Introduction to DNA viruses
Terje Dokland
dokland@uab.edu
BBRB 311, T: 996-4502
• Replication strategies
• Evolutionary and structural relationships
DNA virus taxonomy (“traditional” scheme)
Group I - dsDNA virus families
Order Caudovirales
Myoviridae - bacteriophage T4
Podoviridae - bacteriophage P22
Siphoviridae - bacteriophage λ
Group II - ssDNA families
Inoviridae
Microviridae bacteriophage φX174
Geminiviridae
Circoviridae porcine circovirus
Nanoviridae
Parvoviridae Parvovirus B19
Unassigned families:
Ascoviridae
Adenoviridae Human Adenovirus C
Asfarviridae African swine fever virus
Baculoviridae
Coccolithoviridae
Corticoviridae
Fuselloviridae
Guttaviridae
Herpesviridae HSV, Varicella Zoster, Epstein-Barr
Iridoviridae Chilo iridescent virus
Lipothrixviridae
Nimaviridae
Papillomaviridae HPV
Phycodnaviridae PBCV-1
Plasmaviridae
Polyomaviridae Simian virus 40, JC virus
Poxviridae Cowpox (Vaccinia), smallpox
Rudiviridae
Tectiviridae bacteriophage prd1
Mimivirus (unassigned)
Group III - RNA/DNA families
Caulimoviridae Cauliflower mosaic virus
Hepadnaviridae Hepatitis B virus
http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htm
http://en.wikipedia.org/wiki/DNA_virus
Sizes of DNA viruses
• Circovirus
genome: ssDNA, 1.7 kb
7 genes
capsid diameter: 17 nm
• Adenovirus
genome: dsDNA, 30-38kbp
30-40 genes
capsid diameter: 90-100 nm
• Mimivirus
genome: dsDNA, 1.2 Mbp
911 genes
capsid diameter: 600 nm
• The largest virus, Mimivirus, has a 1.2Mbp dsDNA genome with 911 genes.
• Mycoplasma genitialium, a small cell, has a 580kbp genome with 470 genes…
• Size of cells: Mycoplasma: <500nm; E. coli: 1-5µm; Eukaryotes: 10–100µm
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DNA virus life cycles
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Many families of viruses with double-stranded (ds)
or single-stranded (ss) DNA genomes.
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infect eukaryotes, prokaryotes and archea
vertebrates, invertebrates
few DNA viruses in plants (only gemini: ssDNA)
Structural and evolutionary relationships
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within ds/ssDNA viruses
across biological domains (prokaryotes, eukaryotes,
archea)
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Enveloped or non-enveloped
DNA viruses can use replication (DNA > DNA) and
transcription (DNA > RNA) machinery of host
Most replicate and at least partially assemble in
nucleus
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except Poxviruses (+ ASFV, mimi- & iridoviruses)
May integrate into host genome (by recombination)
DNA virus life cycles
• DNA viruses generally follow the normal path of DNA > mRNA > protein
- Hepadnaviruses (and Caulimoviruses) use an RNA intermediate
• Early and late phases: Late phase starts with the replication of the DNA genome.
Immediate
Early
Transcription regulation
Early
DNA replication
Reverse transcription
Late
Assembly
General DNA virus (Herpesvirus)
Hepatitis B virus (Hepadnaviridae)
Specific challenges for DNA viruses
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DNA viruses can utilize cellular replication and
transcription machinery (DNA/RNA polymerase)
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May require infection of actively growing cells
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a prolonged period with no virus production, possibly
followed by reactivation
virus exists in a plasmid state in the host cell (HSV)
integration into the host genome (HPV)
Need to enter nucleus (because that’s where the
replication and transcription machinery is)
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Eukaryotic cells only replicate their DNA in S phase
Many cells are frozen in G1 or terminally differentiated
If no replication occurs then virus cannot be replicated either
Some viruses actively promote cell growth (transformation)
Others produce their own proteins for DNA replication
Viral latency
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no need for a viral RNA-dependent RNA/DNA polymerase
for replication
except Poxviruses (+ASFV & Iridoviruses)
entry of intact virus or uncoating in cytoplasm
enter during mitosis
Need to exit from nucleus
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pass trough nuclear envelope or lyse the cell
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DNA replication
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DNA replication requirements:
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A template
A primer (DNA or RNA)
DNA polymerase
Accessory proteins (helicase, RNA
nuclease, primase, ss binding protein…)
DNA replication is 5’ > 3’
Leading strand vs lagging strand
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viral genomes may use RNA primers, DNA
hairpins or terminal proteins for priming DNA
synthesis
What to do at the ends?
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DNA will get shorter and shorter
Eukaryotes use telomerase
Prokaryotes have circular genomes (no ends)
3’
5’
5’
3’
3’
5’
5’
3’
– viruses have circular genomes or
use special terminal proteins
Getting access to the cellular
DNA replication machinery
The nuclear envelope represents a barrier for the virus to get access to the cellular replication
machinery.
Solutions:
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Entry intact
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2.
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use nuclear localization signals (NLS)
Ejection of DNA at nuclear envelope
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e.g. Parvoviruses are small enough (<50nm) to get through the nuclear pore complex (NPC) intact
Others are partially unfolded before entry through NPC
Disassembly in cytoplasm and transport of genome/protein complex
e.g. herpes- and adenoviruses (too large to pass through NPC)
Compare to tailed bacteriophages: ejection of DNA through cell wall
Some DNA viruses replicate in the cytoplasm
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Pox-, Asfa- (ASFV), irido- and mimi-viruses
very large, complex viruses
need to bring all the enzymes required for DNA replication and transcription
Few plant DNA viruses. Dual problem of cell wall and nucleus?
Small, ssDNA viruses: Parvoviridae
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5,500 nt linear, self-priming ssDNA
18-26 nm naked, T=1 icosahedral virion (60 copies of capsid protein)
B19 erythrovirus: causes “fifth disease” (rash, fever); arthritis in adults
Several species on animals (cats, dogs, cattle, pigs, minks…)
Also adeno-associated virus (AAV) in humans
5’
3’
B19 cryo-EM reconstruction (Chipman et al. 1996, PNAS 93, 7502-6)
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Erythema infectiosum (fifth disease)
characteristic “slapped
cheek” appearance
• Fifth disease is caused by B19 parvovirus
• Mild symptoms in children (rash, fever, clears in 1-2 weeks)
• In adults can lead to polyarthritis
• B19 replicates in actively growing erythroid precursor cells (bone marrow)
• No vaccine available
Parvovirus life cycle
• Parvoviruses need to infect actively growing cells
• Enter nucleus intact (small size)
• Exit nucleus/cells by lysis
Parvovirus replication
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Parvovirus structure
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Parvoviruses contain 60 copies of capsid protein
Parvovirus capsid protein is similar to F protein of
ssDNA Bacteriophage φX174
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The “viral capsid fold” -- 8-stranded antiparallel
β-sandwich (“jelly roll”)
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first viral fold described, in plant RNA viruses and
subsequently Picornaviridae (rhino and polio)
B19 (red) and FPV (blue) [Kaufmann et al 2004 PNAS 101, 11628-33]
Bacteriophage φX174 [Dokland et al 1998 Acta Cryst D54, 805-16]
Papovaviruses:
Polyoma- and Papillomaviridae
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Polyomavirus:
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45nm capsid “T=7” organization of 72 VP1 pentamers
5,000 bp circular dsDNA genome, 5 genes
Large T and small t antigens—transforming proteins
Papillomavirus:
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50-55nm capsid “T=7” organization of 72 L1 pentamers
Circular, dsDNA genome, 8,000 bp, 9-10 genes
Causes warts, cervical cancer
Life cycle of polyomavirus
SV40
• Polyomavirus only replicates in S phase of cells
• T antigen stimulates entry into S phase (host cell specific)
• T antigen also required to recruit DNA polymerase to replication origin
• Integration of viral genome (non-permissive cells) may lead to transformation
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Polyomavirus replication
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Replication mode also known as “theta” replication
Uses host DNA pol but requires large T to recruit it to origin
Similar to replication of bacterial genomes
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Also used by ds/ssDNA bacteriophages
– bi-directional, RNA primers, leading and lagging strand synthesis
Polyomavirus structure
• The polyomavirus(SV40) VP1 capsid protein contains a β-sandwich domain
- suggesting a relationship to parvo- and picornaviruses ?
• The orientation of the β-sandwich is perpendicular to the viral surface
• VP1 is organized into pentamers; 72 pentamers form the viral shell (360 copies of VP1)
- in parvo- and picornaviruses the orientation is tangential and the protein is organized into dimers or
pentamers/hexamers (180 sandwich domains per shell)
Adenoviruses
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TP
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Naked (non-enveloped) capsid
30-38 kbp linear dsDNA genome, inverted terminal repeats, 30-40 genes
5’
3’
3’
5’
TP
A 55kDa 5’ terminal protein (TP) acts as initiator for DNA synthesis
Ad encodes its own DNA-dependent DNA polymerase
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(even though this function is found in the host -- compare with RNA viruses)
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Adenoviral conjunctivitis
Adeno nuclear entry
Adenoviruses use a 5’ terminal protein to
prime DNA replication
• There is no lagging strand synthesis in adenovirus, and no DNA/RNA primers are involved
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Adenovirus structures
fibre knob
• Capsid protein (hexon), pII, is a trimer
• Each pII monomer has two β-sandwich domains, giving a quasi-sixfold structure
(arranged on a pseudo-T=25 icosahedral lattice)
hexon
Herpesviruses
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Large dsDNA viruses
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Enveloped virions 100-300 nm in diameter:
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120–230 kbp circular dsDNA
At least >70 ORFs, no splicing
icosahedral nucleocapsid core
amorphous tegument layer
envelope with glycoproteins
Numerous human pathogens:
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Herpes simplex virus (HSV)
Cytomegalovirus (HCMV)
Varicella zoster virus (VZV; chickenpox)
Epstein-Barr virus (EBV; glandular fever)
Kaposi sarcoma-related virus (KSV)
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Herpesvirus
life cycle
Step 1 (immediate early):
• Penetration and release of DNA in nucleus
• Expression of Immediate Early proteins
(transcription factors)
Step 2 (early):
• Expression of DNA polymerase and other
enzymes required for DNA replication
• Construction of nuclear factory
• Genome replication
Step 3 (late):
• Synthesis of structural proteins
• Assembly of capsid (in nucleus)
• DNA is packaged into preformed procapsids,
similar to the process in bacteriophages
• Construction of cytoplasmic factory (“Assembly
compartment”)
• Budding and release of mature nucleocapsids
through the nuclear envelope
• Tegumentation occurs in nucleus and cytoplasm
• Tegumented capsid buds into membraneous
compartments
• Final assembly and release by exocytosis
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Assembly and DNA packaging in herpesviruses
resemble tailed dsDNA bacterophages
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DNA replication via rolling-circle mechanism
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Formation of procapsid precursor, using a scaffolding protein
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DNA packaging through a portal
Packaging of a concatemeric DNA substrate using a terminase protein
Cytoplasmic DNA viruses: the
exception to the rule
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Some families of DNA viruses replicate in the cytoplasm:
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Poxviridae – smallpox, vaccinia (cowpox) …
Asfaviridae – African Swine fever virus (ASFV)
Iridoviridae – insects and lower vertebrates
Phycodnaviridae – Paramecium bursaria Chlorella virus (PBCV), infects
Chlorella unicellular algae
– Mimivirus – amoeba
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These viruses need to synthesize all the enzymes required for
DNA replication and transcription
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Replication and assembly takes place in “viral factories” in the
cytoplasm
– consequently, they are large (180–300kbp) and complex (>200 proteins)
The Poxviruses
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Many members, infecting vertebrates and invertebrates, divided in
several genera:
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Orthopoxvirus (Variola, Vaccinia, monkeypox)
Parapoxvirus (orf; sheep and goat poxvirus)
Avipoxvirus (bird viruses)
Molluscipoxvirus (Molluscum contagiosum)
(NB: chickenpox is not a poxvirus!)
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Genome: 134-360kbp dsDNA, terminally redundant, inverted repeats
DNA replication is self-primed (hairpin) and leads to the formation of DNA concatemers
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Poxviridae Phylogeny
Smallpox (Variola)
Orf (sheep and goat pox)
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Figure 2 Structural changes in viral factories of VV-infected cells
membrane-enclosed
replication complex (early
phase)
Viral Factory
Biology of the Cell
www. biolcell.org
www.biolcell
.org
Biol.
Biol . Cell (2006) 97, 147-172
Evolutionary relationships between viruses
• Structurally related viruses are
found in all domains of life,
suggesting that
– viruses are ancient,
or that
– viruses have evolved the ability
to jump between very diverse hosts
• These relationships are only
apparent when you look at
capsid structures (large scale
organization and/or high resolution
structures)
• Sequences are extremely diverse
Bamford DH et al (2005) Curr Opin Struct Biol 15, 655-663
DNA viruses: Things to consider
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What challenges does the virus face and what strategies does it employ to resolve these
challenges?
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How does it get into the cell?
How does it get into the nucleus?
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How does it replicate its DNA?
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linear vs. circular DNA
primers?
What does the virus need to replicate itself?
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What cellular functions can and/or does it use?
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Where does it find those functions?
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dsDNA viruses can take advantage of the cellular DNA replication and transcription machinery
most dsDNA viruses replicate in the nucleus
What functions does it supply?
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intact or disassembled?
some dsDNA viruses supply DNA polymerases and enzymes involved in DNA synthesis – why?
Cells only replicate their DNA during S phase. Many cells are halted in G1. How does the
virus deal with this?
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infect actively growing cells (parvo)
activate the cells (polyoma)
viral latency (herpes)
co-infect with helper virus (AAV)
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Literature and resources
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Murray et al. 2005. Medical Microbiology, 5th ed. (Elsevier
Mosby) Chapters 6 and 52-56.
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Strauss E.G. and Strauss J.H. 2001. Viruses and human
disease. Academic Press.
Shors, T. 2008. Understanding viruses. Jones and Bartlett.
Voet and Voet. Biochemistry. Chapter 31: DNA replication.
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http://en.wikipedia.org/wiki/DNA_virus
http://pathmicro.med.sc.edu/mhunt/dna1.htm
http://www.virology.net/garryfavwebindex.html
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http://www.tulane.edu/~dmsander/Big_Virology/BVFamilyIndex.html
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