1. EM of purified papillomavirus particles Papovaviruses

2. Papovaviridae Name from: Papilloma Polyoma Vacuolating agent Suffix “oma” means swelling or tumor Two subfamilies: Papillomavirinae, Polyomavirinae Not very important as lethal human pathogens, but cause warts and in some cases cancer The most common viral STD Highly restricted host ranges

3. Major papovavirus diseases Papillomavirus: –Most important of papovaviruses –More than 100 distinct strains identified to date –Cause common and genital warts, sometimes associated with cancer Polyomaviruses: –JC Virus Infect only humans Common, causes disease only in immunosuppressed –BK Virus Also found in immunosuppressed Both JC and BK associated with cancer –Role of SV40 in human cancer still debated

4. Papovaviridae properties Simple T=7 particles, 45-55 nm, no envelope Small dsDNA genomes, 5-7 kbp 3 capsid proteins subunits Host-derived histones account for 20% of viral protein

5. PolyomavirusPapillomavirus Genome double-stranded circular DNA ~5kbp Uses overlapping genes and both strands of DNA to pack all 6 genes into a tiny space genome ~5000 nucleotides double-stranded circular DNA ~8kbp Uses overlapping genes and one strand of DNA to pack at least 12 genes into 8kbp Genome ~ 8000 nucleotides Morphol ogy Noneveloped ~45 nm diameter icosohedral, skew, T= 7 3 capsid proteins Nonenveloped ~52-55nm diameter icosohedral, skew, T= 7 2 capsid proteins Other Differen ces Subfamily specific antigens Papillomavirus can't be grown in culture, while polyomaviruses are often grown in culture Pathoge nesis Most are asymptomatic, although can be oncogenic in hamsters Cause various types of warts, epidermodysplasia verruciformis Represe ntative Viruses JC Virus Associated with Progressive multifocal leukoencephalopathy, found mainly in the elderly and immunocompromised BK Virus Results in mild respiratory illness in kids Found in some tumors Simian Virus 40 A completely sequenced animal virus used frequently as a cloning vector. At least 62 strains of Human Papillomaviruses Widespread Cause growths or warts Many are associated with cancer Summary from Robert Siegel, Stanford U.

6. Papillomavirus properties No tissue culture system available, so relatively poorly studied Productively infect only fully differentiated squamose epithelial cells (dead end cells) Result in warts, sometimes become malignant All ORFs located on one strand; all transcripts from that strand 70% of genome for transformation and plasmid maintenance

7. Viral capsid Minor capsid component transformation Transcriptional transactivation episomal persistence transformation high copy Papillomavirus genome organization Linear representation of circular genome

8. A. EM of particles and B. Circular representation of Human papillomavirus genome. Note that transcription proceeds from only one of the two strands.

9. Polyomavirus properties SV40 the best known Infectious closed circular DNA 5.2-5.3 kbp Infection: –Establish lytic infection in permissive cells, may transform non-permissive cells Entry –By receptor-mediated endocytosis –Virions enter nucleus for genome expression and replication

10. Polyomavirus properties Transcription of T-antigens before replication –Large T Induces cellular DNA synthesis Required for transformation and maintenance off transformed state Required for viral DNA synthesis (has ATPase and helicase activities Regulates its own synthesis and that of other T antigens –Small T Regulation of viral DNA synthesis –Middle T Required for transformation

11. Polyomavirus replication Occurs 12-15 hours post-infection Proceeds bidirectionally from origin Large T is the only viral protein involved in DNA replication Other replication-associated proteins are from host

12. Polyomavirus late transcription Late mRNA: – transcribed after DNA replication –transcribed from the strand complementary to the strand used for early RNA transcription –transcribed from progeny, not parental genomes –transcribed in much greater amounts than early –encodes three structural proteins, by differential splicing

13. Polyomavirus assembly and release Assembly –Takes place in the nucleus –Viral genome complexed with histones encapsidated into preformed particles –Particle assembly process is mediated by host chaperone sp70 and Large T Release –Initially by exocytosis of virus-containing vesicles –Later by cell death and lysis

14. A. SV40 has a distinctive T=7 structure made up of three proteins. Circular dsDNA inside is complexed with histone proteins. B. Genome organization is compact; transcription bidirectional from ORI. Transcription of early T-antigens is from input DNA; transcription of late structural proteins is from newly synthesized DNA.

15. A.EM of replicating SV40 DNA, showing unwinding during bidirectional replication. B.Schematic of bidirectional replication from single origin of replication (Ori)

16. A.Continuous DNA synthesis from Ori, RNA primed (primase)5’>3’ B.Discontinuous DNA synthesis toward Ori, also RNA primed, also 5’>3” (Fig 9.2, Principles of Virology) Semidiscontinuous DNA synthesis from SV40 bidirectional origin

17. SV40 infection cycle 1.Entry and release of core 2.Entry of DNA/nucleosome complex to nucleus 3-5.Synthesis and alternative splicing of early (T) transcripts; proteins synthesized 6-7.LT imported back to nucleus where it initiates DNA synthesis with host enzymes and potentiates late gene transcription 8-12. Late gene mRNAs synthesized, spliced, exported, translated, and products imported back to nucleus for particle assembly and release by unknown mechanism