Replication of DNA Virus Genomes. Lecture 7 Virology W3310/4310 Spring 2013

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1 Replication of DNA Virus Genomes Lecture 7 Virology W3310/4310 Spring 2013

2 It s all about Initiation Problems faced by DNA replication machinery Viruses must replicate their genomes to make new progeny 2

3 Virus Genomes Require Special Copying Mechanisms Parvovirus Herpesvirus Adenovirus Polyomavirus 3

4 DNA Replication Replication requires expression of at least one virus protein, sometimes many DNA is always synthesized 5-3 via semiconservative replication Replication initiates at a defined origin using a primer The host provides other proteins 4

5 What s the Host for? Viruses Can t do it Themselves Viruses are parasites Enzymes and scaffolds Simple viruses conserve genetic information - always hijack more host proteins Complex viruses encode many, but not all proteins required for replication 5

6 DNA Replication Genomes come in a wide assortment of shapes and sizes Replication yields progeny - a switch for gene regulation - an important regulatory event Always delayed after infection 6

7 Outcomes of DNA Replication Lytic infection - new progeny - high copy number Latent infection - stable assimilation in host at low copy number - virus genomes may be episomal or integrated 7

8 Requirements for DNA Replication Ori recognition for initiation - binding to an AT-rich DNA segment Priming of DNA synthesis - RNA - Okazaki fragments - DNA - hairpin structures - protein - covalently attached to 5 end Elongation Termination 8

9 Requirements for DNA Replication Viruses don t replicate well in quiescent cells Induction of host replication enzymes and cell cycle regulators Virus encoded immediate early and early gene products 9

10 Where Does the Polymerase Come From? Small DNA viruses do not encode an entire replication system -encode proteins that orchestrate the host -Papillomaviridae, Polyomaviridae, Parvoviridae Large DNA viruses encode most of their own replication systems -Herpesviridae, Adenoviridae, Poxviridae 10

11 Virus Encoded Proteins Origin Binding Protein, Helicases and Primase DNA polymerase and accessory proteins Exonucleases Thymidine kinase, RR, dutpase 11

12 Replication Occurs at Replication Centers! DNA templates and rep proteins Form at discrete sites ND10 s (PML bodies) Polymerases, ligases, helicases, topoisomerases Pre Post 12

13 Getting Started at Viral Origins AT-rich DNA segments recognized by viral origin recognition proteins - seed assembly of multiprotein complexes Some viruses have one ori others up to three - used for different purposes Often associated with transcriptional control regions 13

14 Virus Genomes TR p5 p19 p40 TR Ori 14

15 How to supercoil DNA 15

16 What Do Oris Look Like? 16

17 Origin Recognition Proteins Polyoma Tag binds specifically to DNA Papilloma E1 binds to ori in presence of E2 AAV Rep68/78 binds at ends and unwinds DNA, also involved in terminal resolution Adenovirus ptp binds at terminus and recruits DNA polymerase Herpesvirus UL9 protein recruits viral proteins to AT-rich ori s and then unwinds DNA 17

18 dsdna Virus Genomes 18

19 ssdna Genomes Circoviridae Parvoviridae 19 ITR ITR

20 Hepadnavirus Life-Cycle What to do with a molecule that looks this way 20

Two Basic Modes of Replication Replication Fork Leading Strand displacement (primer) 5 5 3 Papillomaviruses Polyomaviruses

21 Two Basic Modes of Replication Replication Fork Leading Strand displacement (primer) Papillomaviruses Polyomaviruses Herpesviruses Retroviral Proviriuses Lagging Primer 3 Adenoviruses (protein) Parvoviruses (DNA hairpin) Poxviruses (hairpin

22 Polyomavirus Replication Forks Initiation from a single ori, requires expression of Tag Replicate as covalently closed circles Leading strand replication occurs via extension from an RNA primer Lagging strand replication is delayed until the replication fork has moved - also uses RNA primers - creates discontinuities How to fill in the gaps? 22

23 Properties of T T is a species-specific DBP/OBP -preinitiation complexes do not form in the wrong species -failure to interact with DNA polα-primase Binds and sequesters cell cycle regulators -causes cells to enter S phase - WHY? T synthesis is autoregulated -protein is heavily modified -controls DNA binding -promotes cooperativity -affects unwinding of DNA 23

24 T antigen a multifunctional virus-specified early protein 24

25 What s the Ori Core Sequence? Late promoter Between pe and pl LT binding sites, SP1 sites AT rich Nucleosome free - Why? 25

26 Polyomaviruses Covalently closed circular, double stranded DNA Bidirectional replication Leading Lagging Lagging 26 Leading

27 Leading vs. Lagging Leading strand DNA synthesis is continuous Lagging strand DNA synthesis is discontinuous Direction of synthesis off of either template strand is the same 27

28 Initiation of DNA Synthesis LT Binding A/T site II EP 1 ATP + LT Two LT hexamers bind 28

29 Initiation of DNA Synthesis 2 Conformational change of EP sequence Binding distorts early palindrome unwinding origin 29

30 Initiation of DNA Synthesis ATP ADP + Pi 3 RPA Binding of Rpa occurs 30

31 Initiation of DNA Synthesis Two LT hexamers bind Binding distorts early palindrome unwinding origin Binding of Rpa occurs 31

32 DNA Synthesis Initiates at a Ori Unique Origin RE Site Ori RE Site RE Site How do you know that replication is bidirectional? 32

33 The Problem How to connect the Okazaki fragments 33

34 The Leading Strand Is Easy Presynthesis complex pol α, T and Rp-A Rf-C binds 3 OH along with PCNA and pol δ -Rf-C a clamp loading protein -Allows entry of PCNA on DNA -Causes release of pol α Form sliding clamps along DNA Continuous copying of parental strand 34

35 The Lagging Strand - Not So Easy 1st primer and Okazaki fragment made by pol α-primase complex DNA is copied from the replication fork toward the origin Multiple initiations are required to replicate the template strand Both leading and lagging strands move in the same direction! Which moves, the DNA or the complex? -Template has to move, otherwise...? 35

36 Unwinding at the Ori SSBP 36

37 Cellular Proteins Required for Polyomavirus DNA Replication 37

38 DNA Synthesis by Polyomaviridae is Bidirectional Leading strand presynthesis complex 38

39 DNA Synthesis by Polyomaviridae is Bidirectional Rf-C binds 3 R-D pcna is next Lagging strand Polα/primase PCNA RFC 39

40 DNA Synthesis by Polyomaviridae is Bidirectional 40

41 DNA Synthesis by Polyomaviridae is Bidirectional Remove RNA, fill gaps, seal 41

42 Polyomaviridae DNA Synthesis Leading strand Rf-C binds 3 R-D pcna is next presynthesis complex Lagging strand 42 Remove RNA, fill gaps, seal

43 The DNA Replication Machine Lagging strand Leading strand 43

44 The Replication Machine DNAi_replication_vo1-lg.wmv 44

45 Problems in Replication Catenated molecules 45

46 Termination - the End Separate daughter molecules from replication complex Topos relax and unwind supercoils - relieve torsional stress caused by unwinding - unwinding leads to overwinding throughout the rest of the molecule Topo II decatenates - separating daughter molecules by cleaving and resealing the replicated molecules 46

47 DNA Priming Priming via a specialized structure Parvoviruses self prime, form a template primer Their genomic DNAs are ss of both + and - polarity and they contain Inverted Terminal Repeats start here - but how to get to the end? 47

48 AAV Replication is Continuous ITR A dependovirus! No pol α, uses Inverted Terminal Repeat to self-prime Requires pol δ, Rf-C and PCNA Rep78/68 proteins are required for initiation and resolution - endonuclease, helicase, binds 5 terminus No replication fork! 48

49 Replication of Adenovirus Genome Strand displacement synthesis Utilizes a protein primer Origins at both ends Assembly of ptp into a preinitiation complex activates covalent linkage of dcmp to a S residue in ptp by viral DNA pol Semiconservative DNA replication from different replication forks 49

50 Protein Priming Adenoviridae - -Precursor to terminal protein (ptp) -Ad DNA pol links α-phosphoryl of dcmp to OH of a S residue in ptp -Added only when protein primer is assembled with DNA pol into preinitiation complex at ori -3 OH primes synthesis of daughter strand 50

51 Protein Priming Displaced ss Template DNA 51 ITRs Single-stranded DNA Template

52 Herpes Simplex Virus HSV 2 oris and a unique oril sequence Four equimolar isomers of virus genome DNA enters as linear molecule converts to circle Virus dissociates host ND10s Replicates as a rolling circle 52

53 Initiation of Herpesvirus DNA Replication UL US Circularization DNA ligase IV/ XRCC4 Host proteins are responsible for circularization 53

54 HSV Gene Products Required for Replication UL5, 8 and 52 - form primase UL42 - a processivity protein UL9 - Origin Binding Protein UL29 - SS DNA Binding Protein UL30 - DNA polymerase Necessary but not sufficient! 54

55 Herpesvirus DNA Replication OBP ss DBP ss DBP Helicase-Primase HSV Polymerase Processivity Protein 55

56 Rolling Circle Replication Cleavage Packaging Sites 56

Viral DNA replication

Viral DNA replication Lecture 8 Biology 3310/4310 Virology Spring 2017 The more the merrier --ANONYMOUS Viral DNA genomes must be replicated to make new progeny Parvovirus Retrovirus Poliovirus VII Hepatitis