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Bacteriophage Families with a detailed description of Models Phages Inoviridae – M13

Bacteriophage Families with a detailed description of Models Phages Inoviridae – M13. Viro102: Bacteriophages & Phage Therapy 3 Credit hours NUST Centre of Virology & Immunology. LECTURE 9:. M13: Genome. Replication of RF. Required for replication of RF.

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Bacteriophage Families with a detailed description of Models Phages Inoviridae – M13

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  1. Bacteriophage Families with a detailed description of Models PhagesInoviridae – M13 Viro102: Bacteriophages & Phage Therapy 3 Credit hours NUST Centre of Virology & Immunology LECTURE 9:

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

  3. M13 genome exist in two forms: • Replicative form (RF), dsDNA • whereas the positive (+) strand ssDNA form of the genome is referred to as the infective form (IF). • The replicative form serves as a template to synthesize the (+) strand and for transcription of the phage genes.

  4. M13 life cycle Replication: A: Second strand synthesis: • Plus(+) strand viral genome, is the template for synthesis of the complementary minus(-) strand. • Host RNA pol synthesizes an RNA primer, the 3’ end of which is extended by host DNA pol III, synthesizing the (-) strand. • The primer is removed, the gap is filled & ds, circular, supercoiled replicative form (RF) generated.

  5. Stage: 1 Replication

  6. M13 Life cycle • Multiplication of RF • RF is replicated by a rolling circle mechanism. • Phage encoded pII, a nicking-closing enzyme, nicks RF at specific site on the viral (+)strand. • DNA pol III elongates the (+) strand, & the original (+) strand is displaced. • After a round of replication is completed, pIIcleaves & circularizes the displaced (+) strand, which is converted to RF.

  7. Multiplication of RF; Rolling circle replication

  8. M13 Life cycle • Conversion of RF to viral(+) strand DNA for packaging is initiated by binding of pV • pV dimers binds cooperatively to (+) strand & convert the circular ssDNA into a filament like pV:DNA complex • pV:DNA complex is translocated to the inner membrane for assembly of phage particles

  9. Stage:3Amplification of ssDNA; RF into ssDNA

  10. Animations • http://www.eko.uj.edu.pl/mycorrhiza/mikro/perry_1413.swf

  11. Bacteriophage Families

  12. ФX174 Electron micrograph of Microviridae family member ФX174 Microviridae • Group II (ss circular DNA). • Genome size ranges from 4.6 – 6.1 kb. • Tailless icosahedral bacteriophages. • Diameter is 25 – 27 nm.

  13. ФX174: Genome Rolling circle replication initiation & termination • 11 genes Beneficial for growth of phage Capsid morphogenesis Phage maturation Phage assembly Core protein Major coat protein Major spike protein Minor spike protein Required for Host autolysin activation

  14. ФX174: Life Cycle • Virus particle binds to target cell. Its tail fibers recognize LPS of the host cell & the capsid injects DNA core into cell. • Host polymerase convert the (+)ssDNA viral genome into a covalently closed dsDNA called replicative form DNA I (RFI). • Early viral genes are transcribed by host RNA polymerase, producing viral replication proteins.

  15. Cont’d • Viral protein A cleaves RFI DNA strand at the origin of replication and covalently attaches itself to the DNA, generating RFIImolecule. • (+)strand replication occurs by rolling circle, which is converted to dsDNA by host polymerase. • Late viral genes are transcribed by host RNA polymerase.

  16. Procapsid assembly in the cytoplasm. • Viral protein C binds to replication complex, inducing packaging of neo-synthesized (+)DNA into procapsids. • Procapsids are matured in host cytoplasm • Viral lysozyme attacks peptidoglycan wall, lysing the cell & releasing mature virions.

  17. ФX174 release from Cell • Ф X174 does not encode an endolysin or holin and has no way to attack pre-existing bacterial wall structures. • So, how does it escape the cell? • This phage interferes with bacterial enzymes that make precursors of peptidoglycan. • The lack of coordination between peptidoglycan synthesis and bacterial wall construction leaves the wall weak, leading to its ultimate collapse due to osmotic pressures from within.

  18. ФX174 release from Cell • Ф X174 protein E, associated with the bacterial membrane, blocks the activity of MraY, a bacterial protein that catalyzes the transfer of peptidoglycan precursors to lipid carriers so that the precursors can be transported through the membrane • As the cell disintegrates, Ф X174 particles are released to find new, unsuspecting hosts.

  19. ФX174: FACTS • The ФX174 (or phi X) bacteriophage was the first DNA-based genome to be sequenced. • This work was completed by Fred Sanger and his team in 1977. • In 2003 it was reported that the whole genome of phi X 174 had been assembled synthetically from scratch.

  20. Thank YOU !!

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