Lecture 15: DNA Virus Genome Replication
Flint et al., Chapter 9
BSCI437
General introduction
 Viral DNAs must be replicated efficiently in infected cells to provide genomes of
assembly into progeny virions.
 Typically requires at least 1 (usually many) viral proteins.
 Therefore, 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 nubers of new viral DNAs.
DNA replication: general principles.
 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.
 Accessory proteins required for initiation and elongation
 A primer is always required. Unlike RNA polymerases, there is no initiation de
novo
DNA synthesis by the cellular replication machinery (Fig, 9.1)
 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.
Semidiscontinuous DNA synthesis (Fig. 9.2)
 DNA synthesis is always 5’  3’.
o DNA helicase activity required to unwind duplex DNA
o DNA ligase required to glue together the newly synthesized DNA
fragments.
o Topoisomerases: required to resolve supercoiling (twists and knots)
incurred during replication.
 Leading strand: toward the 3’ side of the origin. DNA synthesis is continuous.
 Lagging strand: toward the 5’ side of the origin. DNA synthesis is discontinuous.
o Requires priming using Okazaki fragments: short pieces of RNA made by
Pol primase.
o Once primed, DNA Pol. takes over until it reaches the next piece of newly
synthesized DNA on that strand.
o RNase H required to degrade Okazaki fragment.
MECHANISMS OF VIRAL DNA SYNTHESIS
All viruses face the same problems:
1. Origin recognition and unwinding
2. Priming
3. Elongation
4. Termination
5. Resolution of intermediates.
Origin recognition and unwinding: SV40 as a model (Fig. 9.3, 9.4)
SV40 origin: Fig. 9.3 requires specific sequences: AT rich element, 2 LT protein
binding sites, perfect and imperfect palindromes.
Recognition and unwinding (Fig. 9.4)
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 (Fig. 9.5, steps 1, 2)
1. Pol-primase synthesizes RNA primers along leading strand.
2. Replication factor C (RfC) binds 3’OH groups
3. Proliferating cell nuclear antigen (Pcna) recruited onto template
4. DNA Pol recruited, leading strand DNA elongation begins
Chain elongation: Lagging strand synthesis (Fig. 9.7)
1. DNA pol-primase lays down Okazaki fragment
2. RfC-Pcna-DNA-Pol complex begins elongation on 3’ OH groups of RNA
primers
Chain termination and resolution (Fig. 9.8)
Termination occurs when DNA Pol encounters dsDNA.
Resolution:
 Unwinding a portion of a closed, wound structure creates a topological problem in
that it causes another region to become over-wound. Can resolve this be
creating either single or double stranded breaks, allowing unwinding to occur.
This is done by DNA Topoisomerase I or II.
 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 their own DNA or from specific viral proteins.
Priming via DNA – Parvoviruses (Fig. 9.9)
 Viral genomes have Inverted Terminal Repeats (ITR)
 3’ end of genome primes elongation to 5’ end
 Completion requires formation of a nick, and replication of ITR
Priming via Protein – Adenoviral DNA (Fig. 9.10)
 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
o Loss of Rb function associated with retinoblastomas in children/young
adults
 Many viruses encode proteins that inactivate Rb protein
 Allows uncontrolled DNA synthesis
o 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.
 Strategies:
o Replication as part of cellular genome: retroviruses
o Replication as a minichromosome (episome): herpesviruses,
papillomaviruses.
 Can be regulated to provide for limited or unlimited replication (Fig. 9.20)