1. Lecture 15: BSCI437 DNA Virus Genome Replication Flint et al., Chapter 9

2. 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.

3. DNA replication: general principles 1.Always template directed 2.5’  3’ synthesis 3.Semiconservative 4.Begins at specific sites: origins of replication 5.Stops at specific sites: termini 6.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

4. 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. Fig. 9.1 Note semi-conservative replication

5. Transmission electronmicrograph of replication bubbles

6. 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.

7. 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 Pol  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.

8. 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.

9. SV40 origin of replication SV40 origin requires specific sequences: AT rich element, 2 LT protein binding sites Perfect and imperfect palindromes.

10. 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.

11. Chain elongation: Leading strand synthesis Pol  -primase synthesizes RNA primers along leading strand. Replication factor C (RfC) binds 3’OH groups Proliferating cell nuclear antigen (Pcna) recruited onto template DNA Pol  recruited, leading strand DNA elongation begins

12. Chain elongation: Lagging strand synthesis DNA pol  -primase lays down Okazaki fragment RfC-Pcna-DNA-Pol  complex begins elongation on 3’ OH groups of RNA primers. Leading strandLagging strand

13. 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.

14. 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.

15. 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.

16. Priming via DNA – Parvoviruses Viral genomes have Inverted Terminal Repeats (ITR)

17. Priming via DNA – Parvoviruses 3’ end of genome primes elongation to 5’ end

18. Priming via DNA – Parvoviruses Completion requires formation of a nick, and replication of ITR

19. 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.

20. All you need is an –OH group to prime nucleic acid synthesis An NTP Serine

21. 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

22. 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.

23. 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

24. 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

25. 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.

26. LIMITED REPLICATION OF VIRAL DNA DNA replication must be limited in viral Latent Infections Allows for long-term infection.

27. LIMITED REPLICATION OF VIRAL DNA Strategies: –Replication as part of cellular genome: retroviruses

28. 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)