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1 Graduate School of Bioscience and Biotechnology, and 2 Integrated Research Institute, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
3 National Institute of Longevity Sciences, 36-3 Gengo, Moriyama-cho, Obu, Aichi 474-8522, Japan
4 Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We and others have established a defined system for VLP assembly/disassembly. VLPs expressed in bacteria or in insect cells can be disassembled into VP1 pentamers by the addition of DTT and EGTA (Brady et al. 1977, 1978, 1979), and VP1 pentamers can be reassembled into VLPs under appropriate conditions (Salunke et al. 1986). Assembly of purified pentamers is highly sensitive to the conditions used and can result in various higher-order structures. In the presence of 2 M (NH4)2SO4 and 2 mM CaCl2, for example, 40-nm spherical particles are formed (Kanesashi et al. 2003), while in the presence of 1 M NaCl, 20-nm spherical particles, which probably represent T = 1 structures composed of 12 VP1 pentamers, are formed (Kanesashi et al. 2003; Nilsson et al. 2005). Under different conditions, surprisingly long tubular structures of 40–45 nm in width and up to approximately 1 µm in length are formed (Kanesashi et al. 2003; see Fig. 3A).
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The viral genome is packaged into SV40 particles through multiple interactions with the N-terminal region of VP1 (Soussi 1986; Li et al. 2001) and the C-terminal region of VP2/3 (Griffith et al. 1992; Clever et al. 1993). Moreover, recombinant VP1 VLPs expressed in bacteria or in insect cells are known to contain endogenous DNA/RNA molecules (Pawlita et al. 1996; Gillock et al. 1997) or exogenously added DNA (Braun et al. 1999; Stokrová et al. 1999). Thus, DNA may conversely affect the process of VP1 assembly, but this possibility has not been explored in detail. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm spherical particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2000 bp is packaged into 40-nm spherical particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.
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Results |
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To explore whether DNA has any effect on the assembly of VP1 pentamers, we performed in vitro assembly reactions in the presence of DNA. A solution containing VP1 pentamers (0.44 µM) and 4729-bp double-stranded circular DNA (1.2 nM) was dialyzed against a buffer at physiological salt concentrations and pH (0.15 M NaCl and 2 mM CaCl2, pH 7) for 16 h and then observed by electron microscopy. In the absence of DNA, VP1 pentamers did not form any higher-order structures other than aggregates (Fig. 1A). In the presence of DNA, however, VP1 pentamers efficiently assembled into 40-nm spherical particles, which appeared to be similar to the 40-nm spherical particles assembled in vivo (Fig. 1A,B). It was thus concluded that DNA facilitates VP1 assembly into 40-nm spherical particles under these conditions in vitro.
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Effect of pH on VP1 assembly
Next, we examined the effect of pH on VP1 assembly in the absence or presence of DNA. In accordance with our previous reports (Kanesashi et al. 2003; Kawano et al. 2006), in the absence of DNA, VP1 pentamers assembled into tubular structures at pH 5 (Fig. 3A, black arrowheads) and into incomplete (partially closed) spherical particles at pH 9 (Fig. 3A, white arrowheads). At other pH values, VP1 pentamers only formed aggregates. In the presence of DNA, VP1 pentamers assembled into 40-nm spherical particles at pH 5–9 (Fig. 3A,B). At pH 4, DNA facilitated the formation of irregular spherical particles with attached thread-like structures, which are similar to those seen at low concentrations of NaCl or CaCl2 (Fig. 2), while at pH 10, DNA had little effect on VP1 assembly. These results demonstrate that DNA facilitates the assembly of VP1 pentamers into complete 40-nm spherical particles over a wide range of pH values.
Effect of DNA structure on VP1 assembly
We next investigated the requirements for the structure, sequence and concentration of DNA in DNA-mediated VP1 assembly. All the subsequent assembly reactions were carried out in 0.15 M NaCl and 2 mM CaCl2 at pH 5, wherein tubular structures were preferentially formed without DNA (Fig. 3A). First, VP1 pentamers (0.44 µM) were incubated with 3.1 nM circular or linear DNA of various lengths (Fig. 4A,B). In the presence of linear 250- or 500-bp DNA, VP1 pentamers mainly formed tubular structures and 25- to 40-nm irregular particles, while in the presence of linear 750-bp DNA, VP1 pentamers mainly assembled into 25- to 40-nm irregular spherical particles. In the presence of linear or circular 1- to 10-kb DNA, VP1 pentamers efficiently assembled into 40-nm spherical particles. Thus, under these conditions, 1 kb or longer DNA is necessary for 40-nm particle formation whether it is linear or circular.
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In order to study VP1–DNA interactions, various assembly reactions were each subjected to sucrose gradient centrifugation, and resulting fractions were analyzed for the presence of VP1 and DNA. VP1 particles prepared in the presence of linear 1- to 5-kb DNA were found to co-migrate with the DNA in fractions 8–10, the so-called VLP fractions (Fig. 4C, panels 3–10, 17), while VP1 particles formed in the presence of 10-kb DNA were found together with the DNA in fractions 5–8, distinct from the VLP fractions (Fig. 4C, panels 1 and 2). In the presence of 250–750 bp DNA, VP1 and DNA were separated into the VLP fractions and the pellet fraction (Fig. 4C, panels 11–16). On the other hand, when DNA, VP1 pentamers, and tubular structures were analyzed separately, the first two were found in top fractions (Fig. 4C, panels 18, 20, 21), and tubular structures were found in the pellet fraction (Fig. 4C, panel 19). These results suggest that various structures formed in the presence of DNA such as 40-nm spherical particles, 25- to 40-nm irregular spherical particles, and tubular structures are all associated with the DNA. As for 10 kb DNA (panels 1 and 2), it may be that a large part of the DNA was not packaged but connected to 40-nm spherical particles, and induced a shift of the sedimentation peak toward that of free DNA (panel 21).
Effect of DNA concentration on VP1 assembly
So far, DNA was used at the concentration of 1.2 (Figs 1–3) or 3.1 nM (Fig. 4), which correspond to DNA : VP1 pentamer molar ratios of 1 : 360 and 1 : 144, respectively. Since 40-nm spherical particles are considered to be comprised of 72 VP1 pentamers, we assumed that 6.1 nM DNA, corresponding to the DNA : VP1 pentamer molar ratio of 1 : 72, might be most suitable for the formation of 40-nm spherical particles. We therefore examined the effect of DNA concentrations on VP1 assembly using circular 4729-bp DNA. While 0.6 nM DNA had little effect on VP1 assembly, DNA at higher concentrations up to 6.1 nM promoted the formation of 40-nm particles in a concentration-dependent manner. In the presence of 61 nM DNA, however, 20-nm particles were formed in addition to 40-nm spherical particles, and in the presence of 610 nM DNA, 20-nm particles predominated. Thus, (sub)stoichiometric amounts of 4729 bp DNA facilitate the formation of 40-nm particles, whereas excessive amounts of DNA induce the formation of 20-nm particles, which probably represent T = 1 structures composed of 12 VP1 pentamers.
From the above results, we cannot determine which of the following parameters directly influences VP1 assembly: DNA length, molar concentration or weight-per-volume concentration (or total mass). For example, in Fig. 4A, wherein DNA was used at a constant molar concentration, it could not be discriminated which of DNA length and weight-per-volume concentration contributed to particle formation, as both parameters varied simultaneously. To address this issue, we performed assembly reactions at three different weight-per-volume concentrations of DNA. As shown in Fig. 5B, 4729 and 250 bp DNAs equally facilitated the formation of 40-nm particles at 10 µg/mL, but not at 2.5 µg/mL. It should be noted that although 250 bp DNA weakly facilitated the formation of 25- to 40-nm irregular particles at 3.1 nM (Fig. 4A), the same DNA strongly facilitated 40-nm particle formation when used at a significantly higher concentration, that is, 10 µg/mL or 61 nM. Conversely, large DNAs such as 48-kb 
phage DNA and 166-kb T4 phage DNA facilitated 40-nm particle formation at subnanomolar concentrations (Fig. 5B), at which 4729 bp DNA had little effect on VP1 assembly (Fig. 5A). Thus, DNA-mediated formation of 40-nm spherical particles does not correlate well with molar concentrations of DNA, but appears to be influenced by weight-per-volume concentrations of DNA. On the other hand, 100 and 50 bp DNAs each facilitated the formation of 20-nm T = 1 particles at 10 µg/mL. Thus, there appears to be a critical DNA length required for 40-nm particle formation, which is somewhere between 100 and 250 bp. Collectively these results suggest that DNA length and weight-per-volume concentration are important for DNA-mediated 40-nm particle formation.
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SV40 wild-type particles, which are similar in size and shape to 40-nm spherical particles, accommodate 5243-bp SV40 minichromosomal DNA, which has a highly compact structure. Given the finding that naked DNA of a certain length interacts with VP1 and promotes their assembly in vitro, we next examined whether and to what extent naked DNA is packaged into 40-nm spherical particles. For this purpose, assembly reactions were carried out with linear or circular DNA of various lengths and subsequently incubated with DNase I in order to remove unpackaged DNA. Prior to DNase I addition, 180 bp DNA was included as a control, and DNase I-treated samples were analyzed by Southern blotting. Linear or circular 2-kb DNA and linear 1-kb DNA in the assembly reactions remained largely intact after DNase I treatment, whereas the 180-bp control DNA added after assembly was susceptible to DNase I (Fig. 6A,B). Linear or circular DNAs of larger sizes were digested into approximately 2 kb fragments by DNase I (Fig. 6A,B). These results suggest that 40-nm spherical particles can accommodate naked DNA of up to about 2 kb. To clearly distinguish the topological states of the circular DNAs used, the same samples were resolved on an agarose gel containing chloroquine (Fig. 6B). As a result, total DNAs were found to contain both relaxed and supercoiled forms, but as stated above, only 2 kb DNA was fully protected from DNase I digestion. Thus, although supercoiled DNA should have a compact structure, DNA superhelicity does not seem to affect packaging efficiency appreciably.
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Time-course of the DNA-mediated particle formation
So far we have used the long incubation time of 16 h because our initial study showed that the duration is sufficient for the formation of various types of VP1 structures (Kawano et al. 2006). However, precise time required for VP1 assembly was not clear. To address this issue, we carried out assembly reactions without or with 5 kb linear or circular DNA for various times (Fig. 7A). In all cases examined, higher-order structures were rarely observed during the first 1 h, except that small aggregates were formed in the presence of DNA (Fig. 7A,B), and assembly reactions appeared to be almost complete within 2 h. These results indicate that DNA has only a small effect on the kinetics of VP1 assembly, and that the formation of different VP1 structures such as 40-nm spherical particles and tubular structures follows a similar time-course. We next asked whether SV40 sequence has any effect on the kinetics of DNA-dependent particle formation. For this purpose, we performed VP1 assembly reactions in the presence of the plasmid pSV40, containing the full-length SV40 genome, or the control plasmid pLAPSN (Fig. 7C,D). In both cases, 40-nm particle formation followed a similar time-course, and assembly reactions were complete within 2 h. Thus, we concluded that any particular sequence of the SV40 genome does not contribute to DNA-dependent particle formation.
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Discussion |
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We did not observe any preference for the SV40 genomic DNA. These data are consistent with previous reports showing that VP1, VP2 and VP3 all possess sequence nonspecific DNA-binding activity (Soussi 1986; Moreland et al. 1991; Clever et al. 1993; Li et al. 2001). According to a series of studies (Dalyot-Herman et al. 1996), however, the SV40 genome contains a DNA element called ses that is important for viral genome packaging. As ses is reported to bind to a cellular transcription factor and VP2/3 proteins (Gordon-Shaag et al. 2002), these factors may be necessary for sequence-specific packaging of the SV40 genome.
DNA-dependent formation of 40-nm particles
Generally, viral particle assembly is driven by intrinsic properties of the coat protein(s). In some cases, however, the assembly process appears to be assisted by other molecules. For example, in tobacco mosaic virus, protein discs composed of a single viral protein pile up to form a helical structure, in which the RNA genome is embedded. Although the viral protein can be polymerized into the helical structure without any RNA component (Schuster et al. 1980), it is also suggested that RNA may perform a scaffolding function during viral assembly (Butler 1999). Another example is adenovirus, which has a complex structure composed of at least 10 different proteins. Structural components of adenovirus such as pentons (fiber structures), the base of the penton capsomere, and the hexon capsomere are each assembled independently and are then brought together. Although the assembly process remains to be elucidated, there appear to be viral scaffolding proteins that are necessary for assembly but are removed from the final particles (Hasson et al. 1989). In other viruses such as polyomavirus, molecular chaperones of host cell origin are reported to be involved in viral assembly (Cripe et al. 1995).
We offer two possible mechanisms by which DNA facilitates the formation of 40-nm spherical particles. The most likely explanation is that DNA acts as a scaffold that increases local concentrations of VP1 and thereby facilitates VP1 assembly into 40-nm particles. An alternative, not mutually exclusive, possibility is that DNA acts as an allosteric effector that changes the tertiary structure of VP1. Whereas DNA-binding activity resides in the N-terminal region of VP1 (Li et al. 2001), its C-terminal region is known to be critical for the formation of higher-order structures (Stehle et al. 1996). X-ray crystallography has shown that the C-terminal region of each VP1 within a VLP participates in one of three interpentameric interactions, 
-'-'', ß-ß' and 
- (Liddington et al. 1991), and consistently, its mutations lead to the formation of different higher-order structures such as T = 1 particles and tubular structures (Yokoyama et al. 2007). Thus, DNA may change the C-terminal structure of VP1 so as to favor the formation of 40-nm particles by interacting with the N-terminal region of VP1.
It was found that DNA-mediated formation of 40-nm particles requires 250 bp or longer DNA at the minimum concentration of 5 µg/mL (Fig. 5B). The strong concentration dependency is probably a reflection of the affinity of the VP1–DNA interaction. According to the results with 4729 bp DNA (Fig. 5A), optimal DNA concentrations for the formation of 40-nm particles appear to be 10–20 µg/mL (3.1–6.1 nM), at which a VP1 pentamer is calculated to bind to every 33 or 66 bp of DNA. We assume, for a number of reasons, that the association of several VP1 pentamers with a single DNA molecule at a relatively short interval is important for the formation of 40-nm particles. Consistent with this idea are the observations (Fig. 5B) that the formation of 40-nm particles does not correlate with molar concentrations of DNA but with weight-per-volume concentrations of DNA (or molar concentrations of base pairs), and that 40-nm particles are not formed in the presence of 100 or 50 bp DNA, which are considered to associate with not more than a few copies of VP1 pentamers. The above idea is further supported by the observation (Fig. 5A) that assembly efficiency of 40-nm particles is decreased by the presence of such excessive amounts of DNA that significantly increase the spacing of VP1 on DNA. At the highest DNA concentration examined in Fig. 5A, for example, it is calculated that only one VP1 pentamer binds to 4729 bp DNA on average. These findings suggest that assembly may be initiated by several VP1 pentamers being associated with DNA in cis at an appropriate interval, and that such subcomplexes may then direct the formation of complete 40-nm particles, possibly by interacting with each other. Time-course analysis (Fig. 7) showed that VP1 aggregates are formed in a DNA-dependent manner at early time points prior to the appearance of 40-nm particles. These aggregates may correspond to the intermediate subassemblies mentioned above.
Formation of various atypical structures
Twenty nanometer particles were formed in the presence of excessive amounts of DNA or in the presence of short, 100 or 50 bp DNA (Fig. 5A,B). Interestingly, under both conditions, DNA is considered to bind to a small number of VP1 pentamers. The formation of 20-nm particles may therefore be facilitated when a weak scaffolding activity of DNA is exerted or when DNA functions only as an allosteric effector.
At 0 M NaCl, VP1 pentamers assembled into 40-nm particles in the absence of DNA (Fig. 2A). Electron microscopic observations, however, indicated that these particles are morphologically different from the DNA-mediated 40-nm particles or native SV40 particles. Notably, the insides of the DNA-independent particles were not stained well with uranium acetate, suggesting that these particles may not be empty but may be filled with some material. Since electrostatic protein–protein interactions are dominant under such a low-salt condition, VP1 pentamers may have assembled onto aggregated VP1 "cores."
As reported previously (Kanesashi et al. 2003), tubular structures were formed in the absence of DNA under two different conditions, that is, at high concentrations of NaCl or at pH 5 (Figs 2 and 3). Since electrostatic protein–protein interactions are probably weak at high salt or at the pH close to the pI of VP1, hydrophobic interactions between VP1 molecules that are normally prevented by the presence of DNA may have occurred under these conditions and led to the formation of tubular structures.
Under various conditions such as at 0 M NaCl, low concentrations of CaCl2, or pH 4, thread-like structures associated with 40-nm spherical particles were often observed in the presence of DNA (Figs 2 and 3). These thread-like structures are most likely due to incomplete DNA packaging and probably represent unpackaged DNA protruding from 40-nm spherical particles, onto which VP1 pentamers assembled. However, it is not clear why these structures were formed only in the few circumstances.
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Experimental procedures |
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Circular DNA of 2000 bp was obtained as follows. An ampicillin-resistance gene fragment was obtained by PCR using pBS-SK(+) and the primers 5'-gaaggcctccaatgcttaatcagtgagg-3' and 5'-cggaattcatgagtattcaacatttccgtg-3'. The sequences of the primers were designed to include AatI and EcoRI restriction sites, respectively (underlined), and the PCR product was digested with these enzymes. A ColE1 ori fragment was obtained by PCR using pBS-SK(+) and the primers 5'-cggaattcgttgttccagtttggaacaag-3' and 5'-cgggatccgcattaagcgcggcggg-3'. The sequences of the primers were designed to include EcoRI and BamHI restriction sites, respectively (underlined), and the PCR product was digested with these enzymes. A pUC ori fragment was obtained by PCR using pBS-SK(+) and the primers 5'-gaaggcctgtagaaaagatcaaaggatcttc-3' and 5'-cgggatccgtgagcaaaaggccagcaa-3'. The sequences of the primers were designed to include AatI and BamHI restriction sites, respectively (underlined), and the PCR product was digested with these enzymes. The three DNA fragments were then ligated to generate 2000-bp circular plasmid DNA. pBS-SK(+), pEGFP-N3 (Clontech, Otsu, Japan), pFastBac1 (Invitrogen, Carlsbad, CA) and hSpt6WT/pFB (Endoh et al. 2004) were used as 3000-, 4729-, 5000- and 10 000-bp circular DNAs, respectively. pSV40, containing the complete SV40 genome (Ishii et al. 1994), and pLAPSN (Clontech) were also used in some experiments. These circular plasmid DNAs were linearized with appropriate restriction enzymes to obtain linear DNAs ranging in size from 2000 to 10 000 bp.
Linear DNA fragments of 50, 100, 250, 500, 750 and 1000 bp corresponding to parts of the EGFP gene were obtained by PCR using pEGFP-N3 (Clontech) and the following primers: for the 50-bp DNA fragment, 5'-ctatatcatggccgacaagc-3' and 5'-tcttgaagttcaccttgatg-3'; for the 100-bp DNA fragment, 5'-gacgacggcaactacaagac-3' and 5'-cgtcctccttgaagtcgat-3'; for the 250-bp DNA fragment, 5'-caactacaacagccacaacg-3' and 5'-aactccagcaggaccatgt-3'; for the 500-bp DNA fragment, 5'-ggacgacggcaactacaa-3' and 5'-gggaggtgtgggaggtt-3'; for the 750-bp DNA fragment, 5'-cagtgcttcagccgctac-3' and 5'-tgagtttggacaaaccacaac-3'; and for the 1000-bp DNA fragment, 5'-gacgtaaacggccacaag-3' and 5'-cgatttcggcctattggt-3'. The phage genome of 48 kb and the T4 (T4GT7) phage genome of 166 kb were also used as large linear DNAs.
Expression and purification of recombinant SV40 VP1 pentamers
SV40 VP1 pentamers were prepared as described previously (Kosukegawa et al. 1996; Ishizu et al. 2001; Kanesashi et al. 2003; Kawano et al. 2006). Briefly, recombinant VP1 was over-expressed in insect Sf-9 cells by using a baculovirus vector, and VLPs were purified by two successive cesium-chloride density gradient centrifugations. VLPs were disassembled to VP1 pentamers by the treatment with EGTA and DTT, and VP1 pentamers were purified through a Superdex 200 gel filtration column (GE Healthcare, Buckinghamshire, England). A peak fraction eluted at approximately 250 kDa was collected.
In vitro assembly of SV40 VP1 pentamers and electron-microscopic observation
Standard assembly reactions were carried out as follows: VP1 pentamers (0.44 µM) were incubated with or without DNA for 30 min at 4 °C and dialyzed against 0.15 M NaCl and 2 mM CaCl2 at various pHs for 16 h at room temperature using a mini-dialysis unit with a molecular weight cut off of 3500 Da (Pierce, Rockford, IL). The solutions at pH 4, 5 did not contain any strong buffer, and the pH was adjusted with HCl. The solutions at pH 6–10 contained 20 mM Tris–HCl, and the pH was adjusted with HCl or NaOH. Samples were negatively stained with uranium acetate and observed using a transmission electron microscope (H-7500, Hitachi, Tokyo, Japan) as described (Kosukegawa et al. 1996; Ishizu et al. 2001; Kanesashi et al. 2003; Kawano et al. 2006). In Figs 1 and 3–5, diameters of 100 randomly selected particles were measured and shown as histograms. In Fig. 7, mean numbers (n = 6) of 40-nm spherical particles formed per grid square (1 x 1 µm) were counted at x50 000 magnification and plotted against time.
Sucrose gradient sedimentation analysis of VLP assemblies
VP1 pentamers (0.44 µM) were incubated with 3.1 nM linear DNA of various lengths for 30 min at 4 °C and dialyzed against 0.15 M NaCl and 2 mM CaCl2 at pH 5 for 16 h at room temperature. Aliquots of the samples (20 µL) were loaded onto 600 µL of 20%–40% sucrose gradient and centrifuged at 237 020 g for 1 h at 4 °C using a Beckman SW55-Ti rotor with an appropriate adaptor. After centrifugation, fractions (55 µL) were collected from the top of the gradient and analyzed by 10% SDS-PAGE and immunoblotting with anti-SV40 VP1 antibody. For detection of DNA, a 10-µL aliquot of each fraction was incubated with 0.42 µg of proteinase K at 55 °C for 1 h, electrophoresed on 1.2% agarose gels, and transferred to nitrocellulose membranes. The blots were probed with 32P-labeled DNA probes for EGFP and ampicillin-resistance genes according to the manufacturer's instructions (BcaBest, Takara, Otsu, Japan).
DNase I treatment of VLP assemblies
VP1 pentamers (0.44 µM) were incubated with linear or circular DNA of various lengths for 30 min at 4 °C and dialyzed for 16 h at 22 °C against 150 mM NaCl and 2 mM CaCl2 at pH 5. Aliquots of the samples (5 µL) were mixed with 180-bp control DNA and incubated with 0.2 unit of DNase I (Worthington, Lakewood, NJ) for 15 min at 37 °C and then with 0.42 µg of proteinase K for 1 h at 55 °C. Subsequently, Southern blot analysis was carried out as described above. Where indicated, gel electrophoresis was carried out using 0.8% agarose gels containing 2 µg/mL chloroquine at 45 V for 9 h in the dark.
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Acknowledgements |
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Footnotes |
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aThese authors contributed equally to the work.
* Correspondence: E-mail: hhanda{at}bio.titech.ac.jp |
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Received: 4 February 2007
Accepted: 8 August 2007
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