part in the host’s “social” life, “biofilms”  Widespread among Gram negative,  Escherichia, Salmonella,  Pseudomonas, Xanthomonas,  Vibrio, Thermus and Neisseria Inoviridae

5.  F pilus specific phage known as f1, M13 and fd  Model system in understanding membrane transactions  Therapeutic recombinant antibodies  Lithium powered Nano-battery Massachusetts Institute of Technology (MIT) as well as scientist around the world, have been using M13 bacteriophage viruses to construct high power lithium ion batteries through modification of coat proteins.  Pathogenesis of Vibrio cholerae, Neisseria meningitidis and Pseudomonas aeruginosa

6. M13: Introduction  Also known as f1 (Filamentous).  Genome length ~6.4 kb.  Non-lytic virus. However a decrease in the rate of cell growth is seen in the infected cells.  Establish permanent infections without lysogeny.  ~300 particles/cell are produced Electron micrograph of M13

7. Structural Proteins  Cartoon Representation of M13  Major protein (p8 = 2700 copies)  Minor proteins (p3, p6, p7, p9 = 5 copies)

8. M13:Structure At amino terminus: Negatively charged region contains the acidic amino acid residues and it forms the outer covering At carboxyl terminus: Positively charged region contains the basic amino acids residues and forms the inner core The central domain of the protein is hydrophobic & this region of proteins interacts to resulting in assembled virions

9. Domain organization of pIII protein. SS: signal sequence, N1, N2, C, domains of pIII; G, glycine-rich linkers; TM, transmembrane helix.

10. involved in assembly M13: Genome Required for replication of RF controls switch from RF replication to progeny Major coat protein Minor protein N- terminus binds to F pilus of host cell Involved in attachment & morphogenesis Required for assembly

11. Life Cycle of M13  Filamentous phage adhere to either of two major life styles  Episomally replicating phage  Temperate phage that are chromosomally integrated  10 13 per mL of culture

12. M13: Life Cycle 1.Attachment & Entry:  M13 phages are 'male-specific', i.e.  They require the F pilus on the surface of E. coli for infection (g3p/g6p on tail fibers interacts with F pillus).  This interaction causes a conformational change that result in expelling the phage DNA.  G8p ends up in the inner cell membrane, where it may possibly be stored and reused to produce new particles.

13. Model of M13 phage infection

14. Phage Receptors  The primary receptors for filamentous phage are pili, long filamentous structures on the surface of bacterial cells.  Three types of pili can serve as primary receptors, including conjugative F, N (or I) and type IV pili  The secondary receptor for phage as diverse as E, coli Ff and V. cholerae CTXφ is the TolQRA complex of inner membrane proteins, highly conserved in Gram negative bacteria

15. M13: Life Cycle

16. 2. Replication:  Infecting (+)strand DNA is converted in ds replicative form (RF) form by host cell enzymes.  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 packaging.

17. 3.Assembly & Release :  Assembly occurs at the inner membrane of the cell.  DNA is extruded into the peri-plasmic space & the g5p coat is replaced by g8p.  g5p is recycled to form further particles.

18.  IV, IX and I protein are involved in phage release process  Secretin Complex  Export is driven by ATP hydrolysis Assembly & Release

19. The pIV multimer forms a barrel like structure with an outer diameter of about 13.5 nm and height of about 12 nm. The barrel is comprised of three “stacked” rings, an N- terminal ring (N-ring), middle ring (M-ring) and C-terminal ring (C-ring)

20. M13: Life Cycle

21. Secretin Systems in Bacteria

22. THANK YOU!