Replication of DNA Virus GenomesLecture 7

Virology W3310/4310Spring 2013

It’s all about Initiation

• Problems faced by DNA replication machinery

• Viruses must replicate their genomes to make new progeny

2

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

4

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

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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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 ori’s 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

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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T antigena multifunctional virus-specified early protein

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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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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 + Pi

3 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 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

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Leading strand presynthesis complex

DNA Synthesis by Polyomaviridae is Bidirectional

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Lagging strand

PCNA RFC

Polα/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 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

48

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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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