Lecture objectives:

Introduction to DNA virusesTerje Dokland, Dept. of Microbiology, UAB

[email protected] 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 familiesOrder Caudovirales Myoviridae - bacteriophage T4 Podoviridae - bacteriophage P22 Siphoviridae - bacteriophage lUnassigned families:AscoviridaeAdenoviridae Human Adenovirus CAsfarviridae African swine fever virusBaculoviridaeCoccolithoviridaeCorticoviridaeFuselloviridaeGuttaviridaeHerpesviridae HSV, Varicella Zoster, Epstein-BarrIridoviridae Chilo iridescent virusLipothrixviridaeMimiviridae MimivirusNimaviridaePapillomaviridae HPVPhycodnaviridae PBCV-1PlasmaviridaePolyomaviridae Simian virus 40, JC virusPoxviridae Cowpox (Vaccinia), smallpoxRudiviridaeTectiviridae bacteriophage prd1

Group II - ssDNA familiesInoviridaeMicroviridae bacteriophage fX174GeminiviridaeCircoviridae porcine circovirusNanoviridaeParvoviridae Parvovirus B19

Group III - RNA/DNA familiesCaulimoviridae Cauliflower mosaic virusHepadnaviridae Hepatitis B virus

http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htmhttp://en.wikipedia.org/wiki/DNA_virus

Families of DNA viruses that infect humans:

Group I - dsDNA virus familiesAdenoviridae – Human Adenovirus C (respiratory disease)Herpesviridae – Varicella Zoster virus (chickenpox)Papillomaviridae – HPV (warts, cervical cancer)Polyomaviridae – JC virus (PML)Poxviridae – Variola virus (smallpox)

Group II - ssDNA familiesParvoviridae – Parvovirus B19 (fifth disease)

Group III - RNA/DNA familiesHepadnaviridae (Hepatitis B virus)

Sizes of DNA viruses

• Circovirusgenome: ssDNA, 1.7 kb2 genescapsid diameter: 17 nm

• Mimivirusgenome: dsDNA, 1.2 Mbp911 genescapsid 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

• Adenovirusgenome: dsDNA, 30-38kbp30-40 genescapsid diameter: 90-100 nm

DNA vs. RNA viruses

RNA• Less stable • Mixture of ss and ds forms;

extensive secondary structure

• ssRNA is flexible; dsRNA is rigid

• Error-prone replication– small genomes

• dsRNA actively degraded by cell– RNA MUST BE

PROTECTED DURING REPLICATION AND ASSEMBLY!

• VIRAL RNA USUALLY CO-ASSEMBLES WITH CAPSID PROTEIN

DNA• Very stable• B-form double helix• dsDNA is rigid• Accurate replication

– large genomes• Protected by cell

• VIRAL DNA IS USUALLY PACKAGED INTO PREFORMED CAPSID SHELLS (PROCAPSIDS)

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– 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 – 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)

– 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

– e.g. Parvoviruses are small enough (<50nm) to get through the nuclear pore complex (NPC) intact

– Others are partially unfolded before entry through NPC2. Disassembly in cytoplasm and transport of genome/protein complex

– use nuclear localization signals (NLS)3. Ejection of DNA at nuclear envelope

– e.g. herpes- and adenoviruses (too large to pass through NPC)– Compare to tailed bacteriophages: ejection of DNA through cell wall

4. 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

5’3’

3’5’

5’3’

3’5’

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)

• 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

characteristic “slapped cheek” appearance

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

• 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

SV40

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• Naked (non-enveloped) capsid, 90nm diameter• 30-38 kbp linear dsDNA genome, inverted terminal repeats, 30-40 genes

• A 55kDa 5’ terminal protein (TP) acts as initiator for DNA synthesis• Adenovirus encodes its own DNA-dependent DNA polymerase

5’3’

3’5’

TPTP

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 diseaseVirus Disease Primary target

cellsSite of latency

Means of spread

Alphaherpesviruses

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

Betaherpesviruses

Cytomegalovirus (HCMV) mononucleosis, birth defects

monocytes, lymphocytes, epithelia

monocytes, lymphocytes

close contact, transfusions, congenital

Gammaherpesviruses

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)

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!)

• 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

360 nm

Smallpox (Variola)

Orf (sheep and goat pox)

(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)

Replication cycle of vaccinia virus

Figure 2 Structural changes in viral factories of VV-infected cells

Biology of the Cell www.biolcell.org Biol. Cell (2006) 97, 147-172

membrane-enclosed replication complex (early phase)

Viral Factory

C = “crescents”

IV = immature virus

IMV=intracellular mature virions

EEV=extracellular enveloped virions

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?

• intact or disassembled? – 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?

• 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