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

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 Minor protein N-terminus binds to F pilus of host cell Required for assembly Involved in attachment & morphogenesis Required for assembly

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.

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.

Stage: 1 Replication

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, pII cleaves & circularizes the displaced (+) strand, which is converted to RF.

Multiplication of RF; Rolling circle replication

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

Stage:3Amplification of ssDNA; RF into ssDNA

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

Bacteriophage Families Cystoviridae Siphoviridae Leviviridae Myoviridae Rudiviridae Inoviridae Fuselloviridae Microviridae Tectiviridae Podoviridae Lipothrixviridae Corticoviridae Plasmaviridae

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

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

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.

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

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.

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.

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.

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.

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