Coassembly of Tobacco Mosaic Virus Coat Proteins into Nanotubes

Letter www.acsami.org

Coassembly of Tobacco Mosaic Virus Coat Proteins into Nanotubes with Uniform Length and Improved Physical Stability Kun Zhou,†,‡ Sabine Eiben,*,§ and Qiangbin Wang*,† †

Key Laboratory for Nano-Bio Interface, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Department of Molecular Biology and Plant Virology, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany S Supporting Information *

ABSTRACT: Using tobacco mosaic virus coat proteins (TMVcp) from both sources of the plant and bacterial expression systems as building blocks, we demonstrate here a coassembly strategy of TMV nanotubes in the presence of RNA. Specifically, plant-expressed cp (cpp) efficiently dominates the genomic RNA encapsidation to determine the length of assembled TMV nanotubes, whereas the incorporated Escherichia coli-expressed cp (cpec) improves the physical stability of TMV nanotubes by introducing disulfide bonds between the interfaces of subunits. We expect this coassembly strategy can be expanded to other virus nanomaterials to obtain desired properties based on rationally designed protein−RNA and protein−protein interfacial interactions. KEYWORDS: tobacco mosaic virus, coassembly, disulfide bond, chimerical scaffold, length-controllability, physical stabilization

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contrast, Escherichia coli (E. coli) and yeasts are widely used for the facile expression of recombinant proteins, which can be used, for example, to introduce addressable functionalities into the viral cp by site-specific genetic engineering. Thus, by introducing chemical bonding or tuning the electrostatic interaction the interfaces of the TMV subunits and disks,23,26,27 the stability of the TMV-like nanotubes could be enhanced. Using the E. coli expression system, introduction of a T103C mutation, in which threonine at position 103 in the inner loop is exchanged to cysteine, resulted in significantly improved structural stability of the TMV-like but RNA-free nanotubes.27,28 Although pure cpec, which lacks the N-terminal acetylation, has been proven unable to package RNA in vitro,22,29 we could recently show that a cpec mutant with a Cterminal His6-tag, could be used for RNA directed assembly into TMV particles in the presence of cpp. This might be attributed to the cooperative effect of protein−protein interfacial interaction.30 In this work, a coassembly strategy is presented to produce hybrid TMV nanotubes with precise length control and improved physical stability using concomitant cp yielded from plant and bacteria. The purified genomic TMV RNA (6395 nt) was employed to guide the coassembly of both kinds of TMVcp

irus-derived hybrid materials have been explored for many applications such as vaccination, drug delivery, catalysis, and nanoelectronics.1−5 Especially, tobacco mosaic virus (TMV), has been at the center of virus research from one century ago and gave rise to profound findings significant in fundamental biology.6 Recently, TMV has received focused attention as a supramolecular building block within the realm of nanotechnology.7−9 The TMV particle possesses a well-defined tubular structure with a length of 300 nm, a channel width of 4 and 18 nm outer diameter. Plant-derived TMV coat proteins (cpp), which harbor an N-terminal acetylation site have a strong affinity toward the characteristic RNA loop (origin of assembly sequence, OAs) of its genomic RNA,10,11 which enables efficient reconstitution of TMV nanotubes with controlled longitudinal length governed by the length of the RNA.12,13 This naturally evolved exquisite scaffold has been extensively utilized as nanoreactor, delivery vehicle, and mineralization platform for applications in catalysis,14,15 biomedicine,16,17 and nanoelectronics.18,19 Despite being successfully employed for different applications in combination with a variety of organic and inorganic materials there are some drawbacks in using in planta produced TMV. First of all, native TMV is not stable at alkaline conditions at values above pH 9.5 as well as at higher temperatures (>65 °C) in solution.20,21 Second, it has been shown that only a small number of genetic modifications of the cp are tolerated during propagation in planta and thus the possibility to add new functions to the virus particle by changing the amino acid composition are limited.22−25 In © XXXX American Chemical Society

Received: April 12, 2016 Accepted: May 18, 2016

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DOI: 10.1021/acsami.6b04321 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Native agarose gel of the coassembled TMV particles of E. coli-derived T103Ccp (right) and plant-derived WTcp (left) mixed with plantderived T158Kcp in the presence of TMV RNA. The increasing ratios of T103Ccp or WTcp (%) in the mixture result in defined bands with gradually increased electrophoretic mobility. The bands referring to T103Ccp-incorporated TMV nanotubes are marked by the red frame. The two kinds of cloudy bands below the red frame correspond to residual T103Ccp/T158Kcp hybrid disks and pure T103Ccp disks/aggregates. On sides of the gel, T158K-TMV and WT-TMV isolated from N. tabacum were included leading to bands corresponding to typical 300 nm nanotubes and headto-tail multimers with lengths of 600, 900, and 1200 nm, respectively.

Figure 2. TEM images of coassembled particles. Different amounts of E. coli-derived T103Ccp from (a) 15 to (b) 30, (c) 40, (d) 50, (e) 65, and (f) 80% in the mixtures all resulted in 300 nm long nanotubes that coassembled with plant-derived T158Kcp by means of genomic TMV RNA.

and hybrid TMV nanotubes with a uniform length of 300 nm and improved physical stability were expected. To evaluate the coassembly efficiency, plant-derived T158Kcp (T158Kcpp, with mutation of the neutral threonine 158 to a positively charged lysine) was applied in combination with E. coli-derived T103Ccp (T103Ccpec). As these cps have different isoelectric points (pI), 5.09 for T103Ccp and 5.41 for T158Kcp (see Figure S1), mixtures at varying ratios will result in differently charged TMV particles that can be distinguished by native agarose gel electrophoresis due to their difference in

electrophoretic mobility. To determine the efficiency with which the T103Ccp was incorporated into the virus tubes, we coassembled wild type cpp (WTcpp), with the same pI of 5.09 as T103Ccpec, with T158Kcpp as reference. As both cp are produced in planta and therefore able to assemble with RNA, their compositional ratio in the nanotubes is solely depending on the initial mixed amount of T158Kcpp and WTcpp. As shown in Figure 1, the increasing ratios of T103Ccpec (%) in the mixture result in clearly defined bands with gradually increased electrophoretic mobility (marked by the red-color B

DOI: 10.1021/acsami.6b04321 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. (a) TEM image of the coassembled T103Ccp hybrid nanotubes demonstrating the high length uniformity. Inset is the statistical distribution of nanotube length. (b) SDS-PAGE analysis showing the dimer band caused by disulfide bonds in the chimerical nanotubes. M, protein molecular weight marker. Lane 1, sample of T103Ccp hybrid nanotubes mixed with loading buffer containing dithiothreitol (DTT) to break disulfide linkage (reducing conditions). Lane 2, sample of T103Ccp hybrid nanotubes mixed with loading buffer without DTT (nonreducing conditions).

the C-terminus in addition to the T103 exchange (T103CT158Kcpec). In the presence of WTcpp we could also realize RNA-controlled hybrid nanotube formation, demonstrating the universality of this coassembly strategy for combining properties of engineered cpec with those of cpp (see Figures S5 and S6). The T103Ccpec/T158Kcpp chimerical nanotubes containing ca. 17% of T103Ccpec (corresponding to 50% in the cp mixture) were purified by isopycnic CsCl density gradient centrifugation and the length of the hybrid particles was measured. Analysis of 150 particles demonstrated a very sharp length distribution around 300 nm (Figure 3a). This proves that because of the presence of T158Kcpp we have obtained chimerical TMV nanotubes incorporating the complete RNA, resulting in nucleoprotein particles with high length uniformity in comparison to our previous RNA-free T103Ccp nanotubes (Figure S7).27 Although the successful incorporation of T103Ccpec in these chimerical nanotubes was confirmed; the formation of disulfide bonds introduced by the mutated cysteine residues of the doped T103Ccp and their stabilizing effect had to be evaluated. As shown in Figure 3b, as we expected, SDS-PAGE under reducing conditions resulted in one cp band in accordance with the molecular weight of 17.5 kDa for the cp monomer (lane 1), whereas under nonreducing conditions two bands appeared (lane 2). With regard to the band intensities, the major band at about 17.5 kDa can be attributed to the monomers of T158Kcpp and the upper band at the position close to 35.0 kDa, twice the molecular weight for a cp, is proposed to be composed of two T103Ccpec cross-linked by a disulfide bond. The occurrence of faint bands below the two target bands is a common feature of nonreducing SDS-PAGE of T103Ccpec dimers, due to asymmetric cleavage of dimer during heating of the gel sample preparation.27 As the formation of disulfide bonds within the T103Ccpec hybrid nanotubes was verified, we investigated their impact on physical stability under alkaline conditions. Figure 4a shows the native agarose gel of T103Ccpec hybrid particles after incubation at different pH values ranging from 7.0 to 11.5 for 5 h, in comparison to also in vitro assembled

frame), indicating the successful formation of hybrid TMV nanotubes. However, without the addition of genomic RNA, only smeared bands corresponding to disks or oligomeric proteins occurred, thereby validating the critical role of RNA on directing the coassembly of T158Kcpp and T103Ccpec into TMV nanotubes (see Figure S2). Although higher incorporation rates of T103Ccpec could be achieved with increasing amounts of T103Ccpec in the coassembly mixture, the yield was compromised. This is likely due to heterogeneity of the T103Ccpec aggregation state obtained from E. coli, part of the cpec is no longer able to form functional disks with the cpp. Consequently, two kinds of cloud-like bands representative of T103Ccpec/T158Kcpp hybrid disks and T103Ccpec disks/ aggregates appeared below the defined bands of TMV nanotubes. In addition, as expected, the in vitro assembled nucleoprotein nanotubes consisting of WTcpp or T158Kcpp alone migrated to the same position as the T158K-TMV or WT-TMV virus particles with the typical 300 nm-length isolated directly from plants and loaded on the outmost lanes of the gel. The additional bands in these lanes with lower mobility are caused by head-to-tail attachment of two or more TMV particles, representing multiples of 300 nm. By measuring the retention factor (Rf) of the nanotube bands of T103Ccpec/ T158Kcpp and the corresponding WTcpp/T158Kcpp particles (see Figure S4), the real incorporation rate of cpec into the hybrid nanotubes was determined. For example, approximately 17% of T103Ccpec, were blended into chimerical nanotubes in the sample containing 50% of heterologous cpec. We further verified the coassembly products by transmission electron microscopy (TEM) analysis, as shown in Figure 2. The characteristic 300 nm TMV nanotubes, indicating the complete packaging of genomic RNA, can be observed in all samples with different proportions of T103Ccpec in the mixtures even up to 80% (Figure 2f). While, without the help of T158Kcpp, T103Ccpec alone failed to incorporate the genomic RNA into nucleoprotein nanotube assemblies (see Figure S3), which further implies the importance of the cooperative effect of protein−protein interfacial interaction for coassembly. For validation of the principle, in addition to the T103Ccpec, we also tested a double mutant containing the T158K mutation at C

DOI: 10.1021/acsami.6b04321 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

behaved similar to WTcpp nanotubes and resulted in complete loss of the band for intact nanotubes also at pH 11.0. In conclusion, we could demonstrate that the incorporation of E. coli-derived T103Ccp into uniform chimerical nanotubes in the presence of genomic RNA depends on the cooperation with plant-derived TMVcp. Despite the heterogeneity of E. coliderived TMVcp derogating the coassembly efficiency, the doping rate of recombinant T103Ccp could be tuned by the applied proportion for combination. Through this coassembly approach, we successfully integrated the programmability of RNA-templated assembly, the cooperation effect of plantderived TMVcp and the structural stabilization of disulfide bonds by E. coli-derived T103Ccp, thereby resulting in hybrid TMV materials with uniform length and enhanced stability. Taking advantage of different expression systems, viral proteins with different posttranslational modifications often differ in properties and functions. On the basis of the understanding of protein−RNA and protein−protein interfacial interactions, the coassembly strategy presented here gives the opportunity to obtain chimerical nanotubular scaffolds with different components for multifunctionalization. We look forward to expanding this work to other virus nanomaterials to acquire diverse functional properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04321. Experimental section, native agarose gel electrophoresis of the coassembly samples without TMV RNA, theoretical isoelectric point of mutated TMVcp, failed assembly of E. coli-derived T103Ccp with TMV RNA, native agarose gelelectrophoresis and TEM images of the coassembly products of E. coli-derived T103C-T158Kcp with plant-derived WTcp, and analysis of the measured retention factor (Rf) values of the hybrid nanotubes (PDF)

Figure 4. Stability assessment of T103Ccp hybrid nanotubes compared to in vitro assembled WTcp nanotubes. (a) Native agarose gel electrophoresis of the samples after incubation at different pH values (7.0, 9.5, 10, 10.5, 11, and 11.5) with (right) or without (left) DTT treatment. The T103Ccp hybrid nanotubes, which coassembled with T158Kcp, resulted in slower migrating bands compared to WTcp nanotubes. (b) Semiquantitative densitometry analysis of the gel electrophoresis results. The percentage of preserved, intact particles after incubation at alkaline pH was determined densitometrically.

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WTcpp particles containing genomic RNA. Obviously, increased pH values are disadvantageous to TMV virions due to alkaline degradation. At a pH of 11.5, both, T103Ccpec hybrid nanotubes and WTcpp nanotubes were totally damaged, resulting in abnormally smeared or aggregated bands. While, WTcpp nanotubes already showed a second band indicating partially fractured nanotube structures at pH 9.5 in agreement with the pH tolerance range of TMV virions in previous studies,20,21 T103Ccpec hybrid nanotubes appeared to remain intact at alkaline pH even up to 10.0. Moreover, even after incubation at pH 11.0, besides some smear, there was still a defined band of nanotubes for the T103Ccpec hybrid sample visible. In order to quantitatively assay the stability of nanotubes against alkaline conditions, we measured the band grayscale intensities of the incubated samples by densitometry analysis in comparison to the control samples at pH 7.0. As shown in Figure 4b, WTcpp nanotubes degraded distinctly with increasing pH, leading to only ca. 35% intact particles at pH 10.5 and nearly completely degraded ones at pH 11.0. However, more than 80% of T103Ccp hybrid nanotubes were intact at pH 10.0, and about 50% retained their structure at pH 11.0, thus corroborating the enhanced physical stability. The effect of the disulfide bonds on structural stability was further supported by experiments executed in the presence of DTT. Under these conditions, T103Ccpec hybrid nanotubes

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the funding from the German Federal State of Baden-Wuerttemberg (scholarship programme supporting research visits of excellent Chinese scientists in the area of nanotechnology, granted to C. Wege as a member of the Network of Competence ’Functional Nanostructures’) to support K. Zhou’s stay in C. Wege’s lab, the DFG-SPP 1569, the Zeiss foundation (ProjekthausNanoBioMater), and National Natural Science Foundation of China (21425103).

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DOI: 10.1021/acsami.6b04321 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX