Lecture 15: BSCI437
•DNA Virus Genome
Replication
•Flint et al., Chapter 9
General introduction
• Viral DNAs must be replicated efficiently in
infected cells to provide genomes for
assembly into progeny virions.
• Typically requires at least 1 (usually many)
viral proteins.
• Replication cannot begin until viral proteins
have been made in sufficient numbers.
• Viral DNA synthesis leads to many cycles of
replication and accumulation of large numbers
of new viral DNAs.
DNA replication: general principles
1.
2.
3.
4.
5.
6.
Always template directed
5’  3’ synthesis
Semiconservative
Begins at specific sites: origins of replication
Stops at specific sites: termini
Catalyzed by DNA-dependent DNA
polymerases.
7. Accessory proteins required for initiation and
elongation
8. A primer is always required. Unlike RNA
polymerases, there is no initiation de novo
DNA synthesis by the cellular
replication machinery
• Replicon: an
autonomously replicating
unit of DNA.
• Contain origins of
replication: where
replication starts.
• Replication is
bidirectional: 5’  3’ on
each strand of DNA
• As nascent DNA chains
are synthesized, can see
“bubbles” in the DNA
caused by outward
extension of replication
forks.
Note semi-conservative
replication
Fig. 9.1
Transmission electronmicrograph of replication bubbles
Semidiscontinuous DNA synthesis
• DNA synthesis is
always 5’  3’.
– DNA helicase activity
required to unwind
duplex DNA
– DNA ligase required
to glue together the
newly synthesized
DNA fragments.
– Topoisomerases:
required to resolve
supercoiling (twists
and knots) incurred
during replication.
Semidiscontinuous DNA synthesis
• Leading strand: toward the
3’ side of the origin (on the
strand that is being
synthesized). DNA synthesis
is continuous.
• Lagging strand: toward the
5’ side of the origin (on the
strand that is being
synthesized). DNA synthesis
is discontinuous.
– Requires priming using
Okazaki fragments: short
pieces of RNA made by Pola
primase.
– Once primed, DNA Pol. takes
over until it reaches the next
piece of newly synthesized
DNA on that strand.
– RNase H required to degrade
Okazaki fragment.
MECHANISMS OF VIRAL DNA
SYNTHESIS
All viruses face the same problems:
1.
2.
3.
4.
5.
Origin recognition and unwinding
Priming
Elongation
Termination
Resolution of intermediates.
SV40 origin of replication
•
•
•
•
SV40 origin requires specific sequences:
AT rich element,
2 LT protein binding sites
Perfect and imperfect palindromes.
Recognition and unwinding
1.
2 hexamers of SV40
encoded LT proteins
bind origin. Binding
requires ATP.
2. LT hexamers change
conformation,
changing the shape
of DNA
3. This is recognized by
cellular Replication
Protein A (RpA)
which has DNA
helicase activity.
Chain elongation: Leading strand synthesis
•
•
•
•
Pola-primase
synthesizes RNA
primers along leading
strand.
Replication factor C
(RfC) binds 3’OH
groups
Proliferating cell
nuclear antigen (Pcna)
recruited onto
template
DNA Polg recruited,
leading strand DNA
elongation begins
Chain elongation: Lagging strand
synthesis
•
DNA pola-primase
lays down Okazaki
fragment
•
RfC-Pcna-DNA-Polg
complex begins
elongation on 3’ OH
groups of RNA
primers.
Leading strand
Lagging strand
Chain termination and resolution: I
• Termination occurs when DNA Pol encounters
dsDNA.
• Resolution:
– Unwinding a portion of a closed, wound structure
creates a topological problem: it causes another
region to become over-wound. Can resolve this by
creating either single or double stranded breaks,
allowing unwinding to occur. This is done by DNA
Topoisomerase I or II.
Chain resolution II
• Termination occurs when DNA Pol encounters
dsDNA.
• Resolution:
– After DNA replication, the two DNA strands are
hopelessly intertwined (catenated). Can resolve this by
creating double stranded breaks, allowing one DNA
molecule to pass through the other. Done by DNA
Topoisomerase II.
Virus-specific priming
•
Many DNA viruses have evolved to
dispense with RNA priming.
•
They can direct priming from either:
1. Their own DNA or from
2. Specific viral proteins.
Priming via DNA – Parvoviruses
• Viral genomes have Inverted Terminal
Repeats (ITR)
Priming via DNA – Parvoviruses
• 3’ end of
genome
primes
elongation
to 5’ end
Priming via DNA – Parvoviruses
• Completion
requires
formation of a
nick, and
replication of
ITR
3’ –OH groups and Nucleic Acid Synthesis
• Nucleolytic
attack by 3’ –
OH group on
the phosphate
group on 5’ end
of an NTP
results in
formation of a
phosphodiester
bond.
All you need is an –OH group to
prime nucleic acid synthesis
An NTP
Serine
Priming via Protein – Adenoviral DNA
• Virus encoded preterminal protein (pTP)
covalently attaches to 3’ end of genome.
• OH group on a pTP serine acts as 3’ OH end to
prime DNA synthesis
MECHANISMS OF EXPONENTIAL
VIRAL DNA REPLICATION
General points
• Uncontrolled DNA replication is bad for
cells  cancer
• Cells express many proteins that inhibit
DNA replication: called Tumor
suppressor genes.
• Viruses must circumvent these controls.
Example: Inactivation of Rb tumor
suppressor protein
• Retinoblastoma (Rb) protein binds
promoters of proteins required for cells
to exit G1 phase and enter S phase
• Rb protein blocks transcription of these
genes
• Result: inhibition of DNA replication
– Loss of Rb function associated with
retinoblastomas and other tumors in
children/young adults
Example: Inactivation of Rb tumor
suppressor protein
• Many viruses encode proteins that
inactivate Rb protein
• Allows uncontrolled DNA synthesis
– Examples include:
• SV40 LT protein
• Papillomavirus E7 proteins
• Adenovirus E1A proteins
Viral DNA replication
independent of cellular proteins
• e.g. Poxviruses
• Large genomes of
Poxviruses encode all
proteins required for
viral DNA synthesis.
• Genomically “expensive”
strategy.
• Virus replication occurs
in foci in cytoplasm
called viral factories.
LIMITED REPLICATION OF
VIRAL DNA
• DNA replication must be limited in viral
Latent Infections
• Allows for long-term infection.
LIMITED REPLICATION OF
VIRAL DNA
• Strategies:
– Replication as part of cellular genome:
retroviruses
LIMITED REPLICATION OF VIRAL DNA
• Strategies:
– Replication as a minichromosome (episome):
herpesviruses, papillomaviruses.
• Can be regulated to provide for limited or unlimited
replication (Fig. 9.20)