Replication of Reverse-Transcribing Hepatitis B Virus

1. Replication of Reverse-Transcribing Hepatitis B Virus

2. Family Hepadnaviridae • Hepatitis – inflammation of liver (injury, chemical/drug, infectious agent) • DNA virus – tropism for hepatocytes • Unusual genome architecture and mode of replication (DNA pol;reverse transcription) • Difficult to grow in cell culture • Found in: • Humans (HBV) • Animals (Woodchuck hepatitis virus) • Plants (Cauliflower mosaic virus)

3. Genus: Hepadnavirus • Mammalian, avian • Hepatitis B virus (HBV) – “serum” hepatitis, virus found and transmitted in human blood & body secretions, close contact • Woodchuck hepatitis virus (WHV) • Duck hepatitis B virus (DHBV)

4. HBV • Envelope – HBS Ag (3 surface gp), 35-45 nm • Spherical inner core nucleocapsid – HBC, HBE • Incomplete viral forms (HBS Ag) found in blood: • spherical, fiber • initially identified as “Australian Ag” • 25 nm Dane particle

5. HBV Genome: dsDNA • Small, circular, not closed, partially ds DNA • Full-length (-)DNA strand, 3.2 kb (5’ end has covalently linked viral protein) • Partial (+)DNA strand, 1.7 – 2.8 kb (5’ end has covalently linked viral RNA) • Two 11 bp direct repeat sequences (DR1, DR2) required for DNA replication • Terminally redundant ends (circularize)

6. HBV Genome: Four ORF • Overlapping genes, no splicing, 10 proteins • ORF C – 2 core proteins: • HBC, HBE • ORF P – 2 proteins for DNA replication: • DNA pol (RT/RNase) • protein primer (-)DNA synthesis • ORF S – 3 surface envelope gp: • HBS (L, M, S) • ORF X – 3 proteins: • regulatory, transactivation

7. HBV: Entry / Uncoating • Receptor-mediated endocytosis • Partial uncoating, viral DNA pol completes dsDNA synthesis • Viral dsDNA enters nucleus • Ligate free ends, convert to covalently closed circular dsDNA • Associate with cell histones, similar to episome DNA

8. HBV Genome Replication • 3.4 kb mRNA transcript (larger than genome), template for DNA genome synthesis • Viral protein primer for (-)DNA strand synthesis by DNA pol (RT) • Internal start site on mRNA • As DNA synthesis occurs, RNA strand degraded by RNase-H (RT) • When DNA reaches end, “jumps” to other end to complete synthesis • Terminal repeat sequences important for strand recognition • Short viral RNA sequence primer for (+)DNA strand synthesis by RT

9. HBV Assembly/Release • Core particles form in nucleus • Viral DNA genome inserted before synthesis of (+)DNA finished • Complete (-)DNA strand, with partial (+)DNA strand • Assembly with cell envelope in ER/Golgi • Virus and small number of incomplete membrane particles (HBS) released and found in blood

10. HBV and Hepatocellular Carcinoma (HCC) • HBV infection increases risk (200x) for HCC • Following infection, HBV DNA persists as episome or integrates into host cell DNA • Modulated by viral X protein (trans-activator) • Chronic HBV infection with continued virus replication, over time, results in liver damage and cancer • Host unable to clear virus by immune defense (no appearance of HBS antibody)

11. Reading • Chapter 21 – Hepadnaviruses: Variations on the Retrovirus Theme • Questions: 3, 4

12. Class Discussion – Lecture 12 • 1. What is the evidence that HBV is a retrovirus? • 2. Why does hepadnavirus infection sometimes lead to liver cancer?

13. Chapter 25: Viruses and the Future – Problems and Promises • “Imagination is more important than knowledge.”·Albert Einstein

14. PROBLEMS: Emerging Viruses • HIV, Influenza, HCV – pandemics, high mortality • New, changing strains • Breakdown of public health – war, depression, poverty • Social changes – legal, moral, truths • It’s a small world – global economy, international travel, interrelationships • Environmental change – habitat, weather, industry, pollution • Are there solutions to these problems?

15. PROBLEMS:Bioterrorism and Fear • Classes of Bioterrorism Agents • Class A – Ebola Virus, Smallpox virus • Class B – Encephalitis Viruses • Class C – Sin Nombre Virus • What is our best protection?

17. “I am not dreaming of Utopia, only of a world in which problems are not resolved by force but by intelligence, good will and equity; • a world in which killing, no matter the reason, and the destruction of a fellow man’s life or home, is a crime, a world in which our youth will not have to spend their best years studying organized manslaughter, • in which neither force nor megatons nor poison gases will decide a nation’s standing but the sum of its knowledge, its ethics, the gifts it makes to mankind, the happiness it gives to men, the measure in which it lifts human life.” -Albert Szent-Gyorgyi

18. “Do not be overcome by evil, but overcome evil with good.” • Paul the Apostle (c.5 – c.67)

19. PROMISES:“Trojan Horse” Virus • Use VSV (good) to overcome HIV (evil) • Recombinant VSV: • Remove VSV envelope gene • Insert cell CD4, CXCR4 genes (cell receptors for HIV); express as envelope proteins on VSV • Novel VSV will attach to HIV gp120 • In cell culture, novel VSV infects and kills HIV-infected cells

20. PROMISES:Phage Factor • Phages produce lysins (enzymes) to exit bacteria • Use lysins to treat bacteria infection (strep throat, endocarditis, meningitis, pneumonia) • 2012 Clinical Trials: CF-301 lysin against MRSA (methicillin-resitant Staphylococcus aureus) • Scientific American.com/aug2012/phage

21. PROMISES:Going Viral • Use M13 bacteriophage to generate electricity • Piezoelectricity: mechanical energy into electrical energy • Biomolecules such as protein and nucleic acids are piezoelectric when compressed • Press down on virus film attached to electrodes to produce electricity • Apply to cell phone, pacemaker, etc. • Safe, cheap, easy to create

22. Why Study Virology? • Viruses important members of our biosphere • Biology “writ small” • Principles learned • Apply to all life

23. “The future is a world limited by ourselves.” • Maurice Maeterlinck (1882-1949) • Nobel Laureate in Literature

24. MICR 401 Final Exam • Tuesday, Dec. 4, 2012 • 1:30 – 3:00pm • Papovavirus thru Hepadnavirus • Case Study #9-15 • Lecture & Discussion Questions, Reading & Chapter Questions • Exam: • Objective Questions (MC, T/F, ID) • Short Essay Questions

25. QUESTIONS???

26. ALL I REALLY NEED TO KNOW

27. ALL I REALLY NEED TO KNOW I LEARNED IN KINDERGARTEN • Robert Fulghum

28. ALL I REALLY NEED TO KNOW • about how to live and what to do and how to be I learned in kindergarten. • Wisdom was not at the top of the graduate-school mountain, but there in the sandpile at Sunday School.

29. These are the things I learned: • Share everything. • Play fair. • Don’t hit people. • Put things back where you found them. • Clean up your own mess.

30. Don’t take things that aren’t yours. • Say you’re sorry when you hurt somebody. • Wash your hands before you eat. • Flush.

31. Warm cookies and cold milk are good for you. • Live a balanced life – learn some and think some and draw and paint and sing and dance and play and work every day some.

32. Take a nap every afternoon. • When you go out into the world, watch out for traffic, hold hands, and stick together.

33. Be aware of wonder. • Remember the little seed in the Styrofoam cup: • The roots go down and the plant goes up and nobody really knows how or why, but we are all like that.

34. Goldfish and hamsters and white mice and even the little seed in the Styrofoam cup – they all die. • So do we.

35. And then remember the Dick-and-Jane books and the first word you learned – the biggest word of all – • LOOK.

36. LOOK • Fulghum, Robert. • 1988. All I Really Need To Know I Learned In Kindergarten: Uncommon Thoughts On Common Things. New York: Villard Books. • 2003. 15th Anniversary Edition. All I Really Need To Know I Learned In Kindergarten: Reconsidered, Revised & Expanded, With Twenty-Five New Essays. New York: Ballantine Books.

37. ALOHA Kauai, Hawaii