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Uncatalyzed assembly of spherical particles from SV40 VP1 pentamers and lineardsDNA incorporates both low and high cooperativity elements
Santanu Mukherjee a, Stanislav Kler b, Ariella Oppenheim b, Adam Zlotnick a,c,⁎a Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USAb Department of Hematology, Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israelc Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47401, USA
⁎ Corresponding author. Department of Molecular andUniversity, Bloomington, IN 47401, USA. Fax: +1 812 8
The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined thesynergism between pentamer–pentamer interaction and pentamer–DNA interaction using a minimal systemof purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and sizeexclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did notsurvive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migrationwas slower and tubes were replaced by 200 Å diameter particles and excess free pentamer. At high VP1/DNAratios, a progressively larger fraction of particles was similar to 450 Å diameter virions. VP1 association withDNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexesappear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation eventbased on non-specific pentamer–DNA interaction that controls assembly and the ultimate capsid geometry.
Simian virus 40 (SV40), a member of the polyomaviridae, is a non-enveloped DNA virus with 5.2 kb double stranded DNA genome that iscompacted by histones. The virus capsid is composed of threestructural proteins VP1, VP2, and VP3. VP1, the major capsid protein,forms the contiguous capsid of SV40. Similar to papillomaviruses,SV40 capsids are composed of 72 pentamers of VP1 arranged on aT=7 icosahedral lattice, where pentamers fill both pentavalent andhexavalent sites (Baker et al., 1991; Rayment et al., 1982; Stehle et al.,1996). The architecture of VP1 is the eight stranded antiparallel betabarrel common to many small non-enveloped icosahedral viruses(Rossmann and Johnson, 1989). Stable pentamers of VP1 have beenexpressed and purified from virions and several expression systems(Kosukegawa et al., 1996; Sandalon and Oppenheim, 1997). VP2 andVP3 are encoded by the same open reading frame, except VP2transcription begins at an earlier start codon and includes all of VP3 asits C-terminal domain. VP2/VP3 is located on the interior of the capsidin a concavity at the VP1 fivefold axis.
Empty capsids can be assembled in response to low pH, ionicstrength, calcium, and/or an oxidizing environment (Ishizu et al., 2001;Sandalon and Oppenheim, 1997). The pentamer–pentamer interactionsinvolve exchange of C-terminal segments (Liddington et al.; Stehle etal.). However, assembly can be influenced by DNA in the absence of
Cellular Biochemistry, Indiana56 5710.
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chaperones (Tsukamoto et al., 2007). Though VP2 and VP3 are oftenreferred to as DNA-binding proteins, all three capsid proteins bind DNAnonspecificallywith high affinity (Clever, Dean, andKasamatsu; Li et al.;Soussi). VP1 binds DNA with a nanomolar dissociation constant, aninteraction believed to be mediated by the N-terminal 15 amino acids(Clever et al., 1993; Li et al., 2001).
It has been suggested from in vitro studies that regulated capsidassembly is generally a chaperone-assisted process (Chromy, Pipas,and Garcea; Kanesashi et al.; Mukherjee et al.; Sandalon andOppenheim). However, in this paper we use a simplified system ofVP1 pentamers and DNA without the complication of a catalyticchaperone to identify the roles of protein–protein and protein–DNAinteraction in the disassembly and assembly of virus-like particles(VLPs). We report here that empty VLPs that are formed in vivo areextremely fragile in a low ionic strength, reducing environment.Conversely, VP1 assembly can be regulated by a dsDNA substratethough surprisingly high ratios of VP1 per DNA are required to initiateassembly.
Results
Dissociation of VP1 VLPs
In general, reversibly assembled virus capsids are held together byrelatively weak interactions (Zlotnick, 2003). This generalizationimplies that relatively mild conditions should be sufficient todissociate SV40 virus-like particles (VLPs) to obtain pentamers forreassembly. To facilitate testing conditions, we used 90° light
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scattering to observe dissociation. Light scattering is proportional to theweight-averagemolecularweight of solute. The initial VLP preparationscontain 45 nm diameter particles and 20 nm diameter particles,presumably with T=7 and T=1 symmetry, respectively. Theirdissociation should result in a substantial decrease in light scattering.We found that VP1 VLPs were readily dissociated by treating VLPs with2mMDTT and 5mMEDTA together in a low ionic strength pH8.9 buffer(Fig. 1a). Transmission electron microscopy (TEM) of dialyzed samples(Fig. 1d) suggests that VLPs were completely dissociated to individual“star shaped” pentamers. The fivefold symmetry of some pentamers isevident in micrographs. Dissociation to pentamers is consistent withearlier studies (Kosukegawa et al., 1996; Sandalon and Oppenheim,1997; and references therein).
EDTA alonewas sufficient for breakdown of particles, demonstratedby an 80% decrease in light scattering in 10 min. However, the effect ofDTT and EDTA on dissociation was synergistic. The decrease in lightscattering induced by EDTA without DTT was much slower, even at
Fig. 1. Dissociation of virus-like particles. (a) Dissociation of particles to pentamersobserved by light scattering at 320 nm. Data shown here are 3.7 μMVP1 pentamer. VLPsin 0.1 M NaCl were mixed with dissociation buffer (50 mM Tris–Cl, pH 8.9) containingDTT and/or EDTA at 1:1 ratio. A rapid decrease in light scattering was observed inpresence of 2 mM DTT and 5 mM EDTA, together, and leveled off within 90 s. Bycomparison addition of EDTA alone (final concentration of 25 mM) resulted in muchslower dissociation kinetics. No change of light scattering was observed in presence ofDTT even at high concentrations, 30mM. These light scattering trajectories are averagesof three experiments (standard deviation is b10% at all points). (b) Size-exclusionchromatography VLPs (thick line) and dissociation products using a 20-ml Sephacryl S-500 column. VLPs elute at 10 ml. VLPs were dissociated by dialysis in 20 mM Tris–Cl pH8.9, 50 mM NaCl, 2 mM DTT and 2 mM EDTA. Dissociated material (thin line) ispolydispersed; the major peak, presumably pentamers, elute at 17.5 ml. The peak at theend of the chromatograph is due to salt in the VLP buffer. DTT adheres to the columnpacking and elutes after the salt schlieren peak. (c and d) Negative stained transmissionelectron micrographs of SV40 VP1 before and after dissociation, respectively.
higher EDTA concentrations (25 mM) (Fig. 1a). We did not observe anychange in light scattering with DTT alone up to a concentration of30mM. This result is consistentwith the assertion that calcium is criticalto VLP stability (Colomar et al., 1993; Liddington et al., 1991; Sandalonand Oppenheim, 1997; Stehle et al., 1996). The limited effect of DTT issurprising. If a large fraction of possible disulfides were formed, thecapsidwould be covalently crosslinked and resistant to dissociation.Wesurmise that the contribution of S–S bonds to capsid stability issignificantly less than that of the C-arms and Ca++ and that only afraction of possible disulfide bonds are present in a given capsid.
Size-exclusion chromatography (SEC) of dissociation reactionswas consistent with EM and light scattering data. In dissociationbuffer, a single pentamer peak was seen with no evidence of VP1monomer or capsid peaks (not shown). For subsequent assemblyexperiments, VP1 pentamers were prepared by dialyzing VLPs againstdissociation buffer (20 mM Tris HCl, pH 8.9, 50 mM NaCl, 2 mM DTTand 5 mM EDTA). However, the high pH of dissociation buffer wasnecessary to minimize aggregation. At neutral pH (20 mM MOPS, pH7.2, 150 mM NaCl) samples of pentamer showed a distinct pentamerpeak and a broad peak corresponding to larger complexes, indicativeof re-association and/or aggregation (Figs. 1b). However, no VLP wasobserved in these samples by TEM (see Figs. 1b–d). Dissociation topentamers is consistent with earlier work on polyomaviruses(Friedmann, 1971; Kosukegawa et al., 1996).
We examined assembly of empty particles induced by Ca++ (5 to30 μM), lowpH(pH4.6 to 7), andhigh concentrations of (NH4)2SO4 (0.15to 2.0M). In all three cases, beyond a threshold (≥10 μMCa++,≤pH 4.8,≥0.75 M (NH4)2SO4) assembly kinetics were extremely fast, based onlight scattering, and led to heterogeneous assembly products (T=1capsids,T=7capsids, irregular particles, tubes, and aggregates) based onelectron microscopy and size exclusion chromatography (data notshown). With Ca++ in particular there was substantial aggregation,results that are consistent with earlier studies (Kanesashi et al., 2003;Salunke et al., 1986, 1989).More importantly, in all three cases therewasa sharp break between conditions where there was no assembly andwhere 100% of the VP1was assembled into one polymer or another. Theabsence of unreacted pentamer, the speed of the reaction, theheterogeneity of products, and the tendency to aggregate are allhallmarks of kinetically trapped reactions (Endres and Zlotnick, 2002;Johnsonet al., 2005; Zlotnick, 1994), thuswe feltwewereunlikely to gainfurther insight into SV40 assembly paths by re-examination of thesereactions. In contrast, the structurally related papillomaviruses showwell-behaved in vitro assembly kinetics (Casini et al., 2004;Mukherjee etal., 2008).Wenote that these threemeans of inducing SV40 assembly arelikely to proceed by the same mechanism: minimizing electrostaticrepulsion of carboxyl groups at the Ca++ binding site.
VP1–DNA interaction
We wanted to test the hypothesis that DNA could contribute toassembly by raising the local concentration of VP1 and/or by acting asscaffold to restrain the geometry of VP1 interactions. To minimize therole of strong and essentially irreversible interactions on DNA-mediated assembly, we examined protein–DNA interactions underconditions where capsids do not form on their own: 50 mMMOPS pH7.2, 150 mM NaCl, 2 mM DTT, with no calcium. To minimize the role(s) of higher-order DNA structure on assembly, we used a 600 bp DNApolymer (50 nM polymer or 30 μM Bp) as a substrate for titrationswith VP1 pentamer. Pentamer–DNA interaction was observed by acombination of native agarose gel electrophoresis and electronmicroscopy.
We anticipated that VP1 would retard migration of DNA throughthe gel (Johnson et al., 2004; Mukherjee et al., 2006). At low ratios ofVP1/DNA, less than six pentamers per polymer, we observed nochange in DNA migration (Fig. 2a). In contrast to this result, electronmicrographs showed numerous long tubes with very little free
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pentamer in the background (Figs. 2b, c). Tube diameter was variablebut centered around 40 nm. Over a broad range of low ratios (notshown) we observed no change in the intensity or migration of theDNA band in agarose gels. Because long nucleoprotein tubes do notgenerally enter a gel while there was no decrease in the DNA bandfluorescence, we suggest that these large structures do not surviveelectrophoresis. There is a relatively sharp transition at about sixpentamers per polymer above which no tubes were visible inmicrographs. At ratios from about 6 to 18 pentamers per polymerthere was a noticeable broadening of the DNA band in the gel andmicrographs were dominated by amixture of free pentamer and small20 nm diameter particles (Fig. 2d). As the concentration/saturation ofVP1 increased, binding appeared to show strong positive cooperativ-ity. Amarked change in the rate of migrationwas observed at the ratioof 36:1 with a major band migrating halfway between the well andfree DNA and a minor band migrating at a much slower rate,coincident with the appearance of 45 nm diameter particles in a densebackground of pentamer mixed with 22 nm particles (Fig. 2g). At aratio of 72:1 the change in migration approached saturation with theslowest migrating band prevalent and electron micrographs weredominated by 45 nm particles with a dense background of pentamer(Fig. 2h). Further increase in the ratio did slow the migration of thebroadly smearing band and eliminated a number of fast migrating,weakly staining, minor bands. Micrographs of the higher ratioreactions were qualitatively similar to the 72 pentamer per DNAreaction except with a denser background of pentamer. A workinginterpretation of the gel is that 22 nm particles generate the band thatis prevalent in the 36:1 lane and is faintly visible in lower (down to12:1) and higher ratios. The ethidium bromide staining of this bandimplies that at least some of the 22 nm particles have DNA.
DNA substrate as used in these studies was to support assemblywithout introducing the issue of DNA quaternary structure. The lengthof the substrate needed to obtain assembly was determinedempirically. Shorter double stranded DNA oligomers of 28, 43, and66 base pairs did not support particle formation or an electrophoreticmobility shift. A 100 bp oligomer supported a shift in gel migration atsimilar ratios of VP1 per oligomer (i.e. six times the ratio of VP1 perbasepair), but stained relatively poorly. Particles based on the 100-mer were not observed in TEM, but that may be a function of thebackground of excess pentamer. On the other hand, much longerrelaxed plasmid DNA yielded results that were substantially identicalto those using the 600-mer.
VP1–DNA stoichiometry of T=7 reassembled particle
How much dsDNA was packaged in these in vitro assemblyexperiments? Normal SV40 capsids contain a highly compacted 5.2 kbviral minichromosome, but plasmids as large as 17 kb can be packagedin vitro (Kimchi-Sarfaty et al., 2003). Here we are packaging a stifflinear DNA polymer that is approximately 200 nm long into a capsidwith an internal diameter of less than 40 nm. While the DNA polymeris short compared to the length of the chromosome, it is four times the50 nm persistence length of dsDNA and five times the internaldiameter of the T=7 capsid. To address the question of how the DNAis accommodated within these SV40 capsids we determined the
Fig. 2. Assembly of VP1 on DNA. (a) Titration of a 600 bp dsDNA by VP1 pentamers.Reactions of 50 nM DNA 600-mer with various VP1 pentamer to DNA molar ratios in50mMMOPS, pH 7.2, 150mMNaCl were equilibrated for 24 h prior to separation on by0.6% agarose gel electrophoresis. The ethidium bromide stained gel shows migration ofthe 600 bp DNA band is affected by increasing VP1. Molecular weight markers areshown on the left lane (marked as M). (b–i) Negative stained transmission electronmicrographs of complexes assembled at various VP1 pentamer to 600 bp DNA ratios:(b) 1 pentamer to 1 DNA, (c) 3:1, (d) 6:1, (e) 12:1, (f) 18:1, (g) 36:1, (h) 72:1 and (i)144:1. Scale bar=100 nm. At ratios ≥18:1, excess pentamer, visible at highermagnification, partially obscures images. The scale bar in panel (d) applies to panels (d)through (i).
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number of DNA 600-mers packaged within the particles observed inour titration.
To determine the DNA content of capsids we evaluated theabsorbance at 260 and 280 nm as assembly reaction products weresubjected to size exclusion chromatography. We focused our effortson the 72 pentamer 600-mer reaction because (i) there was a highyield of 45 nm particles as judged by EM and electrophoresis, (ii) theamount of free pentamer was minimal, also based on EM and (iii) nofree DNA was visible in electrophoresis experiments. Thus, any DNAeluting from the column likely to be packaged in SV40 virus-likeparticles with little contamination from free pentamer, which elutesmuch later in the column (Fig. 1b). At the peak for the 45 nm particles,residual 22 nm particles are likely to be at most a minor contaminant.
Standards of empty VP1 capsids and DNA 600-mer werechromatographed separately to accurately determine the A260/A280
ratio for each (and correct for light scattering and differences incanonical ratios due to optics) (Figs. 3b, c). The A260/A280 absorbanceratio for empty VP1 capsid was 0.91±0.05. The A260/A280 ratio forDNA was 1.78±0.12. Using these ratios and published extinctioncoefficients for DNA (13,200 M-1 cm-1 per base pair) and an aminoacid content-based estimation for VP1 (33,000 M-1 cm-1) wecalculated ɛ260 for the DNA 600-mer and the VP1 pentamer are7.92×106 and 0.15×106 M-1 cm-1, respectively. (The ɛ260 value used
Fig. 3. VP1–DNA stoichiometry in assembled particles. Seventytwo pentamers/DNAassembly reactions (3.6 μM VP1) were equilibrated for 24 h and analyzed by size-exclusion chromatography using a 20-ml Sephacryl S-500 column. Elution wasdetected at both 260 nm (thin line) and 280 nm (thick line). (a) A 72 pentamers/DNAreaction, (b) control of empty VLPs where the 260 nm chromatograph is obscured bythe thicker line and (c) the 600-bp DNA control. VLPs, DNA and assembled particlescome out of the column at the same position (10 ml). The OD260/OD280 ratios were1.3±0.16 (mean±standard deviation) for VP1–DNA assembled particles (n=3),while it was 0.9±0.05 for empty VLPs (n=4) and 1.8±0.12 for 600 bp DNA (n=4).
for the DNA was 600×13,200 per base pair; the ɛ260 for pentamer isfrom 5×.91×33,000 per monomer.) The ɛ280 values for DNA 600-merand VP1 pentamer are 4.45×106 and 0.16×106 M-1 cm-1, respec-tively. The A260/A280 ratio for the VLP peak of 72:1 VP1/DNA assemblyreaction was intermediate value of 1.32±0.16, consistent withelution of a nucleoprotein complex. Assuming that the absorbancewas the weighted sum of absorbances for VP1 and dsDNA, wecalculated the molar ratio of protein and nucleic acid.
Thus, for 0.1 μM 600-mer there would be 2.5 μM pentamer, i.e. amolar ratio of 1 to 25. If the 45 nm virus-like particles are in fact T=7structures comprised of 72 pentamers, there are on average 2.9 copiesof the 600-mer per capsid.
Discussion
We have investigated the role of protein–protein interactions inthe disassembly and re-assembly of SV40 VP1: dissociation of purifiedVLPs, that lack DNA and reassociation of VP1 pentamers induced byDNA. Dissociation required EDTA, supporting the assertion that Ca++
is required for stability of empty capsids (Ishizu et al., 2001; Sandalonand Oppenheim, 1997); reducing environment played an ancillaryrole in dissociation but was not required (Fig. 1a). More surprising isthe role that DNA was able to play in assembly in the absence Ca++.
Without DNA, VP1 requires Ca++, low pH, and/or high ionicstrength for reassembly (Kanesashi et al.; Li et al., 2003). However,such reassembly reactions lead to pleiomorphic mixtures of assemblyproducts: T=1 particles, T=7 particles, tubes, and irregular struc-tures (Kanesashi et al., 2003; Salunke et al., 1986, 1989, and ourunpublished results). On the other hand, as demonstrated here, DNAstimulated in vitro self-assembly of regular structures in a concen-tration dependent manner under conditions that were inadequate forforming empty VLPs (e.g. the 50mMMOPS, pH 7.2, 150mMNaCl usedin this study). The effect of DNA is consistent with strong interactionbetween isolated VP1 pentamers and DNA (Li et al., 2001; Soussi,1986). The variation in assembly product morphology at different VP1to DNA ratios suggests that distinctly different protein–proteininteractions are regulated by the amount of protein bound to theDNA. By using relatively short pieces of DNA, we gain insight into thedifferential roles of protein–protein and protein–DNA interaction inthe nucleation of SV40 assembly. While the primary role for DNA inthis study appears to be to support nucleation, it also presumablycontributes to capsid stability.
We propose a model of DNA-induced nucleation and assemblywhere binding to DNA is not directly coupled to protein–proteininteraction. In general, virus capsid assembly reactions are initiated bya nucleation step. Nucleus formation is limiting because of kinetics orthermodynamics. However, nucleation of SV40 capsid formationrequires a high density of VP1 on the DNA. Because VP1 pentamersbind DNA with nanomolar dissociation constant (Li et al., 2001), wecan assume that even at our lowest VP1 concentrations, the VP1 isDNA-bound. Thus at low saturation, there appears to be an absence ofinteraction between VP1 pentamers, allowing them to be spread overa DNA molecule, without capsid formation. If there were strongprotein–protein interactions supporting assembly at low levels ofDNA saturation by VP1, one would expect that VP1 pentamers wouldcluster, leaving some DNA saturated and some bare (Epstein, 1978;Kowalczykowski et al., 1986). With SV40 VP1, we observe that anexcess of protein is required initiate assembly of spherical particles onDNA. Thus, nucleation of assembly occurs at high VP1 concentration,or more accurately high densities of VP1 on DNA. This requirement forhigh VP1 suggests that nucleation is either based on very weakinteractions and/or involves several pentamers (i.e. a high order
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reaction). Subsequent assembly proceeds with high apparent coop-erativity resulting in the abrupt transition to 45 nm capsid.Nucleation-dependent assembly is consistent with our previousstudies (Mukherjee et al., 2007), where VP1 concentration neededto exceed a threshold to assemble in a highly cooperative manner.Conversely, in assembly of empty capsids of the structurally similarhuman papillomavirus 16 a dimeric nucleus was observed (Casini etal., 2004). The nucleation barriermay serve to promote specificity: ourprevious studies suggest a role for the packaging signal, ses, and hostproteins, e.g. Sp1, in the formation of the SV40 nucleation center invivo (Gordon-Shaag et al., 1998).
The abovemodel is consistent with our observations. At the lowestprotein concentrationsweobserved a variety of tube-like structures byEM. These are reminiscent of the tubes formed by DNA 600-mersmixed with the capsid protein of Cowpea Chlorotic Mottle Virus(CCMV), another spherical virus (Mukherjee et al., 2006). UnlikeCCMV tubes, conditions where we observe VP1 tubes do not correlatewith loss of the free DNA band in agarose gel electrophoresis nor withaccumulation of very slow migrating DNA that barely enters the gel.Thus, we suggest that the VP1 tubes do not survive electrophoresis,which in turn requires they are either fragile or the off-rate fordissociation is extremely fast. It is likely that at the lowest bindingdensities, multivalent pentamers ionically crosslink several DNAmolecules to support bundling and tube formation; there are fiveDNA-binding domains per VP1 pentamer. Tube formation is consistentwith strong but labile VP1–DNA interaction (i.e. a low dissociationconstant coupledwith fast on- and off-rates), but notwith interactionssupported by a network of VP1–VP1 interaction as in CCMV.
At progressively higher VP1 pentamer to DNA ratios first 20 nmparticles and then 45 nm particles were dominant species. Based ontheir regular morphology, it is likely that they are symmetrical VLPs.Their diameters correspond to T=1 particles of 12 pentamers andT=7 particles of 72 pentamers (Salunke et al., 1986, 1989). Bothtypes of particles required DNA for formation and relatively highsaturation of the 200 nm long DNA by 15 nm diameter pentamers.Another consideration is that in order to form a spherical particle,protein–protein interactions of the correct geometry must be strongenough to overcome the stiffness of the underlying DNA. Thus, athigher binding densities, where excess DNA is not available forbundling, the average number of pentamers bound per DNA supportformation of 20 nm particles. Because 20 nm particles are notobserved in the absence of DNA, we assume that at least one DNAmolecule was associated with each particle during its assembly. Atvery high binding densities, a higher average number of pentamersper DNA support formation of the 45 nm particles.
The barrier to capsid assembly thatwe observe in this in vitro systemis indicative of nucleated assembly. If this barrier is primarily kinetic, it isconsistent with in vitro the chaperone-mediated assembly observed forempty polyomavirus capsids (Chromy et al., 2003). Our data suggestthat there is also a significant thermodynamic component. In vivo, theneed to recruit high concentrations of VP1 and/or chaperones canfunction as biological quality control, decreasing inappropriate assem-bly of particles that are empty or package the wrong nucleic acid.
Materials and methods
Materials
SV40 VP1 VLPs were produced in Spodoptera frugiperda (Sf9)insect cell and purified as previously described (Mukherjee et al.,2007). Sf9 cells, infected with baculovirus expressing VP1 from thepolyhedrin promoter at moi 10, were harvested 72 h post infection.Nuclear extracts were isolated and VLPs were purified by repeatedstirred-ultrafiltration under Argon using XM300 membrane (Milli-pore). For assembly experiments, pentamers were obtained bydissociating VLPs by two cycles of dialysis, each 1.5 h, first against
20 mM Tris–Cl, pH 8.9, 50 mM NaCl, 2 mM DTT, 5 mM EDTA at 4°followed by dialysis against 20mMTris–Cl, pH 8.9, 50mMNaCl, 2 mMDTT, 2 mM EDTA at 4 °C. The dialysate was centrifuged at 13,000 rpmfor 30 min at 4° to sediment aggregated protein. Clear supernatantwas quantified by absorbance using an extinction coefficient ofɛ280=32890 M-1 cm-1 per VP1 monomer. This extinction coefficientwas based on amino acids composition (Pace et al., 1995).
The dsDNA substrate used in this paper is the same 600 nucleotidepcr product previously described (Mukherjee et al., 2006). For quan-tifying bound DNA by absorbance we used ɛ260=13,200 M-1 cm-1
per bp (Sinden, 1994) and ɛ260=7.92×106 M-1 cm-1 per 600-mer.
Light scattering
For dissociation experiments, purified VLPs were first dilutedfivefold in deionized water to a concentration of 7.4 μMVP1 pentamerin 0.1 M NaCl. Samples in a black-masked 50-μl fluorescencemicrocuvettes (Hellma, Forest Hills, NY) were mixed at 1:1 ratiowith dissociation buffer (100 mM Tris–Cl, pH 8.9 containing DTT and/or EDTA). Light scattering were observed at 320 nm excitation andemission in a SPEX-Fluoromax-2 fluorometer (Instruments S.A. Inc.,Edison, NJ) (Zlotnick et al., 1999). Light scatter of buffers wassubtracted from the signal which was normalized against theobserved light scatter of undiluted VP1 prior to dissociation.
Analysis of nucleoprotein complexes
For native agarose gel electrophoresis, samples were electrophor-esed through 0.6% agarose for 1 h using constant voltage of 100 V.Following electrophoresis, gels were stained with ethidium bromide(Johnson et al., 2004). For direct visualization of samples bytransmission electron microscopy, samples were adsorbed to freshlyglow discharged formvar-carbon coated copper grid (EM sciences) for3 min, and stainedwith 2% uranyl acetate. Samples were visualized onan H-7600 transmission electron microscope (Hitachi, Tokyo)equipped with AMT 2 K CCD camera (Advanced MicroscopyTechniques, Danvers, MA).
Size exclusion chromatography was performed using a 20-mlSephacryl S-500 column (Amersham-Pharmacia Biotech, Piscataway,NJ) connected to a Shimadzu HPLC. For VP1–DNA assembly reactions,reaction mixtures were equilibrated for 24 h and injected in to acolumn pre-equilibrated in assembly buffer (20 mM MOPS, pH 7.2150 mM NaCl). Peaks were observed by absorbance at 260 and280 nm, and integrated using Shimadzu software.
Acknowledgments
This work was supported by United States–Israel BinationalScience Foundation Grant 2005050 to AZ and AO. SK received supportfor experimental studies in United States from the FulbrightFoundation.
References
Baker, T.S., Newcomb, W.W., Olson, N.H., Cowsert, L.M., Olson, C., Brown, J.C., 1991.Structures of bovine and human papillomaviruses. Analysis by cryoelectronmicroscopy and three-dimensional image reconstruction. Biophys. J. 60 (6),1445–1456.
Casini, G.L., Graham, D., Heine, D., Garcea, R.L., Wu, D.T., 2004. In vitro papillomaviruscapsid assembly analyzed by light scattering. Virology 325 (2), 320–327.
Chromy, L.R., Pipas, J.M., Garcea, R.L., 2003. Chaperone-mediated in vitro assembly ofpolyomavirus capsids. Proc. Natl. Acad. Sci. U. S. A. 100 (18), 10477–10482.
Clever, J., Dean, D.A., Kasamatsu, H., 1993. Identification of a DNA binding domain insimian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268, 20877–20883.
Colomar, M.C., Degoumois-Sahli, C., Beard, P., 1993. Opening and refolding of simianvirus 40 and in vitro packaging of foreign DNA. J. Virol. 67 (5), 2779–2786.
Endres, D., Zlotnick, A., 2002. Model-based analysis of assembly kinetics for viruscapsids or other spherical polymers. Biophys. J. 83, 1217–1230.
204 S. Mukherjee et al. / Virology 397 (2010) 199–204
Epstein, I.R., 1978. Cooperative and non-cooperative binding of large ligands to a finiteone-dimensional lattice. A model for ligand–oligonucleotide interactions. Biophys.Chem. 8 (4), 327–339.
Friedmann, T., 1971. In vitro reassembly of shell-like particles from disrupted polyomavirus. Proc. Nat'l. Acad. Sci. U.S.A. 68, 2574–2578.
Gordon-Shaag, A., Ben-Nun-Shaul, O., Kasamatsu, H., Oppenheim, A.B., Oppenheim, A.,1998. The SV40 capsid protein VP3 cooperates with the cellular transcription factorSp1 in DNA-binding and in regulating viral promoter activity. J. Mol. Biol. 275 (2),187–195.
Ishizu, K.I., Watanabe, H., Han, S.I., Kanesashi, S.N., Hoque, M., Yajima, H., Kataoka, K.,Handa, H., 2001. Roles of disulfide linkage and calcium ion-mediated interactions inassembly and disassembly of virus-like particles composed of simian virus 40 VP1capsid protein. J. Virol. 75 (1), 61–72.
Johnson, J.M., Willits, D., Young, M.J., Zlotnick, A., 2004. Interaction with capsid proteinalters RNA structure and the pathway for in vitro assembly of Cowpea ChloroticMottle Virus. J. Mol. Biol. 335, 455–464.
Johnson, J.M., Tang, J., Nyame, Y., Willits, D., Young, M.J., Zlotnick, A., 2005. Regulatingself-assembly of spherical oligomers. Nano. Lett. 5 (4), 765–770.
Kanesashi, S.N., Ishizu, K., Kawano, M.A., Han, S.I., Tomita, S., Watanabe, H., Kataoka, K.,Handa, H., 2003. Simian virus 40 VP1 capsid protein forms polymorphic assembliesin vitro. J. Gen. Virol. 84 (Pt. 7), 1899–1905.
Kimchi-Sarfaty, C., Arora, M., Sandalon, Z., Oppenheim, A., Gottesman, M.M., 2003. Highcloning capacity of in vitro packaged SV40 vectors with no SV40 virus sequences.Hum. Gene Ther. 14 (2), 167–177.
Kosukegawa, A., Arisaka, F., Takayama, M., Yajima, H., Kaidow, A., Handa, H., 1996.Purification and characterization of virus-like particles and pentamers produced bythe expression of SV40 capsid proteins in insect cells. Biochim. Biophys. Acta 1290(1), 37–45.
Kowalczykowski, S.C., Paul, L.S., Lonberg, N., Newport, J.W., McSwiggen, J.A., von Hippel,P.H., 1986. Cooperative and noncooperative binding of protein ligands to nucleicacid lattices: experimental approaches to the determination of thermodynamicparameters. Biochemistry 25 (6), 1226–1240.
Li, P.P., Nakanishi, A., Shum, D., Sun, P.C., Salazar, A.M., Fernandez, C.F., Chan, S.W.,Kasamatsu, H., 2001. Simian virus 40 Vp1 DNA-binding domain is functionallyseparable from the overlapping nuclear localization signal and is required foreffective virion formation and full viability. J. Virol. 75, 7321–7329.
Li, P.P., Naknanishi, A., Tran, M.A., Ishizu, K., Kawano, M., Phillips, M., Handa, H.,Liddington, R.C., Kasamatsu, H., 2003. Importance of Vp1 calcium-binding residuesin assembly, cell entry, and nuclear entry of simian virus 40. J. Virol. 77 (13),7527–7538.
Liddington, R.C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T.L., Harrison, S.C., 1991.Structure of simian virus 40 at 3.8-A resolution. Nature 354, 278–284.
Mukherjee, S., Pfeifer, C.M., Johnson, J.M., Liu, J., Zlotnick, A., 2006. Redirecting the coatprotein of a spherical virus to assemble into tubular nanostructures. J. Am. Chem.Soc. 128 (8), 2538–2539.
Mukherjee, S., Abd-El-Latif, M., Bronstein, M., Ben-nun-Shaul, O., Kler, S., Oppenheim,A., 2007. High cooperativity of the SV40 major capsid protein VP1 in virusassembly. PLoS ONE 2 (1), e765.
Mukherjee, S., Thorsteinsson, M.V., Johnston, L.B., DePhillips, P., Zlotnick, A., 2008. Aquantitative description of in vitro assembly of human papillomavirus 16 virus-likeparticles. J. Mol. Biol. 381, 229–237.
Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., Gray, T., 1995. How tomeasure and predict themolar absorption coefficient of a protein. Protein Sci. 4 (11), 2411–2423.
Rayment, I., Baker, T.S., Caspar, D.L., Murakami, W.T., 1982. Polyoma virus capsidstructure at 22.5 A resolution. Nature 295, 110–115.
Salunke, D.M., Caspar, D.L., Garcea, R.L., 1986. Self-assembly of purified polyomaviruscapsid protein VP1. Cell 46 (6), 895–904.
Salunke, D., Caspar, D.L., Garcea, R.L., 1989. Polymorphism in the assembly ofpolyomavirus capsid protein VP1. Biophys. J. 56, 887–900.
Sandalon, Z., Oppenheim, A., 1997. Self-assembly and protein–protein interactionsbetween the SV40 capsid proteins produced in insect cells. Virology 237 (2),414–421.
Sinden, R.R., 1994. DNA Structure and Function. Academic Press, San Diego.Soussi, T., 1986. DNA-binding properties of the major structural protein of simian virus
40. J. Virol. 59, 740–742.Stehle, T., Gamblin, S.J., Yan, Y., Harrison, S.C., 1996. The structure of simian virus 40
refined at 3.1 A resolution. Structure 4 (2), 165–182.Tsukamoto, H., Kawano, M.A., Inoue, T., Enomoto, T., Takahashi, R.U., Yokoyama, N.,
Yamamoto, N., Imai, T., Kataoka, K., Yamaguchi, Y., Handa, H., 2007. Evidence thatSV40 VP1–DNA interactions contribute to the assembly of 40-nm spherical viralparticles. Genes Cells 12 (11), 1267–1279.
Zlotnick, A., 1994. To build a virus capsid. An equilibrium model of the self assembly ofpolyhedral protein complexes. J. Mol. Biol. 241 (1), 59–67.
Zlotnick, A., 2003. Are weak protein–protein interactions the general rule in capsidassembly? Virology 315, 269–274.
Zlotnick, A., Johnson, J.M., Wingfield, P.W., Stahl, S.J., Endres, D., 1999. A theoreticalmodel successfully identifies features of hepatitis B virus capsid assembly.Biochemistry 38 (44), 14644–14652.
