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Introduction to DNA viruses Terje Dokland, Dept. of Microbiology, UAB BBRB 311, T: 996-4502 Lecture objectives: Provide.

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Presentation on theme: "Introduction to DNA viruses Terje Dokland, Dept. of Microbiology, UAB BBRB 311, T: 996-4502 Lecture objectives: Provide."— Presentation transcript:

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2 Introduction to DNA viruses Terje Dokland, Dept. of Microbiology, UAB BBRB 311, T: 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

3 The families of DNA viruses Group I - dsDNA virus families Order CaudoviralesCaudovirales Myoviridae - bacteriophage T4Myoviridae Podoviridae - bacteriophage P22Podoviridae Siphoviridae - bacteriophage Siphoviridae Unassigned families: Ascoviridae Adenoviridae Human Adenovirus C AsfarviridaeAsfarviridae African swine fever virus Baculoviridae Coccolithoviridae Corticoviridae Fuselloviridae Guttaviridae Herpesviridae HSV, Varicella Zoster, Epstein-Barr IridoviridaeIridoviridae Chilo iridescent virus Lipothrixviridae MimiviridaeMimiviridae Mimivirus Nimaviridae Papillomaviridae HPV PhycodnaviridaePhycodnaviridae PBCV-1 Plasmaviridae Polyomaviridae Simian virus 40, JC virus Poxviridae Cowpox (Vaccinia), smallpox Rudiviridae TectiviridaeTectiviridae bacteriophage prd1 Group II - ssDNA families Inoviridae MicroviridaeMicroviridae bacteriophage  X174 Geminiviridae CircoviridaeCircoviridae porcine circovirus Nanoviridae Parvoviridae Parvovirus B19 Group III - RNA/DNA families Caulimoviridae Cauliflower mosaic virus Hepadnaviridae Hepatitis B virus Families of DNA viruses that infect humans: Group I - dsDNA virus families Adenoviridae – 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 families Parvoviridae – Parvovirus B19 (fifth disease) Group III - RNA/DNA families Hepadnaviridae (Hepatitis B virus)

4 Sizes of DNA viruses Circovirus genome: ssDNA, 1.7 kb 2 genes capsid diameter: 17 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 Adenovirus genome: dsDNA, 30-38kbp genes capsid diameter: nm

5 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)

6 Typical DNA virus life cycle Typical steps include: Entry Uncoating Nuclear entry Replication Assembly Release Strauss and Strauss (2002) Viruses and human disease

7 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

8 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

9 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 NPC 2.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

10 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 5’3’5’3’ 3’5’3’5’ 5’3’5’3’ 3’5’3’5’

11 Small, ssDNA viruses: Parvoviridae 5,500 nt linear, self-priming ssDNA, 3 ORFs 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 5’5’ 3’3’ B19 cryo-EM reconstruction (Chipman et al. 1996, PNAS 93, )

12 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

13 Parvovirus life cycle Parvoviruses need to infect actively growing cells Enter nucleus intact (small size) Exit nucleus/cells by lysis

14 Parvovirus replication Strauss and Strauss (2002) Viruses and human disease

15 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

16 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

17 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

18 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

19 Adenoviruses Naked (non-enveloped) capsid, 90nm diameter kbp linear dsDNA genome, inverted terminal repeats, genes A 55kDa 5’ terminal protein (TP) acts as initiator for DNA synthesis Adenovirus encodes its own DNA-dependent DNA polymerase 5’3’5’3’ 3’5’3’5’ TP

20 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

21 Adenoviral conjunctivitis

22 Adenovirus nuclear entry ? Only DNA/protein complex enters nucleus (Exits from the nucleus by cell lysis)

23 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

24 Herpesviruses Large dsDNA viruses –120–230 kbp circular dsDNA –At least >70 ORFs, no splicing Enveloped virions nm in diameter: –icosahedral nucleocapsid core –amorphous tegument layer –envelope with glycoproteins Numerous human pathogens

25 Herpesvirus disease VirusDiseasePrimary target cells Site of latency Means of spread Alphaherpesviruses Herpes simplex 1 (HSV-1)cold soresmucoepithelial cells neuronsclose contact Herpes simplex 2 (HSV-2)genital ulcersmucoepithelial cells neuronsclose contact (STD) Varicella-zoster virus (VZV)chickenpox, shingles mucoepithelial cells neuronsrespiratory 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 saliva Kaposi’s sarcoma-related virus (KSV) tumorslymphocytesB cellsclose contact (STD)

26 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

27 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

28 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

29 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

30 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: kbp dsDNA, terminally redundant, inverted repeats DNA replication is self-primed (hairpin) and leads to the formation of DNA concatemers 360 nm

31 Smallpox (Variola)

32 Orf (sheep and goat pox)

33 (Moss, B. "Poxviridae: the viruses and their replication" Fields Virology. Eds. D.M. Knipe and P.M. Howley. Philadelphia: Lippincott Williams & Wilkins. pp. 2849– ) Replication cycle of vaccinia virus

34 Figure 2 Structural changes in viral factories of VV-infected cells Figure 2 Structural changes in viral factories of VV-infected cells Biology of the Cell Biol. Cell (2006) 97, Biology of the Cell Biol. Cell (2006) 97, membrane-enclosed replication complex (early phase) Viral Factory C = “crescents” IV = immature virus IMV=intracellular mature virions EEV=extracellular enveloped virions

35 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)

36 Literature and resources Murray et al Medical Microbiology, 5th ed. (Elsevier Mosby) Chapters 6 and Strauss J.H. and Strauss E.G Viruses and human disease. Academic Press. Shors, T Understanding viruses. Jones and Bartlett. Voet and Voet. Biochemistry. Chapter 31: DNA replication.


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