M13 and Mu Virus Structure and Life Cycle

M13 and Mu Virus Structure and Life Cycle

1. Life Cycle of M13 & Mu Viruses Shashank Patil 171233, M.Sc. Biotechnology-I

2. I ntroduction Filamentous structure, Single stranded positively charged DNA with Protein capsid. Found useful in g enetic engineering experiments. Gronenborn and Messing experiment- Assaying of β -Galactosidase. Proves one of the best vector for single stranded DNA synthesis.

3. Structure of M13 Virus Helical symmetry-Unusual character. ssDna acts as template strand. Size of the genome is small – 6.4 kb. Phage length – 895 nm, thickness – 6 nm, external core – 2.5 nm. Entire genome packed with GP8 proteins. Attachment end – five GP3 & GP6 proteins. Free end – five GP7 & GP9 proteins. IG region – b/n II & IV region – sequences for viral packaging & +/- strand synthesis.

4. Electron micrograph of the filamentous M13 phage Structure of M13 virus

5. Structure of M13 genome

6. Replication Cycle 4 stages of replication – Attachment, Penetration, Replication & Production of new viruses. Attachment – Binding of the phage to the bacterial surface. Usually F – Plasmid `Male’ bearing sex pili. Penetration – Introduction of viral genome. GP6 & GP3 proteins bind to pilus and GP8 to plasma membrane by conformational changes. Other proteins help in the replication and post – replication processes.

7. Replication – 2 types, positive & negative strands. B & C – ori sites for negative strand D & E – ori sites for positive strand Negative strand synthesis – ssDNA with coated ssB proteins – super coiled. RNAP binds to the C region. IG region acts as a recognition site by hairpin loop formation. dsDNA/RF DNA is synthesized. Positive strand synthesis – RF forms undergo rolling circle mechanism to produce the positive strand. GP2 binds to D region. As the ssDNA is produced, viral protein binds and stops this mechanism.

8. mRNA thus produced from RF form further produces viral proteins. Increased GP5 produces nick in the DNA – in the IG region. ssDNA produced. GP1 creates ion channels on the surface of the plasma membrane and GP4 help in the assemblage of DNA. GP5 binds to DNA to avoid the damages from host nucleases. GP8 covers the whole strand to protect it. GP4 also helps to bind the DNA to all the proteins. GP7 & GP9 – first addition. GP3 & GP6 – last addition. Thus without harming the host cell mechanism, the newly formed DNA passes out through the plasma membrane.

9. Life cycle of Mu virus

10. Introduction Known as Mu phage or Mu bacteriophage. Since it affects Enterobacteria, also called as Enterobacteria phage. Named after its ability to cause mutations in life cycle. Replicates through transposition of gene sequences. B road host range – research areas. Temperate phage – displays both lytic and lysogenic phases.

11. Structure of Mu virus Icosahedral head, tail and 6 tail fibers. Helical symmetry and devoid of envelope. Head – 54 nm, pro-late in shape. Capsids are hexagonal in shape. 152 capsomers. Long tail, rigid and thick & consists an axial canal, base plate and fibers. Sheath composed of stacked rings, upon contraction which becomes shorter and thicker.

12. Scanning Phage Mu Electron Micrograph of Mu

13. Genome of Mu virus Non-segmented, single, linear dsDNA. 37 kb long but actual length is 35.6 kb. Rest genome – host DNA. Completely sequenced genome – 37,611bp long. DNA integration at one point of host chromosome helps in lysogeny of the host cell.

14. Life Cycle of Mu virus Infection through attachment – Virus attaches on the surface of the host bacteria. Penetration – genome is inserted to the DNA of the host cell through cut and paste mechanism. Protected from host restriction by acetylation of Adenine residues. After the infection, phage Mu enters either lytic or lysogenic cycle – dependent on repressor protein Rep. Linear genome is replicated as a part of the host DNA.

15. Host DNA acts as a target site for the binding site after the duplication of 5 base pairs. Staggered cuts are made, which permits the integration of the viral DNA. Single strands are converted to double strands – by Mu integration. Requires transposase – gene A product. Mu repressor – Gene C product - not activated – lytic pathway. Can’t enter if lysogeny is introduced. Mu DNA is replicated by repeated transposition of multiple sites on host genome. Viral protein synthesis – Protein C expression – head and tail formation.

16. Cell is lysed – mature phage particles produced. Accumulation of repressor protein – transcription of Mu integrated DNA prevented – Lysogenic state.

17. References Brock Biology of Microorganisms – Madigan. Martniko. Stahl. Clark – 13 th Edition. Website – NCBI Biologydiscussion.com Viralzone.expassy.org Mol.biol4masters.masters.grkraj.org Utminers.utep.edu