Replication of DNA Virus GenomesLecture 7
Virology W3310/4310Spring 2013
Its all about Initiation
Problems faced by DNA replication machinery
Viruses must replicate their genomes to make new progeny
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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 proteins4
Whats the Host for?Viruses Cant 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 dont 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
ND10s (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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Virus Genomes
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p5 p19 p40TR TR
Ori
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 oris and then unwinds DNA
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dsDNA Virus Genomes
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ssDNA Genomes
Circoviridae
Parvoviridae
ITR ITR19
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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Replication Fork Strand displacement (primer)
PapillomavirusesPolyomavirusesHerpesvirusesRetroviral Proviriuses
Adenoviruses (protein)Parvoviruses (DNA hairpin)Poxviruses (hairpin
Primer53
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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?22
Properties of T
T is a species-specific DBP/OBP
-preinitiation complexes do not form in the wrong species
-failure to interact with DNA pol-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 antigena multifunctional virus-specified early protein
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Whats 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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Initiation of DNA Synthesis
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Two LT hexamers bind
LT BindingA/T site II EP
ATP + LT1
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Binding distorts early palindrome unwinding origin
Initiation of DNA Synthesis
Conformational change of EP sequence2
Initiation of DNA Synthesis
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Binding of Rpa occurs
ATP
ADP + Pi3 RPA
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 , T and Rp-A Rf-C binds 3OH along with PCNA and pol
-Rf-C a clamp loading protein
-Allows entry of PCNA on DNA
-Causes release of pol
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 -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
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Leading strand presynthesis complex
DNA Synthesis by Polyomaviridae is Bidirectional
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Lagging strand
PCNA RFC
Pol/primaseRf-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
Polyomaviridae DNA Synthesis
Leading strand presynthesis complex
Lagging strandRf-C binds 3 R-D pcna is next
Remove RNA, fill gaps, seal42
The DNA Replication Machine
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Leading strand
Lagging strand
The Replication Machine
http://www.hhmi.org/biointeractive/media/DNAi_replication_vo1-lg.wmv
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Problems in Replication
Catenated molecules45
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 , uses Inverted Terminal Repeat to self-prime Requires pol , 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 -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
-3OH primes synthesis of daughter strand
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Protein Priming
ITRs Single-stranded DNA Template
Displaced ss Template DNA
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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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Herpesvirus DNA Replication
OBP ss DBPHelicase-Primase
HSV PolymeraseProcessivity Protein
ss DBP
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Rolling Circle Replication
Cleavage Packaging Sites
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