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Replication of DNA Virus GenomesLecture 7
Virology W3310/4310Spring 2012
1
Virus Genomes Require Special Copying Mechanisms
Parvovirus
Herpesvirus
Adenovirus Polyomavirus
2
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
3
What’s the host for?
• 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
4
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
5
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
6
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
7
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
8
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
9
Virus Encoded Proteins
• Origin Binding Protein, Helicases and Primase
• DNA polymerase and accessory proteins
• Exonucleases
• Thymidine kinase, RR, dUTPase
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Replication Occurs at Replication Centers!
• DNA templates and rep proteins
• Form at discrete sites ND10’s (PML bodies)
• Polymerases, ligases, helicases, topoisomerases
11
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
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Virus Genomes
13
How to supercoil DNA
14
What Do Oris Look Like?
15
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
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dsDNA Virus Genomes
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ssDNA Genomes
Circoviridae
Parvoviridae
ITR ITR18
Hepadnavirus Life-Cycle
What to do with a molecule that looks this way
19
Two Basic Modes of Replication
20
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?
21
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
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T antigen
23
What’s the Ori Core Sequence?
Between pE and pL
AT richLT binding sites, SP1 sites
Nucleosome free - Why?
Late promoter
24
Polyomaviruses
• Covalently closed circular, double stranded DNA
Bidirectional replication
Leading
Leading
Lagging
Lagging25
Leading vs. Lagging
• Leading strand DNA synthesis is continuous
• Lagging strand DNA synthesis is discontinuous
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Initiation of DNA Synthesis
27
Two LT hexamers bind
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Binding distorts early palindrome unwinding origin
Initiation of DNA Synthesis
Initiation of DNA Synthesis
29
Binding of Rpa occurs
ATP
ADP + Pi
Initiation of DNA Synthesis
Two LT hexamers bind
Binding distorts early palindromeunwinding origin
Binding of Rpa occurs
30
DNA Synthesis Initiates at a Unique Origin
RE Site
Ori
OriRE Site RE Site
How do you know that replication is bidirectional?
31
The ProblemHow to connect the Okazaki fragments
32
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
33
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......?
34
Unwinding at the Ori
SSBP
35
Cellular Proteins Required for Polyomavirus DNA Replication
36
DNA Synthesis by Polyomaviridae is Bidirectional
Leading strand presynthesis complex
Lagging strandRf-C binds 3’ R-D pcna is next
Remove RNA, fill gaps, seal37
DNA Synthesis by Polyomaviridae is Bidirectional
38
Leading strand presynthesis complex
DNA Synthesis by Polyomaviridae is Bidirectional
39
Lagging strandRf-C binds 3’ R-D pcna is next
40
DNA Synthesis by Polyomaviridae is Bidirectional
41
DNA Synthesis by Polyomaviridae is Bidirectional
Remove RNA, fill gaps, seal
The DNA Replication Machine
Leading strand
Lagging strand
42
The Replication Machine
http://www.hhmi.org/biointeractive/media/DNAi_replication_vo1-lg.wmv
43
Problems in Replication
Catenated molecules44
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
45
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?
46
• 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!
AAV Replication is ContinuousITR
47
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
48
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 -Semiconservative DNA replication from different replication forks
49
Protein Priming
ITRs Single-stranded DNA Template
Displaced ss Template DNA
50
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
51
Initiation of Herpesvirus DNA Replication
52
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!
53
54
Herpesvirus DNA Replication
OBP ss DBPHelicase-primase
PolymeraseProcessivity
55
Rolling Circle Replication
Cleavage Packaging Sites
Assay for DNA Replication
56