Introduction to DNA viruses
Terje Dokland, Dept. of Microbiology, UAB
dokland@uab.edu
BBRB 311, T: 996-4502
Lecture objectives:
• Provide an overview of the properties of viruses with DNA
genomes, with emphasis on human pathogens
• Understand the specific challenges faced by DNA viruses
– and advantages of having a DNA genome
• Understand the diversity of solutions to these challenges
– Replication strategies
– Viral life cycles
– Relationship between virus and host
• Gain insight into how these mechanisms affect viral pathogenesis
The families of DNA viruses
Group I - dsDNA virus families
Order Caudovirales
Myoviridae - bacteriophage T4
Podoviridae - bacteriophage P22
Siphoviridae - bacteriophage l
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
Mimiviridae Mimivirus
Nimaviridae
Papillomaviridae HPV
Phycodnaviridae PBCV-1
Plasmaviridae
Polyomaviridae Simian virus 40, JC virus
Poxviridae Cowpox (Vaccinia), smallpox
Rudiviridae
Tectiviridae bacteriophage prd1
Group II - ssDNA families
Inoviridae
Microviridae bacteriophage fX174
Geminiviridae
Circoviridae porcine circovirus
Nanoviridae
Parvoviridae Parvovirus B19
Families of DNA viruses that infect humans:
Group I - dsDNA virus families
Adenoviridae
– Human Adenovirus C (respiratory disease)
Group III - RNA/DNA families
Herpesviridae
– Varicella Zoster virus (chickenpox)
Caulimoviridae Cauliflower mosaic virus
Hepadnaviridae Hepatitis B virus
Papillomaviridae – HPV (warts, cervical cancer)
Polyomaviridae
– JC virus (PML)
Poxviridae
– Variola virus (smallpox)
Group II - ssDNA families
Parvoviridae
– Parvovirus B19 (fifth disease)
Group III - RNA/DNA families
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
2 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
DNA vs. RNA viruses
RNA
DNA
•
•
•
•
Very stable
B-form double helix
dsDNA is rigid
Accurate replication
–
•
•
•
•
large genomes
Protected by cell
•
Less stable
Mixture of ss and ds forms;
extensive secondary
structure
ssRNA is flexible;
dsRNA is rigid
Error-prone replication
–
•
VIRAL DNA IS USUALLY
PACKAGED INTO
PREFORMED CAPSID
SHELLS (PROCAPSIDS)
•
dsRNA actively
degraded by cell
–
•
small genomes
RNA MUST BE
PROTECTED DURING
REPLICATION AND
ASSEMBLY!
VIRAL RNA USUALLY
CO-ASSEMBLES WITH
CAPSID PROTEIN
Typical DNA virus life cycle
Typical steps include:
•
Entry
•
Uncoating
•
Nuclear entry
•
Replication
•
Assembly
•
Release
Strauss and Strauss (2002) Viruses and human disease
DNA virus life cycles
•
DNA is protected by the cell and is
transported to the nucleus
– Many viruses have specific mechanisms for
getting the DNA into the nucleus
•
DNA viruses can use replication (DNA >
DNA) and transcription (DNA > RNA)
machinery of host
– DNA replication is accurate –> large genomes
– No need for a viral RNA/DNA polymerase
•
Most replicate and at least partially assemble
in nucleus
– except Poxviruses (+ ASFV, mimi- &
iridoviruses)
•
Proteins are synthesized in the cytoplasm
and imported into the nucleus
Specific challenges for DNA viruses
•
May require infection of actively growing cells
–
–
–
–
–
•
Viral latency
–
–
–
•
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)
–
–
–
•
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
except Poxviruses (+ASFV & Iridoviruses)
entry of intact virus or uncoating in cytoplasm
enter during mitosis
Need to exit from nucleus
–
pass through nuclear envelope or lyse the cell
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:
1.
Entry intact
–
–
2.
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
–
3.
use nuclear localization signals (NLS)
Ejection of DNA at nuclear envelope
–
–
4.
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
–
–
–
Pox-, Asfa- (ASFV), irido- and mimi-viruses
very large, complex viruses
need to bring all the enzymes required for DNA replication and transcription
DNA replication
• DNA replication requirements:
–
–
–
–
A template
DNA polymerase
A primer (DNA or RNA)
Accessory proteins (helicase, RNA
nuclease, primase, ss binding protein…)
• DNA replication is unidirectional
–
From 5’ to 3’
• Leading strand vs lagging strand
–
Viral genomes may use RNA primers, DNA
hairpins or terminal proteins for priming DNA
synthesis
• What to do at the ends?
–
–
–
–
DNA will get shorter and shorter
Eukaryotes use telomerase
Prokaryotes have circular genomes (no ends)
Viruses have circular genomes or use special
terminal proteins
3’
5’
5’
3’
3’
5’
5’
3’
Small, ssDNA viruses: Parvoviridae
•
•
•
•
•
5,500 nt linear, self-priming ssDNA, 3 ORFs
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)
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)
– rash is caused by immune response
• 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
Strauss and Strauss (2002) Viruses and human disease
Papovaviruses:
Polyoma- and Papillomaviridae
•
Polyomavirus:
–
–
–
–
•
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
JC and BK virus of humans (normally mild)
Papillomavirus:
–
–
–
50-55nm capsid “T=7” organization of 72 L1 pentamers
Circular, dsDNA genome, 8,000 bp, 9-10 genes
Causes warts, cervical cancer
Polyomavirus replication
•
•
•
Replication mode also known as “theta” replication
Uses host DNA pol but requires “Large T antigen” to recruit it to origin
Similar to replication of bacterial genomes
– bi-directional, RNA primers, leading and lagging strand synthesis
•
Also used by ds/ssDNA bacteriophages
Strauss and Strauss (2002) Viruses and human disease
Life cycle of polyomavirus
SV40
• Polyomavirus only replicates in S phase of cells
• Large T antigen stimulates entry into S phase (host cell specific) in permissive cells
– T antigen also required to recruit DNA polymerase to replication origin
• In non-permissive cells, integration of the viral genome may lead to transformation
Papillomavirus infection
• HPV infects epithelial tissue (skin or
mucosal)
• Infection may cause warts (stimulation
of cell growth in granular layer)
• HPV may cauce cervical carcinoma by
integrating into the host genome,
expression of E6 and E7 oncogenes
– inactivate tumor suppressor genes
Adenoviruses
•
•
TP
•
•
Naked (non-enveloped) capsid, 90nm diameter
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
Adenovirus encodes its own DNA-dependent DNA polymerase
Adenovirus disease
•
Adenoviruses are very common
–
–
–
–
–
•
•
5-10% of respiratory disease in children
worldwide and non-seasonal
acute respiratory disease (ARD) (Ad 4, 7) in military rectruits
conjunctivitis “shipyard eye” (Ad 8)
gastroenteritis (Ad 11, 12)
Symptoms: high fever, sore throat, aches, conjunctivitis
Species specific
– human adenoviruses only infect humans
•
Transmission from person-to-person
– Respiratory, fecal-oral, close contact
– virus is resistant to inactivation by acid, dehydration and detergents
•
Site of infection:
– epithelia of respiratory tract, intestinal tract, urinary tract, conjunctiva
•
•
Virus may spread to and persist for a long time in lymphoid tissues
No vaccine is currently in use
– Vaccination of military recruits discontinued in 1996
Adenoviral conjunctivitis
Adenovirus nuclear entry
?
Only DNA/protein complex enters nucleus
(Exits from the nucleus by cell lysis)
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
Herpesviruses
• Large dsDNA viruses
– 120–230 kbp circular dsDNA
– At least >70 ORFs, no splicing
• Enveloped virions 100-300 nm in
diameter:
– icosahedral nucleocapsid core
– amorphous tegument layer
– envelope with glycoproteins
• Numerous human pathogens
Herpesvirus disease
Virus
Disease
Primary target
cells
Site of
latency
Means of
spread
Herpes simplex 1 (HSV-1)
cold sores
mucoepithelial
cells
neurons
close contact
Herpes simplex 2 (HSV-2)
genital ulcers
mucoepithelial
cells
neurons
close contact
(STD)
Varicella-zoster virus (VZV)
chickenpox,
shingles
mucoepithelial
cells
neurons
respiratory and
close contact
mononucleosis,
birth defects
monocytes,
lymphocytes,
epithelia
monocytes,
lymphocytes
close contact,
transfusions,
congenital
Epstein-Barr virus (EBV)
mononucleosis
(glandular fever),
lymphoma
B cells
B cells
saliva
Kaposi’s sarcoma-related
virus (KSV)
tumors
lymphocytes
B cells
close contact
(STD)
Alphaherpesviruses
Betaherpesviruses
Cytomegalovirus (HCMV)
Gammaherpesviruses
Herpesvirus
life cycle
Stage 1 (immediate early):
• Penetration and release of DNA in nucleus
• Expression of Immediate Early proteins
(transcription factors)
Stage 2 (early):
• Expression of DNA polymerase and other
enzymes required for DNA replication
• Construction of nuclear factory
• Genome replication
Stage 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
Assembly and DNA packaging in herpesviruses
resemble tailed dsDNA bacterophages
•
DNA replication via rolling-circle mechanism
– leads to the formation of DNA concatemers
•
Formation of procapsid precursor, using a scaffolding protein
•
•
DNA is packaged into procapsid through a portal
Concatemeric DNA substrate is packaged by a terminase complex
Strauss and Strauss (2002) Viruses and human disease
Viral latency
•
Latency is a hallmark of
herpesvirus infections
•
The viral genome exists as an
episome (naked, circular DNA) in
the host cell nucleus
–
–
•
No virus is produced until reactivation
Not the same as persistent infection
(continuous viral production)
E.g. VZV, which causes chickenpox in
children, causes shingles when
reactivated in the adult
Knipe and Cliffe 2008
Cytoplasmic DNA viruses:
the exception to the rule
• Some families of DNA viruses replicate in the cytoplasm:
– 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
• These viruses need to synthesize all the enzymes required for
DNA replication and transcription
– consequently, they are large (180–300kbp) and complex (>200 proteins)
• Replication and assembly takes place in “viral factories” in the
cytoplasm
The Poxviruses
•
Many members, infecting vertebrates and invertebrates, divided in
several genera:
–
–
–
–
Orthopoxvirus (Variola, Vaccinia, monkeypox)
Parapoxvirus (orf; sheep and goat poxvirus)
Avipoxvirus (bird viruses)
Molluscipoxvirus (Molluscum contagiosum)
(NB: chickenpox is not a poxvirus!)
•
•
360 nm
Virion: Large (360nm long axis), brick-shaped, multi-enveloped
Genome: 134-360kbp dsDNA, terminally redundant, inverted repeats
DNA replication is self-primed (hairpin) and leads to the formation of DNA concatemers
Smallpox (Variola)
Orf (sheep and goat pox)
Replication cycle of vaccinia virus
(Moss, B. "Poxviridae: the viruses and their replication" Fields Virology. Eds. D.M. Knipe and P.M. Howley. Philadelphia:
Lippincott Williams & Wilkins. pp. 2849–2883. 2001)
Figure 2 Structural changes in viral factories of VV-infected cells
membrane-enclosed
replication complex (early
phase)
Viral Factory
C = “crescents”
IV = immature virus
IMV=intracellular
mature virions
EEV=extracellular
enveloped virions
Biology of the Cell
www.biolcell.org
Biol. Cell (2006) 97, 147-172
DNA viruses: Things to consider
•
•
What properties of DNA vs. RNA impact the replication strategy of the virus?
What challenges does the virus face and what strategies does it employ to
resolve these challenges?
–
–
How does it get into the cell?
How does it get into the nucleus?
•
–
How does it replicate its DNA?
•
•
•
linear vs. circular DNA
primers?
What does the virus need to replicate itself?
–
What cellular functions can and/or does it use?
•
–
–
dsDNA viruses can take advantage of the cellular DNA replication and transcription machinery
Where does it find those functions?
•
most dsDNA viruses replicate in the nucleus
What functions does it supply?
•
•
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?
–
–
–
–
infect actively growing cells (parvo)
activate the cells (polyoma)
viral latency (herpes)
co-infect with helper virus (AAV)
Literature and resources
• Murray et al. 2005. Medical Microbiology, 5th ed. (Elsevier
Mosby) Chapters 6 and 52-56.
• Strauss J.H. and Strauss E.G. 2002. Viruses and human
disease. Academic Press.
• Shors, T. 2008. Understanding viruses. Jones and Bartlett.
• Voet and Voet. Biochemistry. Chapter 31: DNA replication.
• http://en.wikipedia.org/wiki/DNA_virus
• http://pathmicro.med.sc.edu/mhunt/dna1.htm
• http://www.virology.net/garryfavwebindex.html
•
http://www.tulane.edu/~dmsander/Big_Virology/BVFamilyIndex.html