Introductory biophysics A. Y. 2016-17 11. Viruses
Edoardo Milotti Dipartimento di Fisica, Università di Trieste !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE !"#$%&'()$*+$'$,-./'0*1*&'!"#$%"#&'()#**(& 2-34)% 5+-*&$6%7%/)8%'98 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE F2=.-$*(2&"($G%$-&#H&$&*%;,$/#1#-.&I$&J(/"&#H&,%#*#K#$/3&(?.?3& $&:/(2.)):)$%&.:J$%(#*.L&#H&*=.&!"#$%&'(')% *"+,-. G%#:,&(/&(*1& S#P&"#.1&$&T#%P$)J&5(%:1& (/*.%-."($*.&<)##"1*%.$-&H#%-3&()):1*%$*(/G&*=.&-$M#%& "(HH.%&H%#-&7?&<%:2.(U #%G$/.)).1?&I!"#$%#$%!"&%'(!")*&+%!"(!%,(-$&$%!"&%$.&&'#+*% $#,/+&$$3&(-$G.&H%#-& =**,NOOPPP?()%(?#%GO9/H#F.%5OQ.<,: !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE V(%:1.1&=$5.&<../&P(*=&:1&H%#-&:/-.-#%$<).&*(-.13&$/"&*=.;&=$5.&#H*./&<../& $11#2($*."&*#&12#:%G.1&)(J.&1-$)),#W X#P,#W&).1(#/1&#/&*=.&=$/"&#H&F$%$=&T.)-.13&H%#-&!?&Y.//.%Z1&/&0+."#?&'$*.%($)&H%#-&#/.&#H&*=.1.&).1(#/1&P$1& :1."&*#&[5$22(/$*.\&Y$-.1&]=(,,1&#/&'$;&C^3&CERD?&0%#-&+?&_$W<;N&[!"P$%"&Y.//.%Z1&9/`:(%;a&$&<(2./*./$%;& $/$);1(1\3&V$22(/.&CE&ICRRRL&bBC?& !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE 7=.&*$W#/#-;&#H&5(%:1.1 : +T>&5(%:1.1 : "1+T> : 11+T> : cT>&5(%:1.1 : "1cT> : 11cT>IdL : 11cT>I8L : c.*%#5(%:1.1 : 11cT> Ic7L : "1+T>Ic7L "1&e&"#:<).&1*%$/" 11 e&1(/G).&1*%$/" c7&e&%.5.%1.&*%$/12%(,*(#/ d&e&cT>&2$/&H:/2*(#/&"(%.2*);&$1&-cT> 8 e&cT>&2$//#*&H:/2*(#/&$1&-cT> 9-$G.&H%#-&0?&>?&':%,=;3&[7=.&/$*:%.&#H&5(%:1.1&$1& .*(#)#G(2&$G./*1&#H&5.*.%(/$%;&$/"&K##/#*(2& "(1.$1.1\(/ V.*.%(/$%;&V(%#)#G;3&b%" ."?&>2$".-(2& ]%.11&ICRRRL?&& !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE ]#)(#5(%:13&H%#-&V(%:1Q#%)"!"#$%"#&'()#**(& I=**,NOOPPP?5(%#)#G;?P(12?.":8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CEO5(%:1P#%)"O5(%:1)(1*?,=,L SV40: Simian Vacuolating virus (a DNA virus with an interesting history). Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 >"./#5(%:13&$&2#--#/& 2$:1.&#H&-()"&%.1,(%$*#%;& "(1.$1.1&(/&2=()"%./? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 b&T#<.)&,%(K.&P(//.%1&$-#/G& *=.&$:*=#%1&???&G:.11&P=#ff !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Important topics in the biophysics of viruses • viral shape • thermodynamics of viral self-assembly • dynamics of viral self-assembly • internal capsid pressure • packaging DNA (or RNA) inside capsid • genetic drift and the concept of quasispecies • viruses and the environment • ... Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 On the basis of crystallographic observations, Crick and Watson (1956), and later Caspar and Klug (1962) set basic constraints on possible virus shapes. CW: • viral genome too short to code for a single large protein for the capsid • there must be many small tiles that make up the capsid • unit cell of virus crystals is cubic (Caspar) • observed virus shape is often spherical • observed symmetry constraints (5-3-2 fold symmetry) point to an icosahedral shape Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 0(1)+%2.-* _#%/N&CC&>:G:1*&CRAD3&j.)5$13&k(*=:$/($ T#<.)&]%(K.&(/&X=.-(1*%;&(/&CRlA&mH#%&=(1&".5.)#,-./*&#H& 2%;1*$))#G%$,=(2&.).2*%#/&-(2%#12#,;&$/"&=(1&1*%:2*:%$)& .):2("$*(#/&#H&<(#)#G(2$));&(-,#%*$/*&/:2).(2&$2("8,%#*.(/& 2#-,).W.1\ !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE H%#-&+?&k?&+?&X$1,$%&$/"&>?&i):G3&[]=;1(2$)&]%(/2(,).1&(/&*=.&X#/1*%:2*(#/&#H&c.G:)$%& V(%:1.1\3&X#)"&F,%(/G&S$%<#%&F;-,#1($&#/&n:$/*(*$*(5.&_(#)#G;&V#)?&ooV99&ICRDAL Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Stretching of central region (prolate icosahedra) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Physics of Viral Shells Robijn F. Bruinsma1,2 and William S. Klug3 1Department of Physics and Astronomy, University of California, Los Angeles, California 90095; email: [email protected] 2Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095 3Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, California 90095; email: [email protected] 1. INTRODUCTION In 1955, Fraenkel-Conrat & Williams (1) showed that the rod-like Tobacco mosaic virus (TMV) assembles spontaneously in solutions containing the molecular components of the virus [capsid proteins (CPs) and the RNA genomic molecules]. Because of their highly reproducible size and shape and their precise structural organization, the shells of viruses, or capsids, have since been found to have many applications in materials science. Directed evolution of the CPs allows at- tachment of particular receptor groups on the exterior surface. Functionalized viral shells have been used to create metallic wires, solar cells, batteries, and fuel cells (2). This review discusses the application of methods borrowed from statistical physics, condensed matter physics, soft-matter physics, and elasticity theory to the self-assembly and material properties of viral capsids. 2. THE STRUCTURE OF VIRAL CAPSIDS !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE 2.1. The Cowpea Chlorotic Mottle Virus Figure 1 shows the Cowpea chlorotic mottle virus (CCMV), a roughly spherical virus with a radius of approximately 15 nm that infects the cowpea plant. Like TMV, CCMV self-assembles Access provided by University of California - San Diego on 03/12/15. For personal use only. Annu. Rev. Condens. Matter Phys. 2015.6:245-268. Downloaded from www.annualreviews.org spontaneously (3). The physical properties of CCMV have been extensively studied, and Annu. Rev. Condens. Matter Phys. 2015. 6:245–68 CCMVKeywords is applied extensively in materials and medical science. Figure 1b shows an image of the CCMV capsid, reconstructed by cryotransmission electron microscopy and X-ray diffraction The Annual Review of Condensed Matter Physics is viral capsid, statistical mechanics, elasticity theory, structural phase online at conmatphys.annualreviews.org methods (4, 5). The regular surface structure of the CCMV capsid resembles a crystal and, like transition, Landau theory This article’s doi: a crystal, it can be characterized by its symmetry operations. Figure 1b is oriented along a twofold 10.1146/annurev-conmatphys-031214-014325 symmetryAbstract axis. The capsid also has threefold and fivefold rotation axes. The various rotational Copyright © 2015 by Annual Reviews. symmetryWe review operations the application coincide of statistical with those mechanics, of the icosahedronelasticity theory, (see Figure 1a). Note that Figure 1b All rights reserved hasand the condensed orientation matter of physics the first to the panel assembly of Figure and maturation 1a. of viral capsids.The red arrows in Figure 1b indicate ring-like structures with apparent sixfold and fivefold symmetry known as hexamers and pentamers, respectively. They are composed of six and five CPs, respectively. CCMV capsids have 180 identical CPs that are organized in twelve identical pentamers and twenty identical hexamers. Figure 1c shows schematically245 how the 180 proteins are distributed over the shell. Proteins in symmetry-equivalent positions are indicated by the same color. Sixty pentamer proteins are marked blue. Sixty hexamer proteins are marked green and another sixty red. Finally, Figure 1d shows the assembly diagram of CCMV capsids in a solution of CPs (6). The vertical axis is the ionic strength I (i.e., the salt concentration), and the horizontal axis is the pH (I ∼ 0.1 and pH ∼ 7 under typical physiological conditions). In an aqueous physiological solution, CCMV CPs form dimers at low protein concentrations (typically in the microMolar range). Hexagonal layers or spherocylinders form at higher concentrations. Reducing the pH to below the physiological level leads to the formation of CCMV capsids at higher I values and Access provided by University of California - San Diego on 03/12/15. For personal use only. Annu. Rev. Condens. Matter Phys. 2015.6:245-268. Downloaded from www.annualreviews.org concentric multishells at lower I. The fact that reducing the salinity stimulates aggregation is an indicator that electrostatics play an important role in the interactions between CPs. In the Debye- Hückel (DH) theory of aqueous electrostatics, the range of electrostatic interactions is inversely proportional to I1/2 (7). The reason for the dependence of CCMV capsids on the acidity level is discussed below. If viral RNA molecules are included in the solution, then infectious viruses assemble at reduced pH. In this review, we focus exclusively on empty capsids. 2.2. The Amphiphilic Capsid Protein Figure 2 shows schematically the structure of a typical CP as obtained from X-ray diffraction studies of crystals composed of viruses (5) (maximum resolution currently in the range of an 246 Bruinsma Klug Viral assembly and environment a d 4.0 Shells 28 nm Bilayer 1.0 Multilayer or disc 100, 150 nm Dumbbell 0.1 Tube diameters (nm) bc 16 C4 B3 25 B4 A4 A3 C3 16 C5 A5 A2 B2 25 B9 A1 C12 (M) Ionic strength 0.01 36 B5 C2 12, 22, 55 A9 C9 C1 B1 B10 A12 16, 20, 40, 100 A8 B8 B6 C6 C11 A11 C7 B12 C8 B11 A6 B7 A7 A10 C10 3.0 4.0 5.0 6.0 7.0 pH Figure 1 Structure and assembly of Cowpea chlorotic mottle virus (CCMV). (a) The twofold, threefold, and fivefold symmetry axes of an icosahedron. (b) Cryotransmission electron micrograph, reprinted from Reference 5 with permission from Elsevier. The red arrows indicate pentamers and hexamers. (c) Locations of the capsid proteins in the CCMV shell, reprinted from Reference 5 with permission from Elsevier. Symmetry-equivalent proteins have the same color. (d) Assembly diagram of CCMV capsids, reprinted with permission from Reference 6; copyright 2009, American Chemical Society. The horizontal axis is the pH level. The vertical axis is the salinity. Angstrom). CPs are, like all proteins, composed of chains of amino acids, or residues, linked by chemical bonds, together forming the primary structure of the protein. The polymeric backbone is indicated in Figure 2a as a ribbonEdoardo Milotti that winds- aroundIntroductory biophysics inside the prismatic- A.Y. 2016 outline.-17 The end points of the primary structure of a protein are known as the N and C terminals and are indicated in Figure 2a. Parts of the chain that are highlighted as parallel colored arrows are sections of the main chain whose residues are linked by hydrogen bonds. These b sheets provide rigidity to the spatial structure of the CP. The CPs of a large number of viruses share this particular structural motif, which is known as the jelly roll, even when they may have very different primary amino acid sequences. The top and bottom surfaces of the CP outline are lined with hydrophilic residues, some charged, whereas hydrophobic residues line the vertical sides of the protein. Molecules that have both hydrophobic and hydrophilic exposed groups are known as amphiphiles. The phase diagram of amphiphiles has been extensively studied in the context of soft-matter physics (7), with sur- factants and lipids as the favorite examples. Surfactants and lipids in dilute aqueous solution tend Access provided by University of California - San Diego on 03/12/15. For personal use only. Annu. Rev. Condens. Matter Phys. 2015.6:245-268. Downloaded from www.annualreviews.org to assemble spontaneously and reversibly into aggregates that may be spherical (micelles and vesicles), planar (e.g., the La phase), or three dimensional (e.g., the L3 phase). These aggregates often constitute minimum free energy states, but they are typically delicate and subject to strong thermal fluctuations (7). The spontaneous formation of capsids from CPs in an aqueous solution has some similarity with amphiphilic self-assembly, but there are also important differences that we elucidate below. The hydrophobic edges of the CPs can be matched together so water molecules are expelled from the shared interface. In addition, specific residues are capable of pair formation across the shared interface (10). CPs have very large electrical dipole moments with (for CCMV) approx- imately ten negative charges on the surface facing the exterior of the virus and a similar number of positive charges facing the interior. The net electrostatic charge is, however, modest and changes www.annualreviews.org Physics of Viral Shells 247 A thermodynamic model of viral self-assembly • adhesion energy 1 2 V (θ ) = V (0) + κ θ −θ * 2 ( ) • “mean field” term that accounts for the disk packing density of N disks 2 N ⎣⎡ρmax − ρ(N )⎦⎤ • N-disk Hamiltonian z κ * 2 B 2 H (N ) = NV (0) + ∑(θi, j −θ ) + N ⎣⎡ρmax − ρ(N )⎦⎤ 2 2 i, j 2 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 z κ * 2 B 2 H (N ) = NV (0) + ∑(θi, j −θ ) + N ⎣⎡ρmax − ρ(N )⎦⎤ 2 2 i, j 2 Here z is the mean number of nearest neighbors, and B is proportional to the compression modulus. If is the mole fraction of Φ(N ) N-disk capsids, and S = −kB ∑Φ(N )lnΦ(N ) N is the mixing entropy, then the free energy is F = H (N ) − TS Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 It can also be shown that the minimization of free energy leads to ⎡ ⎤ Φ(N ) = exp ⎣β (µN − H (N ) )⎦ and we can define the onset of self-assembly where half of the disks remain in solution while the others join . It can also be shown that the number N in the dominant capsid structure is a function of T. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 2R6 2R5 R5/R6 = 0.93 ab –2.4 –2.6 N = 12 N = 32 –2.8 12 – E ( N )/ –3.0 32 42 72 –3.2 0 20 40 60 80 N = 42 N = 72 N Figure 5 (a) Capsid energy per particle ɛ(N) E(N)/N as a function of the number of particles. Adapted from Reference 22. (b) Minimum-energy ¼ structures. They correspond to the T 1, 3, 4, and 7 Caspar-Klug icosahedra. ¼ a hexagonal sheet of CPs has preferred curvature radius that is roughly in the right range, the CK icosahedron of choice will assemble. It is not necessary to require the CPs to be programmable. This, of course, does not explain why the T-number structures are so prevalent among viral shells, Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 but that is a question of evolutionary microbiology. 3.3. The Next Level: Self-Assembly Mark II Classical equilibrium assembly theory seems to successfully describe in vitro capsid assembly studies, but there is a problem. Neither the cytoplasm of our cells nor the extracellular fluid surrounding our cells carries, under normal conditions, many free-floating CPs. In an equilibrium description, viruses should disassemble under these conditions, which—unfortunately—they do not: Virus assembly is clearly an irreversible process. The in vitro assembly experiments of empty Access provided by University of California - San Diego on 03/12/15. For personal use only. Annu. Rev. Condens. Matter Phys. 2015.6:245-268. Downloaded from www.annualreviews.org capsids actually show hysteresis and irreversibility as well (23). If, for example, the CP con- centration is reduced back down to below fÃ, then capsids should disappear. This, again, does not happen.4 The mechanism behind the exceptional stability of viral capsids, as compared with traditional self-assembled equilibrium structures composed of amphiphiles, can be illustrated by an elegant model for capsid assembly found by Zlotnick (24) in which the capsid is a dodecahedron as- sembled from twelve regular pentamers with sticky, slanted edges. There are many different 4In order to redissolve self-assembled capsids, it is necessary to change thermodynamic conditions, for example, by increasing the pH. www.annualreviews.org Physics of Viral Shells 253 SAR11: Pelagibacter ubique, et al. Pelagibacter ubique is a member of the SAR11 “unculturable” group of alpha-proteobacteria that predominate the oceanic pelagic ecosystem. This organism, like most SAR11 species, is a free-living, planktonic oligotrophic facultative photochemotroph. It is very small, 0.15 x 0.6um, 1/500th the volume of E.coli, providing a large surface/volume ratio for absorbing trace nutrients and light. SAR11 (Pelagibacter ubique) is the most common prokaryiote in the seas (≈ 1/3 of all prokaryotic cells in surface water). It is really small (about 0.15 µm x 0.6 µm) The 1.3Mbp genome of Pelagibacter Its abundance is counterbalanced by the Pelagipodovirus, which is a bacteriophage virus (or just phage) ubique is extremely streamlined, with no repeated sequences, prophage, &c, and has the smallest known intergenic spacers. However, the genome retains all of the usual metabolic capabilities of alpha- proteobacteria, and is specialized for slow growth, extracting trace dissolved organics, nitrogen, and phosphorous from Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 the open ocean water. LETTER doi:10.1038/nature11921 Abundant SAR11 viruses in the ocean Yanlin Zhao1*, Ben Temperton1*, J. Cameron Thrash1, Michael S. Schwalbach2, Kevin L. Vergin1, Zachary C. Landry1, Mark Ellisman3, Tom Deerinck3, Matthew B. Sullivan4 & Stephen J. Giovannoni1 Several reports proposed that the extraordinary dominance of the Autographivirinae subfamily within the Podoviridae family4 with strik- SAR11 bacterial clade in ocean ecosystems could be a consequence ing conservation of synteny (Supplementary Fig. 3). HTVC011P and of unusual mechanisms of resistance to bacteriophage infec- HTVC019P are divergent from marine cyanophage subgroups and tion, including ‘cryptic escape’ through reduced cell size1 and/or other well-defined Autographivirinae subgroups, showing closer rela- K-strategist defence specialism2. Alternatively, the evolution of tionships to some Autographivirinae prophages (Fig. 2a). HTVC008M high surface-to-volume ratios coupled with minimal genomes con- is most closely related to T4-like myoviruses (Supplementary Fig. 4), taining high-affinity transporters enables unusually efficient meta- but again represents a divergent lineage distinct from the marine cya- bolism for oxidizing dissolved organic matter in the world’s oceans nophage clusters and other T4-like subgroups5 (Fig. 2b). The genome that could support vast population sizes despite phage susceptibi- of HTVC010P contains limited homology to some unclassified podo- lity. These ideas are important for understanding plankton ecology viruses (Supplementary Fig. 5). Single-gene trees of the tail tube B because they emphasize the potentially important role of top-down protein and the head–tail connector protein placed HTVC010P near mechanisms in predation, thus determining the size of SAR11 unclassified podoviruses distantly related to Autographivirinae sub- populations and their concomitant role in biogeochemical cycling. family podoviruses (Fig. 2c, d). Here we report the isolation of diverse SAR11 viruses belonging to We investigated the relative abundances of pelagiphages in a two virus families in culture, for which we propose the name ‘pela- recently published collection of quantitative marine viral metagen- giphage’, after their host. Notably, the pelagiphage genomes were omes from the Pacific Ocean6. The viral metagenomes were prepared highly represented in marine viral metagenomes, demonstrating using chemical flocculation of viral particles to yield sufficient DNA their importance in nature. One of the new phages, HTVC010P, material followed by linker-amplified libraries modified for 454 pyro- represents a new podovirus subfamily more abundant than any sequencing to avoid stochastic amplification biases traditionally asso- seen previously, in all data sets tested, and may represent one of ciated with multiple displacement amplification (MDA)7. Sequences the most abundant virus subfamilies in the biosphere. This dis- of pelagiphage origin were characterized by reciprocal-best-BLAST covery disproves the theory that SAR11 cells are immune to viral (RBB) (see Supplementary Methods) in 27 metagenomes from two predation and is consistent with the interpretation that the success multi-depth coastal-to-open ocean transects and one from Scripps of this highly abundant microbial clade is the result of successfully pier, San Diego, California, USA6, and their abundances were com- evolved adaptation to resource competition. pared to marine cyanophages. Across all viral metagenomic samples, a Four pelagiphages were isolated from seawater samples taken on the total of 183,657 reads (,4.7% of total reads) were successfully assigned Oregon coast and at Bermuda Hydrostation S, using a host culture of to one of the 30 viral genomes used in this study (Supplementary axenic ‘Candidatus Pelagibacter ubique’ HTCC1062. SAR11 isolates do not produce lawns on agar plates, therefore dilution-to-extinction3 was used to purify the viruses (see Supplementary Methods). Infec- abc tions of axenic Pelagibacter cell cultures by the isolated viruses caused sharp declines in host numbers and produced phage particles (Sup- plementary Fig. 1). Pelagiphage HTVC011P, HTVC019P and HTVC- 010P all morphologically belong to the short-tailed Podoviridae family (Fig. 1 a–c). The general biological and genomic features of these four pelagiphages are summarized in Table 1. HTVC011P and HTVC019P 50 nm 50 nm 50 nm have similar morphologies, with capsid sizes of approximately 55 nm, whereas HTVC010P has a smaller capsid (approximately 50 nm in d e diameter). HTVC008M has the typical morphology of the Myo- viridae family with an isometric head and contractile tail structure (Fig. 1d). Transmission electron microscopy also captured a SAR11 host cell filled with HTVC011P immediately before lysis (Fig. 1e). The latent periods of these four pelagiphages ranged from 16 to 24 h (Table 1 and Supplementary Fig. 2). Their burst sizes varied from 9 to 49 (Table 1). The genomes of all four pelagiphages were completely 100 nm 200 nm sequenced, assembled and annotated, yielding genome sequences of 34,892–147,284 base pairs (bp) (Table 1). Phylogenetic analysis of the Figure 1 | Transmission electron microscopy images of isolated pelagiphages confirmed morphological classification of HTVC011P pelagiphages. a, Pelagipodovirus HTVC011P. b, Pelagipodovirus HTVC019P. and HTVC019P as podoviruses, HTVC008M as a myovirus, and c, Pelagipodovirus HTVC010P. d, Pelagimyovirus HTVC008M. e, Host cell of HTVC010P as a divergent podovirus (Fig. 2). Genome annotation ‘Candidatus P. ubique’ HTCC1062 infected with HTVC011P immediately revealed that both HTVC011P and HTVC019P are members of the before lysis. 1Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA. 2Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Wisconsin 53706, USA. 3National Center Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 for Microscopy and Imaging Research, University of California, San Diego, California 92093, USA. 4Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA. from Zhao et al., “Abundant SAR11 viruses in the ocean”, Nature 494 (2013) 357 *These authors contributed equally to this work. 21 FEBRUARY 2013 | VOL 494 | NATURE | 357 ©2013 Macmillan Publishers Limited. All rights reserved • It is estimated that 1 liter of surface marine water contains about 1010 prokaryotes and 1011 viruses. There are as many viruses as there are stars in the galaxy. • Bacterial lysis carried out by viruses may be very important in the global ecosystem because it releases carbon in seawater, and can affect the global carbon cycle. It has been estimated that ocean viruses may turn over as much as 1.5 1014 kg of carbon per year – more than 30 times the standing carbon abundance in marine plankton. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 +($G%$-&#H&*=.&,%#,#1."&)(/J$G.&<.*P../&2)#:"1&$/"&<(#)#G(2$)&$2*(5(*;&(/&*=.&F#:*=.%/& g2.$/3&(/2):"(/G&,#*./*($)&1#:%2.1&#H&2)#:"&2#/"./1$*(#/&/:2).(&I"(-.*=;)&1:)H(".3&1.$& 1,%$;3&1.$&1$)*L?&7=.&"(-.*=;)&1:)H(".&*%$/1H#%-1&(/*#&1:)H$*.&(/&*=.&$*-#1,=.%.a&1:)H$*.& +(-.*=;)&"(1:)H(". $.%#1#)1&2$/&$)1#&<.&,%#":2."&<;&<#*=&5#)2$/#.1&$/"&=:-$/&$2*(5(*(.1? >22#%"(/G&*#&%.2./*&%.1.$%2=3.$/&)(H.&<##1*1&*=.&/:-<.%&#H&"%#,).*1&<;&$<#:*&DBs&#/& $5.%$G.?&]=$G.&$2*(5(*;&(/H):./2.1&*=(1&,%#2.11&$1&P.))? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Vol 438|3 November 2005|doi:10.1038/nature04111 LETTERS Vol 438|3 November 2005|doi:10.1038/nature04111 PhotosynthesisLETTERS genes in marine viruses yield proteins during host infection Photosynthesis1 genes2 in marine viruses1 yield 2 1,3 Debbieproteins Lindell during, Jacob D. host Jaffe infection†, Zackary I. Johnson †, George M. Church & Sallie W. Chisholm Debbie Lindell1, Jacob D. Jaffe2†, Zackary I. Johnson1†, George M. Church2 & Sallie W. Chisholm1,3 Cyanobacteria, and the viruses (phages) that infect them, are (j PSII) during the 8-h latent period before lysis. The former decreased 1,2 significantCyanobacteria, andcontributors the viruses (phages) to the that oceanic infect them, ‘gene are pool’(j PSII) during. This the 8-h pool latent isperiod beforeonly lysis. slightly, The former whereas decreased the latter was constant throughout this period dynamic,significant contributors and the to transferthe oceanic ‘gene of genetic pool’1,2. This material pool is only between slightly, whereas hosts the and latter was constant(Fig. 1b, throughout c), indicating this period that phage infection does not lead to a marked dynamic, and the transfer3–6 of genetic material between hosts and (Fig. 1b, c), indicating that phage infection does not lead to a marked 14–16 17–19 theirtheir phages phages3–6 probablyprobably influences the influences genetic and functional the geneticdecline and in PSII functional performance, as occursdecline in some14–16 in, but PSII not performance, other17–19 as occurs in some , but not other diversitydiversity of both. of both. For example, For photosynthesis example, photosynthesis genes of cyanobac- photosynthetic genes of cyanobac- host–virus systems. photosynthetic host–virus systems. terial origin have been found in phages that infect Prochlorococ- Thus, continued photosynthesis is required for maximum phage terialcus5,7 and originSynechococcus have8,9, thebeen numerically found dominant in phages phototrophs that infectproductionProchlorococ- in our system. Moreover,Thus, photosynthesis continued is sustained photosynthesis is required for maximum phage cusin ocean5,7 and ecosystems.Synechococcus These genes include8,9, thepsbA numerically, which encodes the dominantduring infection phototrophs when a decline in theproduction transcription and in translation our system. Moreover, photosynthesis is sustained photosystem II core reaction centre protein D1, and high-light- of host genes might be expected, suggesting that the expression of ininducible ocean (hli ecosystems.) genes. Here we show These that phagegenespsbA includeand hli genespsbAphage, which photosynthesis encodes genes the might beduring supplementing infection host metabo- when a decline in the transcription and translation photosystemare expressed during II infection core reaction of Prochlorococcus centreand protein are co- D1,lism. To and address high-light- this hypothesis, we determinedof host whether genes these might phage be expected, suggesting that the expression of transcribed with essential phage capsid genes, and that the genes are expressed and, if so, how their expression relates to that of inducibleamount of phage (hli D1) protein genes. increases Here steadily we show over the that infective phagethepsbA homologousand hli hostgenes genes. Using probesphage specific photosynthesis for the phage and genes might be supplementing host metabo- areperiod. expressed We also show duringthat the expression infection of host of photosynthesisProchlorococcushost psbA andandhli genes, are we co- found thatlism. both To of the address phage genes this were hypothesis, we determined whether these phage genes declines over the course of infection and that replication of transcribed (Fig. 2a, b and Supplementary Fig. 1). Using polymerase transcribedthe phage genome with is a function essential of photosynthesis. phage capsid We thus genes,chain reaction and with that reverse the transcriptiongenes (RT–PCR), are expressed we determined and, if so, how their expression relates to that of amountpropose that of the phage phage genes D1 are protein functional increases in photosynthesis steadily over the infective the homologous host genes. Using probes specific for the phage and and that they may be increasing phage fitness by supplementing period.the host production We also of these show proteins. that the expression of host photosynthesis host psbA and hli genes, we found that both of the phage genes were Photosynthesis in cyanobacteria, algae and plants requires two genesphotosystems declines (denoted over PSI and the PSII). course The D1 of and infection D2 proteins and that replication of transcribed (Fig. 2a, b and Supplementary Fig. 1). Using polymerase the(encoded phage by psbA genomeand psbD, respectively) is a function form a heterodimer of photosynthesis. in We thus chain reaction with reverse transcription (RT–PCR), we determined the reaction centre of PSII and bind the components required proposefor photochemistry. that The the D1 phage protein is genes turned over are rapidly functional owing in photosynthesis andto light-induced that they damage may10; thus, be increasingits de novo synthesis phage is required fitness by supplementing for sustained photosynthesis10.High-light-inducibleproteins the(HLIPs) host protect production the photosynthetic of these apparatus proteins. from photodamage by dissipatingPhotosynthesis excess light energy in cyanobacteria,11. algae and plants requires two Numerous cyanophages contain photosynthesis genes (psbA and photosystemsat least one other) (denoted5,7–9 with highly PSI conserved and PSII). amino The acid D1 and D2 proteins (encodedsequences7,12, suggesting by psbA thatand they encodepsbD, functional respectively) proteins that form a heterodimer in may be involved in maintaining host photosynthesis during infec- thetion. Here reaction we used Prochlorococcus centre ofMED4 PSII and and the podovirus bind P-SSP7 the components required for(a T7-like photochemistry. phage5)asamodelsystemtobeginexploringthis The D1 protein is turned over rapidly owing hypothesis. We considered that if these10 genes are involved in host tophotosynthesis, light-induced then the damage amount of phage; thus, production its de might novo be synthesis is required fordependent sustained on photosynthetic photosynthesis performance and,10.High-light-inducibleproteins conversely, host photosynthesis might be compromised by phage infection. We (HLIPs)examined the protect first part of the thisphotosynthetic hypothesis by inhibiting apparatus photosyn- Figure from 1 | Photosynthesis photodamage and phage infection. a, Replication of the phage bythesis dissipating with darkness excess or DCMU light (3-(3,4-dichlorophenyl)-1,1- energy11. genome was assessed by quantifying the phage gene encoding DNA dimethylurea), an inhibitor of electron flow from PSII to PSI, polymerase in host cells kept in the light, transferred to the dark or treated whichNumerous led to a respective cyanophages four- or twofold contain reduction photosynthesis in replication with DCMU genes while (psbA in the light.and Significantly lower (P , 0.05) phage DNA was detected in dark- and DCMU-treated cells after 4 h. b, c, PSII of the phage genome (Fig. 1a).5,7–9 Thus, as in other systems13–15, at least one other) with highly conservedphotochemical amino conversion acid efficiency (F v/F m; b) and PSII functional continued photosynthesis7,12 is necessary for maximal phage replica- sequences , suggesting that they encode functionalabsorption proteinscross-sectional thatarea (jPSII; c) in infected and control cells. The tion. To determine the converse, that is, whether host photosynthesis absorption cross-section remained constant, although there was a 10% mayis influenced be involved by phage infection, in maintaining we measured PSII host photochemical photosynthesisdecline in PSII during conversion infec- efficiency. Error bars indicate the s.d. from tion.conversion Here efficiency we used (F v/F mProchlorococcus) and functional cross-sectionalMED4 and area thebiological podovirus replicates. P-SSP7 1Department of Civil and Environmental5 Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2Department of Genetics, Harvard Medical (aSchool, T7-like Boston, Massachusetts phage 02115,)asamodelsystemtobeginexploringthis USA. 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. †Present addresses: The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02141, USA (J.D.J); Department of Oceanography, University of Hawaii, 1000 Pope Road, Honolulu, Hawaii hypothesis.96822, USA (Z.I.J.). We considered that if these genes are involved in host photosynthesis,86 then the amount of phage production might be dependent on photosynthetic performance© 2005 Nature and, Publishi conversely,ng Group host photosynthesis might be compromised by phage infection. We examined the first part of this hypothesis by inhibiting photosyn- Figure 1 | Photosynthesis and phage infection. a, Replication of the phage thesis with darkness or DCMU (3-(3,4-dichlorophenyl)-1,1- genome was assessed by quantifying the phage gene encoding DNA dimethylurea), an inhibitor of electron flow from PSII to PSI, polymerase in host cells kept in the light, transferred to the dark or treated which led to a respective four- or twofold reduction in replication with DCMU while in the light. Significantly lower (P , 0.05) phage DNA 13–15 was detected in dark- and DCMU-treated cells after 4 h. b, c, PSII of the phage genome (Fig. 1a). Thus, as in other systems , photochemical conversion efficiency (F /F ; b) and PSII functional continued photosynthesis is necessary for maximal phage replica- v m absorption cross-sectional area (jPSII; c) in infected and control cells. The tion. To determine the converse, that is, whether host photosynthesis absorption cross-section remained constant, although there was a 10% is influenced by phage infection, we measured PSII photochemical decline in PSII conversion efficiency. Error bars indicate the s.d. from conversion efficiency (F v/F m) and functional cross-sectional area biological replicates. 1Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. †Present addresses: The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02141, USA (J.D.J); Department of Oceanography, University of Hawaii, 1000 Pope Road, Honolulu, Hawaii 96822, USA (Z.I.J.). 86 © 2005 Nature Publishing Group _$2*.%(#,=$G.&,=(&o&CE^ +?&V#.*&$/"&Y?&t?&V#.*3&[_(#2=.-(1*%;3&^*= ."?\3&Q().;& !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE +?&t##"1.))3&cXF_&['#).2:).&#H&*=.&-#/*=\3&0.<%:$%;&ABBB !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Phage phi X 174 was the first DNA- based genome for which the full DNA nucleotide sequence was determined (in 1977, by F. Sanger and collaborators). Its DNA is 5386 nt long, and is wrapped in a circle. The DNA encodes 11 genes, however since it is so short, the genes actually overlap. In 2003, C. Venter and his group assembled the genome of phi X 174 in vitro, from synthetic nucleotides. Its uncompressed genome has been shown to remain fully functional. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE =&+>&..%?&1&>#!"%3!(+.&< _#%/N&CD&>:G:1*&CRB^3&c("G.5()).3&9T3&4F> C^&&&& C^&&&& 8 +(."N&Ch&Y:/.&CREC3&F$)$-$/2$3&F,$(/ T#<.)&]%(K.&(/&X=.-(1*%;&(/&CR^D&P(*=&Y$-.1&_?&F:-/.%&$/"& Y#=/&S?&T#%*=%#,3&mH#%&*=.(%&,%.,$%$*(#/&#H&./K;-.1&$/"&5(%:1& ,%#*.(/1&(/&$&,:%.&H#%-\ S.&%.2.(5."&$&T#<.)&]%(K.&(/&X=.-(1*%;&(/&CR^D&H#%&=(1&P#%J&#/& 9/*%#":2*#%;&_(#,=;1(213&>?&@?&ABCb 8 9/*%#":2*#%;&_(#,=;1(213&>?&@?&ABCb *=.&*#<$22#&-#1$(2&5(%:13&<.G:/&(/&*=.&CRbB1&$/"&P=(2=&=.& r r 2%;1*$))(K."&(/&CRbh?&@"&%>&;)+$!1(!#)+%)A%!"&%;).&,-.(1% '1)'&1!#&$%)A%!"&%B#1-$%*(B&%#;'&!-$%!)%(%+&C%1&$&(1,"% (''1)(,"%#+%B#1).)* !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE protein tiles. protein coat with about 2130 small bases RNA strand is approximately 6400 It is an RNA virus, and the internal the first virus to be identified, in 1898. The Tobacco Moisaic Virus (TMV) was -long, and is protected by a from W. M. Stanley’s Nobel Lecture (1946) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 from W. M. Stanley’s Nobel Lecture (1946) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Plant Pathology: Problems and Progress, ray Diffraction." In 461. - - (Holton, C. S., ed.), University of Wisconsin Press, Madison, 1958 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 from Franklin, R. E., Caspar, D. L. D. and Klug, A. (1959) "The Structure of Viruses as Determined by X 1908 - Wisconsin, pp. 447 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE , !! "# $% & % & & "' " ( ) %$ " !! " * !! " + % , -%& + #+ ' & + # .+ +! ) + % " + # % " # % " # %$ ./ 0 % / 0, % %& +! 1 " !! ( ! * " %& + ! %$ " %# " * + % " % 2* !!3 # $ " , " # - $ %&& &&&$ ' (& )&& *+ , + - Exp Fluids (2009) 46:477–484 DOI 10.1007/s00348-008-0573-6 RESEARCH ARTICLE0 Flow visualization using tobacco mosaic virus " # 3);&!#;&$%David L. Hu ·ThomasB#1-$&$%,(+%8&%E$&&+FGGG J. Goreau ·John W. M. Bush . " ' +% ) ( ' . $ Received: 26 May 2005 / Revised: 15 September 2008 / Accepted: 17 September 2008 / Published online: 7 October 2008 ' Ó Springer-Verlag 2008 " ! Abstract A flow visualization technique using dilute the birefringent material is placed between crossed polar- solutions of tobacco mosaic/ virus (TMV) is described. izers; the transmission of light generates interference fringe Rod-shaped TMV-particles align with shear, an effect that patterns whose intensities are related to the magnitude of produces a luminous interference pattern when the TMV the strain rate of the material (Wayland 1960). Physically, solution is viewed between crossed polarizers. Attractive such birefringence results from stretching, deformation or features of this technique are that it is both transparent to orientation of chiral polymer-like chains or suspended % "& the naked eye and benign to fish. We use it here to visualize macromolecules (Pih 1980). The effect was used exten- $ ! the evolution and decay of! the flows that they produce. We sively in photoelastic stress analysis in solids, recently 012 & + * ( also report that dilute solutions of Kalliroscope are mod- employed by the biolocomotion community to visualize the erately birefringent and so may similarly be used for forces applied by crawling cockroaches (Full et al. 1995) qualitative in situ flow visualizations. and burrowing worms (Dorgan et al. 2005). Maxwell (1873) developed an analogous technique to visualize fluid !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCDmotion,8CE which became the subject of intense study in the 1950s. Here, we report on a plant-virus based birefringent 1 Introduction technique that may ultimately prove useful to workers in biolocomotion and microfluidics. For many years, engineers have known that certain fluids Reviews of birefringence from the perspective of a and solids become temporarily birefringent or doubly chemist, fluid mechanician and engineer are given by Cerf refractive (able to refract light in two different directions) and Scheraga (1952), Peterlin (1976 ) and Pih (1980), when subjected to shear. This effect is best observed when respectively, and are summarized here. Birefringence was first used to ascertain the shape of particles in colloidal ! Electronic supplementary material The online version of this suspensions (Diesselhorst and Freundlich 1916). Freundlich article (doi:10.1007/s00348-008-0573-6) contains supplementary (1926) found that anisotropic particles such as rods or disks material, which is available to authorized users. align with their long axis parallel to shear. A solution of such particles is birefringent if two conditions are satisfied: the D. L. Hu (&) Department of Mechanical Engineering, particle’s short axis is shorter than the wavelength of light Georgia Institute of Technology, Atlanta, GA, USA and the particle’s refractive index differs from that of the e-mail: [email protected]; [email protected] surrounding solution. Solutions of rods and disks become birefringent under different lighting conditions: specifically, T. J. Goreau Global Coral Reef Alliance, 37 Pleasant Street, for rods, the incident light must be normal to the flow Cambridge, MA 02139, USA direction; for disks, the incident light must be parallel to both the direction of flow and the disk face. Further inves- J. W. M. Bush tigations showed that many synthetic chemicals are Department of Mathematics, Massachusetts Institute of Technology, birefringent, including colloidal suspensions of vanadium Cambridge, MA 02139, USA pentoxide (Humphrey 1922), milling dianthrapyrimidine 123 Exp Fluids (2009) 46:477–484 479 Fig. 2 The experimental apparatus. A dilute suspension of TMV is illuminated with polarized light, and viewed through a crossed polarizer. The arrangement for viewing (a) from the side, and (b) from above Exp Fluids (2009) 46:477–484 479 c 1 c2 24bFig.2 2 The experimental v s 1 s E2 3 ¼À12D À 27D2 4 þ 35apparatus. A dilute suspensionð Þ 2 of TMV is illuminated with where cs =2E sin K0 is the shear rate parallel to the streamline. The theory (1)–(3)polarized has been confirmed light, and exper- viewed imentally by Sutera (1960).through In addition, a crossed a number polarizer. of The investigators have proposed theoriesarrangement allowingfor them viewing to use (a) birefringent data to gather quantitativefrom the information. side, and Binnie (b) from (1945) used the birefringent solutionabove benzopurpurin asEdoardo Milotti a - Introductory biophysics - A.Y. 2016-17 turbulence indicator in pipe flow: he finds that the greatest chromatic intensity appears at Reynolds numbers 1,970– 2,900. Pindera and Krishnamurthy (1978) used lasers to determine the properties of a birefringent fluid. Prados and Peebles (1958) characterized the laminar flow around a cylinder, and Alcock and Sadron (1935) the flow between Fig. 3 A black neon tetra fish flaps its fins in a TMV solution. The pairs of plates and between rotating cylinders. These luminous areas are indicative of the fluid shear generated by its fins studies show that birefringent fluids can be used in a and inhalation by its mouth. Scale bar 1 cm quantitative manner. We now turn to a relatively new application of TMV, its use in visualizing animal-generated camera (Sony, DSC-F707). We obtained side views using a flows. glass tank (length 30 cm, span 1.5 cm, water depth 10 cm, Fig. 2a) and top views using a petri dish (diameter 14 cm, depth 2 cm, Fig. 2b) filled with the TMV solution 2 Experiments (Fig. 2b). Black neon tetra, common tropical fish, were obtained 2 2 We filled the apparatus in Fig. 2 with TMVcs solution1 to fromcs our24 localb pet2 store in New York and housed together v 1 in a filtered, oxygenated,E heated aquarium under3 a 12 h visualize the flows generated by¼À black12 neonD tetraÀ 27 fishD2 4 þ 35 ð Þ (Figs. 3, 4, 5, 6, 7), jets (Fig. 8) and rising bubbles (Fig. 9) light cycle. Fish housing and experiments were approved generated by a syringe. The apparatus was housed in a2 by the NYU committee on animal welfare (protocol num- darkened room, with lighting providedwhere bycs a 250=2 WE tung-sin K0beris 07-1289). the shear While the rate plant parallel virus TMV to has neverthe been sten–halogen lamp (Vision Research,streamline. Inc., North The Star) theoryreported (1)–(3) to has infect been animal confirmed hosts, we took exper- precautions reflected off a white sheet of paper. The birefringent fluid by immersing the fish in a low concentration of TMV was sandwiched between twoimentally crossed linear by polarizers Sutera(18 (1960 parts/million)). In addition, for a short duration a number (min/trial). of At the (polarizing film, 40 by 30 cminvestigators by Edmund Industrial have proposedconclusion theories of the experiments, allowing the fish them appeared to use unaffected Optics; Techspec polarizing lensbirefringent 58 and 37 mm bydata Crystal to gatherby the quantitative TMV solution and information. continued in good Binnie health until the Vision). The color of the fluid, as observed by the polarizer end of their natural lives nearly a year later. farthest from the light source,(1945 was recorded) used by the a digital birefringentThe preparation solution of TMV benzopurpurin for use in flow visualization as a is video recorder (Sony, DCR-TRV950)turbulence and indicator digital still indescribed pipe flow: by Boedtker he finds and thatSimmons the (1958 greatest). Our TMV chromatic intensity appears at Reynolds numbers 1,970– 2,900. Pindera and Krishnamurthy (1978) used lasers123 to determine the properties of a birefringent fluid. Prados and Peebles (1958) characterized the laminar flow around a cylinder, and Alcock and Sadron (1935) the flow between Fig. 3 A black neon tetra fish flaps its fins in a TMV solution. The pairs of plates and between rotating cylinders. These luminous areas are indicative of the fluid shear generated by its fins studies show that birefringent fluids can be used in a and inhalation by its mouth. Scale bar 1 cm quantitative manner. We now turn to a relatively new application of TMV, its use in visualizing animal-generated camera (Sony, DSC-F707). We obtained side views using a flows. glass tank (length 30 cm, span 1.5 cm, water depth 10 cm, Fig. 2a) and top views using a petri dish (diameter 14 cm, depth 2 cm, Fig. 2b) filled with the TMV solution 2 Experiments (Fig. 2b). Black neon tetra, common tropical fish, were obtained We filled the apparatus in Fig. 2 with TMV solution to from our local pet store in New York and housed together visualize the flows generated by black neon tetra fish in a filtered, oxygenated, heated aquarium under a 12 h (Figs. 3, 4, 5, 6, 7), jets (Fig. 8) and rising bubbles (Fig. 9) light cycle. Fish housing and experiments were approved generated by a syringe. The apparatus was housed in a by the NYU committee on animal welfare (protocol num- darkened room, with lighting provided by a 250 W tung- ber 07-1289). While the plant virus TMV has never been sten–halogen lamp (Vision Research, Inc., North Star) reported to infect animal hosts, we took precautions reflected off a white sheet of paper. The birefringent fluid by immersing the fish in a low concentration of TMV was sandwiched between two crossed linear polarizers (18 parts/million) for a short duration (min/trial). At the (polarizing film, 40 by 30 cm by Edmund Industrial conclusion of the experiments, the fish appeared unaffected Optics; Techspec polarizing lens 58 and 37 mm by Crystal by the TMV solution and continued in good health until the Vision). The color of the fluid, as observed by the polarizer end of their natural lives nearly a year later. farthest from the light source, was recorded by a digital The preparation of TMV for use in flow visualization is video recorder (Sony, DCR-TRV950) and digital still described by Boedtker and Simmons (1958). Our TMV 123 >&1(/J&I$L&$/"&$&1#:%2.&I<3&2L&G./.%$*."&<;&$&C8--8"($-.*.%&1;%(/G.&(/1.%*."&(/*#&$&*$/J& #H&"():*.&7'V&1#):*(#/?& I$L 7=.&P=(*.&$%%#P1&"./#*.&*=.&"(%.2*(#/&#H&*=.&H)#P?& I F2$).&<$%&C&2- !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Exp Fluids (2009) 46:477–484 479 Fig. 2 The experimental apparatus. A dilute suspension of TMV is illuminated with polarized light, and viewed through a crossed polarizer. The arrangement for viewing (a) from the side, and (b) from above c 1 c2 24b2 v s 1 s E2 3 ¼À12D À 27D2 4 þ 35 ð Þ 2 where cs =2E sin K0 is the shear rate parallel to the streamline. The theory (1)–(3) has been confirmed exper- imentally by Sutera (1960). In addition, a number of investigators have proposed theories allowing them to use birefringent data to gather quantitative information. Binnie (1945) used the birefringent solution benzopurpurin as a turbulence indicator in pipe flow: he finds that the greatest chromatic intensity appears at Reynolds numbers 1,970– 2,900. Pindera and Krishnamurthy (1978) used lasers to determine the properties of a birefringent fluid. Prados and Peebles (1958) characterized the laminar flow around a cylinder, and Alcock and Sadron (1935) the flow between Fig. 3 A black neon tetra fish flaps its fins in a TMV solution. The pairs of plates and between rotating cylinders. These luminous areas are indicative of the fluid shear generated by its fins studies show that birefringent fluids can be used in a and inhalation by its mouth. Scale bar 1 cm quantitative manner. We now turn to a relatively new application of TMV, its use in visualizing animal-generated camera (Sony, DSC-F707).Edoardo Milotti - Introductory biophysics We obtained- A.Y. 2016 side-17 views using a flows. glass tank (length 30 cm, span 1.5 cm, water depth 10 cm, Fig. 2a) and top views using a petri dish (diameter 14 cm, depth 2 cm, Fig. 2b) filled with the TMV solution 2 Experiments (Fig. 2b). Black neon tetra, common tropical fish, were obtained We filled the apparatus in Fig. 2 with TMV solution to from our local pet store in New York and housed together visualize the flows generated by black neon tetra fish in a filtered, oxygenated, heated aquarium under a 12 h (Figs. 3, 4, 5, 6, 7), jets (Fig. 8) and rising bubbles (Fig. 9) light cycle. Fish housing and experiments were approved generated by a syringe. The apparatus was housed in a by the NYU committee on animal welfare (protocol num- darkened room, with lighting provided by a 250 W tung- ber 07-1289). While the plant virus TMV has never been sten–halogen lamp (Vision Research, Inc., North Star) reported to infect animal hosts, we took precautions reflected off a white sheet of paper. The birefringent fluid by immersing the fish in a low concentration of TMV was sandwiched between two crossed linear polarizers (18 parts/million) for a short duration (min/trial). At the (polarizing film, 40 by 30 cm by Edmund Industrial conclusion of the experiments, the fish appeared unaffected Optics; Techspec polarizing lens 58 and 37 mm by Crystal by the TMV solution and continued in good health until the Vision). The color of the fluid, as observed by the polarizer end of their natural lives nearly a year later. farthest from the light source, was recorded by a digital The preparation of TMV for use in flow visualization is video recorder (Sony, DCR-TRV950) and digital still described by Boedtker and Simmons (1958). Our TMV 123 !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE CRISPR CRISPR stands for “clustered regularly interspaced short palindromic repeats”, and it is a sort of immune system in bacteria. Bacteria use CRISPR sequences, stored in their genome, to identify attacking viruses. They are composed of many small pieces of viral DNA harvested from viruses that attacked in the past, separated by a distinctive repeated sequence used to create the archive. Remarkably, new sequences are added at the beginning of this collection, so we can read the CRISPR sequence to get a history of viruses that have attacked the bacterial population in the past. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 A system of Cas proteins (short for "CRISPR-associated proteins") use this stored information to fight the viruses if they try to infect the bacteria again. The center of this system is the large complex Cascade. It carries an RNA transcript of the CRISPR sequence and searches through the cell for matching viral DNA from an infection. If it finds viral DNA, it unwinds it and mobilizes nucleases to cut it up. The structure shown here includes the Cascade surveillance complex composed of 6 different types of proteins (in different shades of blue) along with the RNA transcript (red) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Cascade works with a team of proteins to build the CRISPR archive and use it to protect against viruses. First, recruiting proteins like Cas1 and Cas2 are needed to chop up viruses during an infection and save appropriately-sized bits in the CRISPR. These pieces are then displayed by Cascade to search for later infections by the virus. If the virus is found, Cas3 is the executioner, taking the infecting viral DNA found by Cascade and destroying it. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Different bacteria and archaea have evolved a number of variations on this general theme. The Cascade complex, termed Type I, displays CRISPR RNA and recruits the executioner Cas3 to chop up the viral DNA. Type III complexes, on the other hand, have the DNA-cutting enzyme as part of the complex. Type II CRISPR systems, such as Cas9 have a surveillance protein and executioner all wrapped up in a single protein chain. The complex shown here includes the CRISPR RNA (red) along with a piece of an attacking viral DNA (yellow). This molecule has recently been used in an experimental approach to curing latent HIV infection. An engineered virus was used to insert Cas9 and an anti-HIV CRISPR into HIV-infected cells, which then chopped up the integrated viral DNA. (from http://pdb101.rcsb.org/motm/181) 0%#-&!?&g?&Q()1#/u1&[k(H.&#/&!$%*=\ !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE 3&k?&Q?& !(1.%)(/G 3&+?&S?&S$))3&0?&>?& 3&t?& '#1(G 3&X?&X$%)1#/3&$/"&!?&F?&'()).%&I."?L?&CRR^?&'#).2:)$%&<(#)#G;& i%.:K.% 3&Y?&+?3&Y?&Q?&+%$J.3&i?&T?& i$%$- H%#-& i:**.% _)$2J3&!?&i?&F,(2.%3&!?& #H&<$2*.%(#,=$G.&7^?&>-.%(2$/&F#2(.*;&H#%&'(2%#<(#)#G;3&Q$1=(/G*#/3&+?X? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 (1968) 18 32 The Caltech Phage Group”, Engineering and Science from R. S. Edgar and W. B. Wood, “Genetics and Development at the Threshold of Life – Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 !).2*%#/&-(2%#G%$,=1&#H&<$2*.%(#,=$G.&7^?&I>L&!W*./"."&*$()&H(<.%1&%.2#G/(K.&*=.&<$2*.%($)&./5.)#,.3&$/"& (*1&,%#)$*. (2#1$=."%$)&=.$"&2#/*$(/1&*=.&CDl3RBb8<,&"1+T>&G./#-.?&c.,%(/*."&P(*=&,.%-(11(#/&#H&'?& Q:%*K3&_(#K./*%:-3&_$1.)3&FP(*K.%)$/"?&I_L&7=.&+T>&G./#-.&(1&".)(5.%."&(/*#&*=.&=#1*&*=%#:G=&*=.& (/*.%/$)&*$()&*:<.3&P=(2=&(1&5(1(<).&,%#*%:"(/G&H%#-&*=.&./"&#H&*=.&2#/*%$2*."&*$()&1=.$*=? I(-$G.1&$/"&*.W*&*$J./&H%#-&!?&F?&'()).%&.*&$)?N&[_$2*.%(#,=$G.&7^&G./#-.?\3&'(2%#<(#)?&'#)?&_(#)?&c.5?&MN IABBbL&lDL !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE The steps of T4 bacteriophage invasion: • phage attaches to E. coli using its six fibers at end of tail • tail enters wall of host (cell wall is degraded using lysozyme from a previous infection) • viral DNA is injected into the host’s cytoplasm (time: 0) • among the early proteins produced are a repair enzyme to repair the hole in the bacterial cell wall, a DNAase enzyme that degrades the host DNA into precursors of phage DNA, and a virus specific DNA polymerase that will copy and replicate phage DNA. (time: +8 min) • synthesis of early protein stops and synthesis of late proteins starts. The late proteins are mainly structural proteins that make up the capsids and the various components of the tail assembly. (time: +13 min) • first phage particle is complete (time: +23 min) • part of the produced lysozyme (a late protein) is used to destroy the cell wall and the infected cell bursts (lysis) and releases more than 100 virus particles (time: +35 min) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 ?(S%H&.81T,/ _#%/N&^&F.,*.-<.%&CRBD3&_.%)(/3&t.%-$/; +(."N&R&'$%2=&CRlC3&]$1$"./$3&X>3&4F> T#<.)&]%(K.&(/&]=;1(#)#G;&#%&'."(2(/.&(/&CRDR&I1=$%."& P(*=&>)H%."&S.%1=.;&$/"&F$)5$"#%&k:%($LN&mH#%&*=.(%& "(12#5.%(.1&2#/2.%/(/G&*=.&%.,)(2$*(#/&-.2=$/(1-&$/"&*=.& G./.*(2&1*%:2*:%.&#H&5(%:1.1\ _#%/&(/&_.%)(/3&=.&=$"&$/&(/*.%.1*&(/&12(./2.&1(/2.& <#;=##"?&S.&1*:"(."&$1*%#,=;1(21&$/"&*=./&-#5."&*#& *=.#%.*(2$)&,=;1(21&":%(/G&=(1&G%$":$*.&1*:"(.1&$*& tv**(/G./?&+:%(/G&=(1&,#1*8"#2&(/&!/G)$/"3&FP(*K.%)$/"& $/"&+./-$%J3&=.&2#/*%(<:*."&*#&*=.&<:%G.#/(/G&*=.#%;&#H& n!+3&$/"&P$1&(/&2#/*$2*&P(*=&12(./*(H(2&G($/*1&1:2=&$1&]$:)(& $/"&_#=%?& +.)<%w2J&-#5."&*#&_.%)(/&(/&CRbA&P=.%.&=.&<.2$-.& $11(1*$/*&*#&k(1.&'.(*/.%?&S.&$%%(5."&<;&*%$(/&#/&*=.& -#%/(/G&#H&_#=%u1&#,./(/G&$""%.11&[k(G=*&$/"&k(H.\3&$& ).2*:%.&P=(2=&2=$/G."&*=.&2#:%1.&#H&=(1&)(H.? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE In Berlin he had further contacts with the local biologists. “Paradoxically, this good intention [having contacts with the biologists] was helped by the rise of Nazism which made official seminars less interesting. A small group of physicists and biologists began to meet privately beginning about 1934. To this group belonged N. W. Timofeeff-Ressovsky (genetics). Out of these meetings grew a paper by Timofeeff, Zimmer, and Delbrück on mutagenesis. A popularization of this paper of 1935 in Schroedinger's little book «What is Life?» (1945) had a curiously strong influence on the development of molecular biology in the late 1940's.” (from the biographical press release for the Nobel prize in Physiology or Medicine awarded to Delbrück in 1969) Throughout his research Delbrück cultivated the hope of finding some aspect of life not reducible to the basic physico-chemical laws, a kind of “complementarity of life” that would push further the concept put forward by Bohr. What he left is a lasting contribution to our understanding of the mechanisms of heredity, and – paradoxically indeed – a framework that seems to bar new physics laws, and effectively utilizes the existing ones. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Delbrück (standing) and Luria in 1941 Petri dish with colonies Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 The experiment of Luria and Delbrück (Genetics 28 (1943) 491). After exposure to phages only few bacteria survive ...is resistance acquired or does it arise from random mutations? Random mutations: resistance Adaptive heritable resistance: virus arises randomly in previous action induces resistance which is generations inherited by offspring phage injection Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Exponential population growth: Bacteria proliferate with average duplication time T, thus the population size at time t is t T ⎛ t ⎞ N(t) = 2 N = N exp ln2 0 0 ⎝ T ⎠ T Luria and Delbrück chose time units such that = 1, and therefore ln2 dN N(t) = N et = N(t) 0 dt Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 A. Hypothesis of acquired immunity Let p be the probability of acquiring immunity after exposure to phages, then the number of bacteria that acquire immunity is a binomial variate. However from experiment we know that few bacteria survive the attack, so p must be small and the binomial distribution can be approximated by a Poisson distribution: therefore – with this hypothesis – the variance of the number of resistant bacteria is equal to the average. Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 In the case of random mutations we expect a much larger variance Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Simulation of Luria-Delbrück experiment 1200 10 generations a = 0.01 1000 800 600 400 number of plates 200 0 0 200 400 600 800 number of resistant colonies Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 B. Hypothesis of random mutations Let a be the rate of mutation that endows resistance: p = a t probability of mutation in time interval Δt m N(t) p = N eta t number of mutated cells in the same ⇡ 0 time interval The number of mutated cells in this time interval is a Poisson variate, therefore var m = m Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Average population of resistant bacteria dρ = [N(t) − ρ(t)]adt + ρ(t)dt new mutations in the time exp growth of the population of interval dt resistant bacteria dρ = N (t)adt + (1− a)ρ (t)dt ≈ N t adt + ρ t dt a ≪1 approximate ( ) ( ) ( ) differential equation Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 The approximate differential equation d⇢ ⇢ = aN(t) dt can be transformed into t d t t e e ⇢ = aN e dt 0 and therefore the mean value of mutated cells is t t ⇢(t)=aN0te + Ce with ⇢(0) = 0 ⇢(t)=atN(t) ) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Variance of the population of mutated cells: ⇢ = ⇢(t) t + aN(t) t m ⇡ var ⇢ var m = m ⇡ the total variance is the sum of the amplification due to variances of independent increments population growth t t t t0 2(t t0) var ⇢(t)= var e d⇢(t0) e dm(t0) 0 ⇡ 0 Z t ⇣ ⌘ Z t 2t 2t0 t0 2t t0 = e e aN0e dt0 = aN0e e dt0 0 0 Z 2t t Z = aN e (1 e ) 0 = aN(t)(et 1) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Variance of the number of resistant bacteria 2 t [N(t)] var ⇢(t) aN(t)(e 1) a t ⇡ ⇡ N0 [N(t)]2 var [⇢(t)] a ⇡ N0 a 1/2 var [⇢(t)] N(t) ⇡ N ✓ 0 ◆ p Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 N (t) ρ (t) ≈ atN (t) = aN (t)ln N0 N (t)2 var ρ (t) ≈ a N0 var ρ (t) 1 1 ~ ≠ ρ (t) ln N (t) ρ (t) Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 k:%($&$/"&+.)<%w2J #<1.%5."&*=$*&*=.&5$%($/2.&#H&*=.&/:-<.%&#H& %.1(1*$/*&<$2*.%($&(1&(/".."&`:(*.&)$%G.&$/"&2#/H(%-1&*=.&=;,#*=.1(1& #H&%$/"#-&-:*$*(#/1& !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE The winners of the 1969 Nobel Prizes stand together before the award presentation by King Gustaf VI Adolf, of Sweden. Left to right are Professor Murray Gell-Mann of the U.S., in physics; Professor Derek H.R. Barton of the U.K., in chemistry; Professor Odd Hassel of Norway, in chemistry; Professor Max Delbruck of the U.S., in medicine; Alfred D. Hershey, of the U.S., in medicine; Salvador E. Luria of the U.S., in medicine; and Professor Jan Tinbergen of the Netherlands, in economics.Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Previous research has shown that genetically modifying the virus to introduce cysteine residues (amino acid with thiol groups) in its coat proteins significantly enhances metal deposition, which is attributed to covalent-like bonds formed between the thiol groups of the cysteines and various metal surfaces ... ... A complementary DNA clone is produced from the wild-type viral RNA, and the specific cysteine codon is introduced in the third position of the coat protein open reading frame. Following this process, RNA transcripts are used to infect the host plant which acts as the factory for mass production of the virus, and finally the TMV1cys is harvested 20 days after inoculation ... Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 Edoardo Milotti - Introductory biophysics - A.Y. 2016-17 !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE F2=.-$*(2&%.,%.1./*$*(#/&#H&*=.&7'V&$11.-<);&$/"&/(2J.)&2#$*(/G&,%#2.11N&*=.&7'V&<(/"1&#/& *=.&G#)"&1:%H$2.&I1*.,&CL3&(*&(1&$2*(5$*."&P(*=&$&,$))$"(:-&2$*$);1*&I1*.,&AL&$/"&(*&(1&H(/$));& 2#$*."&P(*=&/(2J.)&I1*.,&bL? F!'&(-$G.1&$*&*P#&-$G/(H(2$*(#/1&#H& /(2J.)&.).2*%#".1&P(*=&7'V&2#$*(/G1? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE F!'&(-$G.&#H&T(O7(gA&/$/#2#-,#1(*.& .).2*%#".&I*#,L3&2%#1181.2*(#/&7!'& (-$G.&#H&$/&(/"(5(":$)&/$/#%#"& 1=#P(/G&*=.&2#%.O1=.))&/$/#1*%:2*:%.& X%."(*N&4/(5.%1(*;&#H&'$%;)$/"3&X#)).G.& ]$%J 0(/$));&<$**.%(.1&$2=(.5.&$&CB8H#)"& 2$,$2(*;&(/2%.$1.? !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE !"#$%"#&'()#**(&8 9/*%#":2*#%;&<(#,=;1(21&8 >?@?&ABCD8CE Edoardo Milotti - Introductory biophysics - A.Y. 2016-17