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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
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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
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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
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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
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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
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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
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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
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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
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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
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How to supercoil DNA
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What Do Oris Look Like?
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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
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Two Basic Modes of Replication
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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?
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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
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What’s the Ori Core Sequence?
Between pE and pL
AT richLT binding sites, SP1 sites
Nucleosome free - Why?
Late promoter
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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
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Two LT hexamers bind
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Binding distorts early palindrome unwinding origin
Initiation of DNA Synthesis
Initiation of DNA Synthesis
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Binding of Rpa occurs
ATP
ADP + Pi
Initiation of DNA Synthesis
Two LT hexamers bind
Binding distorts early palindromeunwinding origin
Binding of Rpa occurs
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DNA Synthesis Initiates at a Unique Origin
RE Site
Ori
OriRE Site RE Site
How do you know that replication is bidirectional?
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The ProblemHow to connect the Okazaki fragments
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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
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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......?
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Unwinding at the Ori
SSBP
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Cellular Proteins Required for Polyomavirus DNA Replication
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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
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Leading strand presynthesis complex
DNA Synthesis by Polyomaviridae is Bidirectional
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Lagging strandRf-C binds 3’ R-D pcna is next
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DNA Synthesis by Polyomaviridae is Bidirectional
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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
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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
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Initiation of Herpesvirus DNA Replication
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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!
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Herpesvirus DNA Replication
OBP ss DBPHelicase-primase
PolymeraseProcessivity
55
Rolling Circle Replication
Cleavage Packaging Sites
Assay for DNA Replication
56
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