Replication of DNA Virus Genomes

Lecture 7
Virology W3310/4310
Spring 2013

Its all about Initiation

Problems faced by DNA replication

machinery

Viruses must replicate their genomes to

make new progeny

Virus Genomes Require Special

Copying Mechanisms
Parvovirus
Herpesvirus
Polyomavirus

Adenovirus

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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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

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

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

at discrete sites
Form

ND10s (PML bodies)
Polymerases, ligases, helicases, topoisomerases
Pre
Post
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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
TR

TR
p5

p19

p40

Ori

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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 oris and then unwinds DNA
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dsDNA Virus Genomes

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ssDNA Genomes

Parvoviridae
Circoviridae
ITR
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ITR

Hepadnavirus Life-Cycle

What to do with a molecule that looks this way

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Two Basic Modes of Replication

Replication Fork

5
3
Papillomaviruses
Polyomaviruses
Herpesviruses
Retroviral
Proviriuses

Leading

Strand displacement (primer)

5

Primer
3

3
5
Adenoviruses (protein)
Parvoviruses (DNA hairpin)
Poxviruses (hairpin

Lagging

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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 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 antigen
a multifunctional virus-specified early protein

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Whats the Ori Core Sequence?

Late promoter

Between pE and pL
LT binding sites, SP1 sites
AT rich
Nucleosome free - Why?
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Polyomaviruses

Covalently closed circular, double stranded DNA

Bidirectional replication

Leading

Lagging

Lagging

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Leading

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

LT Binding
A/T site II EP
1

ATP + LT

Two LT hexamers bind

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Initiation of DNA Synthesis

2

Conformational change
of EP sequence

Binding distorts early palindrome unwinding origin

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Initiation of DNA Synthesis

ATP
3

RPA

ADP + Pi

Binding of Rpa occurs

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Initiation of DNA Synthesis

Two LT hexamers bind

Binding distorts early palindrome

unwinding origin

Binding of Rpa occurs

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DNA Synthesis Initiates at a

Unique Origin
Ori
RE Site

Ori

RE Site

How do you know that replication is bidirectional?

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RE Site

The Problem

How 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

Leading strand

presynthesis complex

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DNA Synthesis by Polyomaviridae is

Bidirectional
Rf-C binds 3 R-D
pcna is next

Lagging strand

PCNA
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Pol/primase

RFC

DNA Synthesis by Polyomaviridae is

Bidirectional

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DNA Synthesis by Polyomaviridae is

Bidirectional

Remove RNA, fill gaps, seal

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Polyomaviridae DNA Synthesis

Leading strand
Rf-C binds 3 R-D
pcna is next

presynthesis complex
Lagging strand

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Remove RNA, fill gaps, seal

The DNA Replication Machine

Lagging strand

Leading strand

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The Replication Machine

http://www.hhmi.org/biointeractive/media/
DNAi_replication_vo1-lg.wmv
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Problems in Replication

Catenated molecules
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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
genomic DNAs are ss of both + and - polarity
Their
and they contain Inverted Terminal Repeats
start here - but how to get to the end?

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AAV Replication is Continuous

ITR

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!
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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

Displaced ss Template DNA

ITRs
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Single-stranded DNA Template

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
UL

US
DNA ligase IV/
XRCC4

Circularization

Host proteins are responsible for circularization

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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 DBP

ss DBP
Helicase-Primase
HSV Polymerase
Processivity Protein

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Rolling Circle Replication

Cleavage Packaging Sites

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