ВІРУСИ З ол. ДНК
Стадії реплікації ол. ДНК вірусів 1. Перетворення в дволанцюжкову ДНК клітинний процес відновлення? ) 2. Рання транскрипція (клітинні ферменти) 3. Трансляція (регуляторних) білків і “кільцева" оц. ДНК реплікація 4. Пізня транскрипція (як правило, вірус-опосередкованих білків) 5. Синтез пізніх (= структурних) білків 6. "Ізоляція" вірусного генома оц. ДНК 7. Збірка у віріони
ss. DNA Viruses n Bacteria ("bacteriophages", eg. Inoviridae , Microviridae ), n Mammals (Circoviridae , Parvoviridae 細小病毒 科 圓環病毒科 n Birds (circovirus-like organisms), n Plants (Geminiviridae , banana bunchy top-like viruses or Nanoviruses).
Parvovirus n Family: Parvoviridae n Latin parvus means small n ~20 nm in diameter n (0. 02 µm) n n n Single-stranded DNA virus Icosahedral capsid No envelope X-ray crystallographic image of parvovirus
Infection Cycle
Genome Structure n Type: n Autonomous virus: (-) DNA (自動複製能力 ） n eg. Mice minute virus n Defective virus: (+) and (-) DNA in separate virions n Adeno-associated virus n Replication dependents on coinfection with helper virus. Eg. Adenovirus n Terminal hairpins (inverted reapeats)
Adeno-associated virus (AAV); ss. DNA ITR Rep Cap 5 kb ss. DNA, inverted terminal repeats (ITR) Rep gene required for DNA replication Cap gene encodes capsid proteins ITR
Генетична карта парвовірусу (ААВ) Valeriy Polischuk Virology Department Kyiv National Taras Shevchenko University
Replication of Parvovirus n Encodes two protein n Replication and capsid n “Hairpin” structure, serves as the template for conversion into ds. DNA n ss. DNA -> ds. DNA -> ss. DNA
A Model for Autonomous Parvovirus Replication
Autonomous parvovirus Life Cycle From Medical Microbiology, 5 th ed. , Murray, Rosenthal, Kobayashi & Pfaller, Mosby Inc. , 2002, Fig. 56 -2.
Helper dependent parvovirus (AAV) replication Infection with adenovirus Infection without adenovirus Lytic replication AAV DNA integrates into chromosome 19 Superinfect with adenovirus
M 13
Schematic representation of bacteriophage M 13 replication cycle E. coli F-pilus gp 8 DNA gp 5 M 13
Schematic structure of bacteriophage M 13 filament Distal end Gene 7 protein Gene 9 protein Circular ss. DNA Gene 8 protein Gene 6 protein Proximal end Gene 3 protein
M 13 Structure n Dimensions: 6. 5 nm in diameter n Length dependant on genome but wild type approximately 930 nm n Mass 16. 3 MD of which 87% comprised of protein n n Genome: Single stranded circular DNA molecule, covalently closed n Housed in a flexible protein cylinder n
n Proteins n n M 13 Structure Length of phage cylinder comprises 2700 copies of the 50 -amino-acid major coat protein p. VIII At one terminus: 5 copies of 33 AA residue p. VII n 5 copies of 32 AA residue p. IX n n At one terminus: 5 copies of 406 AA residue p. III n 5 copies of 112 AA residue p. VI n
M 13 Gene Functions Gene II Function DNA Replication Amino Acids 410 Mol Wt 46137 X V DNA Replication Binding ss. DNA 111 87 12672 9682 VIII Major capsid protein Minor capsid protein 50 406 5235 42522 VI VII Minor capsid protein 112 33 12342 3599 IX I Minor capsid protein Assembly 32 348 3650 39502 IV XI Assembly 405 108 43476 12424
M 13 (or f 1) phages are filamentous phages that infect E. coli through pili, and are able to produce new virions without lysing the host cell. M 13 has some key structural elements: Circular, single-stranded DNA ~6. 4 kb long 10 genes in the genome.
n n Of these 10 genes, we are concerned with the following three: Gene II: codes for nickase, allows rolling-circle replication Gene III: codes for the pilot protein, which guides the nascent ss. DNA to the membrane, Gene VIII: coat protein, encapsulates the pilot protein and the ss. DNA phage DNA as it extrudes through the membrane n n n n M 13 phages are useful for a number of applications: sequencing mutagenesis probes lambda-ZAP subcloning phage display libraries
