Replication of DNA Virus Genomes - · PDF fileReplication of DNA Virus Genomes Lecture 7...
Transcript of Replication of DNA Virus Genomes - · PDF fileReplication of DNA Virus Genomes Lecture 7...
It’s all about Initiation
• Problems faced by DNA replication machinery
• Viruses must replicate their genomes to make new progeny
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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 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
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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
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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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Pre
Post
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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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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Two Basic Modes of Replication
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Replication Fork Strand displacement (primer)
PapillomavirusesPolyomavirusesHerpesvirusesRetroviral Proviriuses
Adenoviruses (protein)Parvoviruses (DNA hairpin)Poxviruses (hairpin
Primer5’3’
3’5’
5’
3’
Leading
Lagging
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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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
Lagging26
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
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Binding distorts early palindrome unwinding origin
Initiation of DNA Synthesis
Conformational change of EP sequence2
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 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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DNA Synthesis by Polyomaviridae is Bidirectional
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Lagging strand
PCNA RFC
Polα/primaseRf-C binds 3’ R-D pcna is next
Polyomaviridae DNA Synthesis
Leading strand presynthesis complex
Lagging strandRf-C binds 3’ R-D pcna is next
Remove RNA, fill gaps, seal42
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
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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?
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• 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
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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
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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
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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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Host proteins are responsible for circularization
DNA ligase IV/XRCC4
Circularization
UL US
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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