Віруси з олДНК

1.

ВІРУСИ З олДНК

2.

Стадії реплікації олДНК вірусів
1. Перетворення в дволанцюжкову ДНК
клітинний процес відновлення?)
2. Рання транскрипція (клітинні
ферменти)
3. Трансляція (регуляторних) білків і
“кільцева" оцДНК реплікація
4. Пізня транскрипція (як правило,
вірус-опосередкованих білків)
5. Синтез пізніх (= структурних)
білків
6. "Ізоляція" вірусного генома
оцДНК
7. Збірка у віріони

3. ssDNA Viruses

Bacteria ("bacteriophages", eg. Inoviridae,
Microviridae),
Mammals (Circoviridae, Parvoviridae)
Birds (circovirus-like organisms),
Plants (Geminiviridae, banana bunchy top-like viruses
or Nanoviruses).

4. Parvovirus

Family: Parvoviridae
~20 nm in diameter
Latin parvus means small
(0.02 µm)
Single-stranded DNA virus
Icosahedral capsid
No envelope
X-ray crystallographic image of parvovirus

5.

Infection Cycle

6. Genome Structure

Type:
Autonomous virus: (-) DNA
eg. Mice minute virus
Defective virus: (+) and (-) DNA in separate virions
Adeno-associated virus
Replication dependents on coinfection with helper virus.
Eg. Adenovirus
Terminal hairpins (inverted reapeats)

7.

8.

Adeno-associated virus (AAV); ssDNA
ITR
ITR
Rep
Cap
5 kb ssDNA, inverted terminal repeats (ITR)
Rep gene required for DNA replication
Cap gene encodes capsid proteins

9. Генетична карта парвовірусу (ААВ)

Valeriy Polischuk Virology Department Kyiv National Taras Shevchenko University

10. Replication of Parvovirus

Encodes two protein
Replication and capsid
“Hairpin” structure,
serves as the template for
conversion into dsDNA
ssDNA -> dsDNA ->
ssDNA

11. A Model for Autonomous Parvovirus Replication

12.

Autonomous parvovirus Life Cycle
From Medical Microbiology, 5th ed.,
Murray, Rosenthal, Kobayashi & Pfaller,
Mosby Inc., 2002, Fig. 56-2.

13.

Helper dependent parvovirus (AAV) replication
Infection with adenovirus
Infection without adenovirus
Lytic
replication
AAV DNA
integrates into
chromosome 19
Superinfect
with
adenovirus

14.

15.

M13

16.

Schematic representation of bacteriophage
M13 replication cycle
E.coli
F-pilus
gp 8
DNA
gp 5
M13

17.

Schematic structure of bacteriophage M13 filament
Distal end
Gene 7 protein
Gene 9 protein
Circular ssDNA
Gene 8 protein
Gene 6 protein
Proximal end
Gene 3 protein

18. M13 Structure

Dimensions:
6.5nm in diameter
Length dependant on genome but wild type
approximately 930nm
Mass 16.3MD of which 87% comprised of
protein
Genome:
Single stranded circular DNA molecule,
covalently closed
Housed in a flexible protein cylinder

19. M13 Structure

Proteins
Length of phage cylinder comprises 2700 copies of
the 50-amino-acid major coat protein pVIII
At one terminus:
5 copies of 33 AA residue pVII
5 copies of 32 AA residue pIX
At one terminus:
5 copies of 406 AA residue pIII
5 copies of 112 AA residue pVI

20. M13 Gene Functions

Gene
Function
Amino Acids
Mol Wt
II
DNA Replication
410
46137
X
DNA Replication
111
12672
V
Binding ssDNA
87
9682
VIII
Major capsid protein
50
5235
III
Minor capsid protein
406
42522
VI
Minor capsid protein
112
12342
VII
Minor capsid protein
33
3599
IX
Minor capsid protein
32
3650
I
Assembly
348
39502
IV
Assembly
405
43476
XI
Assembly
108
12424

21.

M13 (or f1) phages are filamentous phages that infect E. coli through pili,
and are able to produce new virions without lysing the host cell. M13 has some key
structural elements:
Circular, single-stranded DNA
~6.4 kb long
10 genes in the genome.

22.

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 ssDNA to the membrane,
Gene VIII: coat protein, encapsulates the pilot protein
and the ssDNA phage DNA as it extrudes through the
membrane
M13 phages are useful for a number of applications:
sequencing
mutagenesis
probes
lambda-ZAP subcloning
phage display libraries