2 Structural Virology Fall 2018 Pavel Plevka 3 Class rules •  Turn off anything that beeps or rings. •  Reading any material that is not related to the class, texFng, or checking the internet during the class is rude and will not be tolerated. •  Please refrain from eaFng during class. Having something to drink is fine. •  Ask quesFons - it will help to clarify the issue not only for you but for your peers as well! •  In class discussions, be respecPul of other students' opinions. Classes are recorded for presentaFon at Ostrava University. 4 •  learn elementary molecular virology •  appreciate role of structural informaFon in understanding of biological processes •  learn to read and evaluate scienFfic papers •  learn history of some fundamental discoveries in biology •  be able to evaluate what is an important research quesFon Aims of the course 5 Elementary molecular virology 1. AWachment 2. Entry 3. TranscripFon 4. TranslaFon 5. Genome replicaFon 6. Assembly 7. Exit HIV replicaFon cycle 6 Structural biology in understanding virus infecFon Virus binding to receptor Binding of anFbodies to virus parFcle 7 Read and evaluate scienFfic papers CHALLENGES IN IRREPRODUCIBLE RESEARCH: - more than 50% of drug-related research ar:cles in high profile journals contain irreproducible data Reasons for irreproducibility: - Under supervised students and post-docs - “Publish or Perish” threat - wrong design of experiments – “know your staFsFcs” 8 History of fundamental discoveries WILHELM CONRAD RÖNTGEN (1845-1923) •  1901 Nobel Laureate in Physics discovery of the remarkable rays subsequently named a6er him 9 What is important research quesFon? 1. It asks about something other people care about 2. It builds on what you and others already know 3. It allows you to learn something you don't already know InteresFng X Important What are evoluFonary relaFonships of anaerobic proFsts? What is the probability that current Ebola epidemics is going to spread outside of Africa? 10 What is asked of you: •  Read assigned readings before the day for which they are assigned •  Bring five-sentence essay describing the importance of a scienFfic paper to each lecture •  ParFcipate in discussions •  Submit two mini-assignments •  I am here to help, learning is up to you! 11 Course textbook: 14 copies are available in campus library 12 Viruses and their importance 13 Virosphere! 14 • Phage typing of bacteria • Sources of enzymes (polymerases, T4 ligase, reverse transcriptase) • PesFcides (baculoviruses – lepidoptera, hymenoptera, myxoma virus - rabits) SPECIFICITY! • AnF-bacterial agents (S. aureus phage phi812) • AnF-cancer agents (adeno-associated viruses) • Gene vectors (capsids used as vehicles for gene delivery) • Mass protein producFon Useful viruses 15 •  Phage T2 and E. coli were used to provide evidence that genes are composed of DNA. •  The first enhancers to be characterized were in genes of simian virus 40 (SV40). •  The first transcripFon factor to be characterized was the transplantaFon (T) anFgen of SV40. •  The first nuclear localizaFon signal of a protein was idenFfied in the T anFgen of SV40. •  Introns were discovered during studies of adenovirus transcripFon. •  The role of the cap structure at the 5’ end of eukaryoFc messenger RNA was discovered during studies with vaccinia virus and a reovirus. •  The first internal ribosomal entry site to be discov- ered was found in the RNA of poliovirus. •  The first RNA pseudoknot to be discovered was that in the genome of turnip yellow mosaic virus. Fundamental discoveries in biology Most viruses are 25-400nm in size Units of length 20 Using TMV to increase surface of electrodes © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 21 Methods to study virus structures Wavelength and diffracFon Wavelength comparison of X-rays and visible light 38λ Cellular organisms Viruses dsDNA ssRNA Protein ReplicaFon TranscripFon TranslaFon DNA RNA Protein 25 Viruses are intracellular parasites HIV replicaFon cycle “Virion” – virus parFcle Two forms of virus existence Cell infec:on Are viruses alive? Pro-life An:-life Have genes Do not have cellular structure They evolve No metabolism Subject to natural selecFon Require host cell for pro- creaFon Procreate New viruses assemble from parts 28 Virus definiFon A virus is a very small, non-cellular parasite of cells. Its genome, which is composed of either DNA or RNA, is enclosed in a protein coat. © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 29 • discuss reasons for studying viruses • explain how viruses differ from other organisms • define the term ‘virus’ Learning outcomes © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 30 Methods used in virology © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 31 Virus producFon in eggs 32 Virus producFon honeybee pupae © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 33 Virus producFon in vitro in Fssue culture © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 34 Sterile work with Fssue cultures BSL levels © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 36 Plaque assay for animal viruses HeLa 300 Fs bb./ml, 10.2.14 Fixed auer 72h BEST OF CMC HRV16 from stock 28.11.13, PFU 5.103 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 38 Phage plaque assay 39 DifferenFal centrifugaFon © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 40 Density gradient centrifugaFon 41 SDS-PAGE Israeli acute bee paralysis virus tartrate gradient CsCl gradient CsCl gradient – middle fracFon 1.F tartrate A260=133.083, A280 = 160.379 2.F tartrate A260=36.575, A280 = 28.762 3.F tartrate A260=86.795, A280 = 70.081 4.F tartrate A260=10.029, A280 = 6.267 1.F CsCl A260=111.613, A280 = 63.564 1.F CsCl A260=2.555, A280 = 1.556 1.F CsCl A260=6.836, A280 = 3.918 SDS-PAGE of IAPV 1. Virus purificaFon 3. cryo-EM 4. ReconstrucFon 2. Grid preparaFon 45 Cryo-EM of phage phi812 1. Virus purificaFon 2. CrystallizaFon 3. DiffracFon data 4. Solve structure Crystals of IAPV 0.1 M Cadmium Chloride 0.1 M Na acetate pH 4.5 15 % PEG 400 0.2 M Na/K Phosphate 0.1 M BisTris Propane pH 7.5 20 % PEG (w/v)3350 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 48 Hybridization detection of nucleic acids © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 49 Microarrays © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 50 rtPCR assay © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 51 TCID50 assay © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 52 Sequencing of virus genomes © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 53 • outline methods for ◦ culFvaFon of viruses; ◦ purificaFon of viruses; ◦ detecFon of viruses and their components; ◦ assay of virus infecFvity; ◦ invesFgaFon of virus gene funcFon; • assess the value of virus genome sequencing. Learning outcomes Helical Icosahedral Complex Irregular Virus structures 55 Virus structures Picornavirus virion © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 62 Icosahedron 63 64 65 66 67 E18 Fab E19 Fab 69 70 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 71 72 73 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 74 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 75 76 77 78 79 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 80 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 81 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 82 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 83 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 84 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 85 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 86 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 87 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 88 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 89 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 90 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 91 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 92 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 93 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 94 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 95 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 96 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 97 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 98 © 2012 John Wiley & Sons Ltd. www.wiley.com/college/carter 99