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