Structure. Lecture 4 Virology W3310/4310 Spring 2012

Transcription

1 Structure Lecture 4 Virology W3310/4310 Spring 2012 In order to create something that func2ons properly - a container, a chair, a house - its essence has to be explored, for it should serve its purpose to perfec2on, i.e., it should be durable, inexpensive, and beau2ful. - Walter Gropius 1

2 Func)ons of virion proteins Protec)on of the genome - Assembly of a stable, protecave protein shell - Specific recogniaon and packaging of the nucleic acid genome - InteracAon with host cell membranes to form the envelope 2

3 Func)ons of virion proteins Delivery of the genome - Bind host cell receptors - UncoaAng of the genome - Fusion with cell membranes - Transport of genome to the appropriate site 3

4 Func)ons of virion proteins Other interac)ons with host - With cell components to ensure efficient infecaous cycle - With cell components for transport to sites of assembly - With the host immune system 4

5 Virus par)cles are not inert structures Virions are metastable structures: have not apained minimum free energy conformaaon Gained only when an unfavorable energy barrier is surmounted, following irreversible conformaaonal changes during apachment and entry Viruses are molecular machines that play acave role in delivery of genome 5

6 In other words... To be infecaous, the paracle must be metastable - Must protect the genome (stable) - Must come apart quickly on infecaon (unstable) An infecaous virion is a molecular machine - Has moving parts and does work 6

7 In other words... Virions are spring loaded to uncoat and deliver the genome if and only if the cell provides the proper signals - Energy is put into the structure upon assembly - That potenaal energy is used for disassembly 7

8 Virion structure and func)on Structure (the paracle; the virion) - Created by symmetrical arrangement of many idenacal proteins to provide maximal contact and non- covalent bonding FuncAon (genome delivery) - Structure is NOT permanently bonded together - Can be taken apart or loosened on infecaon to release or expose genome 8

9 Subunit - Single folded polypepade chain Structural unit (protomer, asymmetric unit) - Unit from which capsids or nucleocapsids are built; one or more subunits Capsid (coat) capsa is LaAn for box - Protein shell surrounding genome Nucleocapsid (core) - Nucleic acid - protein assembly within virion Envelope (viral membrane) - Virion - Host cell- derived lipid bilayer InfecAous viral paracle Defini)ons 9

10 PuAng virus par)cles into perspec)ve Nanometer: 10-9 meters Alpha helix in protein: 1 nm diameter DNA: 2 nm diameter Ribosome: 20 nm diameter Poliovirus: 30 nm Mimivirus: 750 nm 10

11 MOLECULAR MACHINERY: A Tour of the Protein Data Bank 3cyt Cytochrome c 1gcn Glucagon 2hiu Insulin 3hhr Human Growth Hormone 1fqy Aquaporin Cholesterol Phospholipid 1mbdMyoglobin 1hrs Ferritin 1e7i Serum Albumin 1msl Mechanosensitive Channel 1grm Gramicidin 2por Porin 1c17+1e79 ATP Synthase 1iat Phosphoglucose 1ald Aldolase Isomerase 1pfk Phosphofructokinase 1cza Hexokinase 1bgy Cytochrome b-c1 Complex 3pgk Phosphoglycerate Kinase 1oco Cytochrome c Oxidase 3gpd Glyceraldehyde-3-phosphate Dehydrogenase 1rfb Interferon 1wsy Tryptophan Synthase 2dnj Deoxyribonuclease 1lz1 Lysozyme 2ptc Trypsin 4hhb Hemoglobin 1bl8 Potassium Channel 7tim Triosephosphate Isomerase 5enl Enolase 3pgmPhosphoglycerate Mutase 1a49 Pyruvate Kinase 1dhf Dihydrofolate Reductase 1f88 Rhodopsin 5rsa Ribonuclease 1smd Amylase 2ohx Alcohol Dehydrogenase 5pep Pepsin 2tsc Thymidylate Synthase 2gls Glutamine Synthetase 1poe Phospholipase 1pth Cyclooxygenase 1igt Antibody 1prc Photosynthetic Reaction Center 1kzu Light-Harvesting Complex 4at1 Aspartate Carbamoyltransferase 1rcx Ribulose Bisphosphate Carboxylase/Oxygenase 1gax Valyl-tRNA Synthetase 1qf6 Threonyl-tRNA Synthetase 1euq Glutaminyl-tRNA 1ttt Elongation Synthetase Factor Tu 1jb0 Photosystem I 4rhv Rhinovirus 1ffy Isoleucyl-tRNA Synthetase 1n2c Nitrogenase 1eiy Phenylalanyl-tRNA Synthetase 1asy Aspartyl-tRNA Synthetase 4tna Transfer RNA 1dar Elongation Factor G info@rcsb.org R ESEARCH C OLLABORATORY FOR STRUCTURAL B IOINFORMATICS RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY S AN DIEGO SUPERCOMPUTER CENTER N ATION AL INSTITUTE OF STANDARDS AND TECHNOLOGY 1efu Elongation Factor 1fjf+1jj2 Ribosome Tu and Ts 2cpl Proline cis/trans Isomerase 1fxk Prefoldin 1aon Chaperonin GroEL/ES 11

12 First recons)tu)on of a virion Purified TMV RNA and coat protein mixed, virus paracles formed spontaneously - TMV: first virus discovered - First virus crystallized - First demonstraaon that RNA serves as geneac material H. Fraenkel- Conrat and RC. Williams, ReconsAtuAon of AcAve Tobacco Mosaic Virus from Its InacAve Protein and Nucleic Acid Components PNAS 41:

13 The tools of viral structural biology Electron microscopy X- ray crystallography Cryo- electron microscopy Nuclear magneac resonance spectroscopy (NMR) Flint volume I, chapter 3, pp

14 Beginning of the era of modern structural virology 1940: Helmuth Ruska used an electron microscope to take the first pictures of virus paracles Ruska, H Die Sichtbarmachung der BakteriophagenLyse im Ubermikroskop. Naturwissenschaagen. 28:45-46). 14

15 Electron microscopy Biological materials have liple inherent contrast: need to be stained NegaAve staining with electron- dense material (uranyl acetate, phosphotungstate), scaper electrons (1959) ResoluAon Å (alpha helix 10 Å dia; 1 Å = 0.1 nm) Detailed structural interpretaaon impossible 15

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17 Linda Stannard, University of Cape Town hpp://web.uct.ac.za/depts/mmi/stannard/linda.html 17

18 Cryo- electron microscopy ( Å) 18

19 Poliovirus + CD155 19

20 X- ray crystallography (2-3 Å for viruses) 20

21 1935: Wendell Stanley crystallized tobacco mosaic virus, showed it remained infecaous. First step towards describing virus structure. Nobel Prize,

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23 Poliovirus,

24 Principles of building virions: Symmetry is key Watson and Crick did more than discover DNA structure : pointed out that most virus paracles were spherical or rod- shaped in the EM Knew paracles were made with many copies of few proteins (geneac economy) Viral proteins have structural properaes that permit regular and repeaave interacaons among them Their seminal contribuaon to virology: - - IdenAcal protein subunits are distributed with helical symmetry for rod- shaped viruses Platonic polyhedra symmetry for round viruses 24

25 The symmetry rules are elegant in their simplicity They provide rules for self- assembly Rule 1: Each subunit has idenacal bonding contacts with its neighbors - The repeated interacaon of chemically complementary surfaces at the subunit interfaces naturally leads to a symmetric arrangement Rule 2: These bonding contacts are usually non- covalent - The reversible formaaon of non- covalent bonds between properly folded subunits leads naturally to error- free assembly and minimizes free energy 25

26 Regular structures form when iden)cal bonds are made between iden)cal subunits Aggregates, clumps, disordered complexes form when non- idenacal bonds form between idenacal subunits 26

27 The repe))on of these interac)ons among a limited number of proteins results in a regular structure Many capsid proteins can self assemble into virus- like paracles (VLPs) The HBV and HPV vaccines are VLPs made in yeast 27

28 TWiV

29 Helical symmetry Coat protein molecules engage in idenacal, equivalent interacaons with one another and with the viral genome to allow construcaon of a large, stable structure from a single protein subunit 29

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33 Enveloped virions of RNA viruses with (- ) ssrna genomes with helical capsids Paramyxoviridae (measles virus, mumps virus) Rhabdoviridae (rabies virus) Orthomyxoviridae (influenza virus) Filoviridae (Ebola virus) These capsids are always called nucleocapsids - The nucleic acid- protein assembly that is packaged within the virion - The nucleocapsid is not the virion 33

34 How can you make a round capsid from proteins with irregular shapes? Clue 1: All round capsids have precise numbers of proteins; mulaples of 60 are common (60, 180, 240, 960) Clue 2: Spherical viruses come in many sizes, but capsid proteins are kda average 34

35 Caspar & Klug s 1962 solu)on They knew from Watson & Crick s work that round capsids are icosahedrons - no other Platonic solids were used Capsid subunits tended to be arranged as hexamers and pentamers The number of capsid subunits followed allowable values of T numbers: 60, 180, 240, 960; nothing in between 35

36 Icosahedral symmetry Icosahedron: solid with 20 faces, each an equilateral triangle Allows formaaon of a closed shell with smallest number (60) of idenacal subunits 36

37 Simple icosahedral capsids Made of 60 idenacal protein subunits The protein subunit is the structural unit InteracAons of all molecules with their neighbors are idenacal (head- to- head, tail- to- tail) 37

38 Adeno- associated virus 2 (parvovirus) 25 nm T=1 60 copies of a single capsid protein How are larger virions built? By adding more subunits 38

39 Triangula)on number, T The number of facets per triangular face of an icosahedron Each facet contains a capsid protein mulamer Combining several triangular facets allows assembly of larger face from same structural unit Capsids with T>1 have a 6-fold axis of symmetry 39

40 Quasiequivalence When a capsid contains more than 60 subunits, each occupies a quasiequivalent posiaon The noncovalent binding properaes of subunits in different structural environments are similar, but not idenacal 40

41 Three modes of subunit packing (orange, yellow, purple) Pentamers & hexamers Bonding interacaons are quasiequivalent: all engage tail- to- tail and head- to- head 180 idenacal protein subunits Figure

42 Nodamura virus nm T=3 180 copies of a single capsid protein 42

43 Poliovirus 30 nm pseudo T=3 60 copies X four capsid proteins 8- stranded anaparallel β- barrel - the structural moaf most commonly found in capsids 43

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45 Structurally complex capsids Largest viruses; disanct components with different symmetries Presence of proteins devoted to specialized roles 45

46 Adenovirus Adenovirus 150 nm T=25 capsid, 240 subunits made of hexons, trimers of viral protein II Fibers at 12 veraces 46

47 Reoviruses T= nm two concentric shells Complex capsids with two icosahedral protein layers VP7 trimers, T=13 VP3 monomers, T=2 47

48 Capsids can be covered by host membranes: enveloped virions Envelope is a lipid bilayer derived from host cell - Viral genome does not encode lipid syntheac machinery Envelope acquired by budding of nucleocapsid through a cellular membrane - Can be any cell membrane, but is virus- specific Nucleocapsids inside the envelope may have helical or icosahedral symmetry 48

49 Viral envelope glycoproteins Integral membrane glycoproteins Ectodomain: apachment, anagenic sites, fusion Internal domain: assembly Oligomeric: spikes 49

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51 Interac)on of viral proteins with viral envelope proteins 51

52 Structured envelopes: Sindbis virus The capsid has T=4 icosahedral symmetry (240 capsid proteins) The 80 envelope spikes (trimers of glycoproteins E1 and E2) ALSO are arranged with T=4 symmetry - anchored to the capsid under the membrane - a structured envelope The cytoplasmic tail of E2 contacts a cleg in the capsid protein, which gives rise to the symmetry in the membrane 52

53 Unstructured envelopes 53

54 Herpesviridae 80 viral genes, >50% encode proteins of 2,000 Å virions Envelope proteins (13) Icosahedral nucleocapsid surrounding DNA genome (4) Tegument (20) - delivery of proteins required early in infecaon 54

55 Herpes simplex capsid Holes for entry and exit of DNA The portal or opening for viral DNA is built at ONE of the 12, 5- fold ver2ces of the T=16 herpesvirus capsid 55

56 The TAILED VIRUSES The majority of bacterial viruses (bacteriophage) employ a molecular device called a tail for recogniaon and apachment to the host cell, penetraaon of the cell envelope, and for DNA transfer from the virus capsid into the host cell cytoplasm The tail is apached at one of the 12 veraces of the capsid (capsid has icosahedral symmetry). The tail is a complex rod - uses helical symmetry in many places - some tails are contrac2le 56

57 Complex virion structures are challenging viral structural biologists t=

58 Other virion components Enzymes - polymerases, integrases, associated proteins - proteases - poly(a) polymerase - capping enzymes - topoisomerase AcAvators, mrna degradaaon, required for efficient infecaon, mrnas Cellular components - histones, trnas, myristate, lipid, cyclophilin A, and many more 58

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