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Sensing & Measurement
Probing viral structure with surface-enhanced Raman spectroscopy
Incorporating gold nanoparticles into viral protein provides a tool for studying viruses.
5 April 2007, SPIE Newsroom. DOI: 10.1117/2.1200702.0672
In recent years, viruses have shown their biological utility as targeted delivery vectors1,2 and as optical probes for biomedical imaging and sensing applications. More recently, their potential use as templates for nanoelectronics has also been explored.3,4 Moreover, the controlled disassembly and reassembly of viruses has been exploited to transfer genes and drugs into cells.1,2 These developments now represent a new paradigm for complex self-assembly on the nanoscale, as understanding these processes will allow fabrication of efficient nanoscale structures while providing novel insights into viral infection mechanisms.
In our work, we use hybrid nanoparticles consisting of a viral protein coat, or capsid, and an incorporated gold nanoparticle (AuNP) to study the conditions responsible for viral (dis-)assembly with surface-enhanced Raman spectroscopy (SERS). The viral capsid is from the mouse polyomavirus (muPy), a small, so-called nonenveloped virus with a double-stranded DNA genome of 5kb (kilobase pairs).5 The muPy capsid consists of one major (VP1) and two minor (VP2/3) proteins. VP1 is sufficient to form the capsid, which can be stabilized by divalent calcium cations and disulfide bonds.6 It can be disassembled by removing the Ca2+ ions with the chelating agent EGTA and reducing the disulfide bonds with dithiothreitol. Conversely, reassembly can be initiated by additing Ca2+ ions and removing the reducing agent. Addition of AuNPs to the reassembly leads to their inclusion into the capsid.
Figure 1. Electron microscopic image (a) and computational reconstruction after recording the electron microscopy tilt series (b) of a 5nm gold nanoparticle (AuNP) incorporated into the capsid of a muPy virus-like particle (VLP). While a conventional transmission electron microscope (TEM) cannot differentiate between gold nanoparticles that are co-localized or unspecifically attached to VLPs, TEM tomography and computer-aided reconstruction can create a 3D image of the VLP. Image (b) clearly shows that the AuNP (pink) is neither fully outside nor inside the capsid, rather occupying the place of a capsomere and sitting squarely in the capsid shell. Image (c) shows that VLPs retain their capacity to enter susceptible cell lines (NIH-3T3) after gold incorporation and can be detected inside cells by electron microscopy. The black arrows point to three VLPs: VLP1 and 2 have been taken up by a cellular organelle, most likely a caveola. VLP1 does not contain a visible AuNP, while VLP2 does. The white arrows highlight the organelle membrane. VLP3 has just attached to the cell membrane.
Structural information on viruses is usually obtained by x-ray crystallography using large amounts of purified virus. However, this technique is not applicable to viruses in vivo. An alternative method is required, such as Raman spectroscopy, a powerful technique good for examining structure and conformational changes in molecules. It is insensitive to water and can probe molecules in solution, e.g., in cells. The Raman spectrum of a typical protein consists of some 30 well-resolved bands in the 500–1750cm-1 region. Normal mode band assignments are commonly used as sensitive and selective structural fingerprints and to probe intra- and intermolecular interactions and dynamics. Time-resolved Raman spectroscopy therefore seems to be the ideal tool to elucidate assembly pathways and structural intermediates in viruses. To date, it has been used to analyze bacteriophages in vitro.7–9 However, the Raman signals of a complex in vivo setting are notoriously weak, hence our use of SERS, which enhances the Raman signal of molecules close to silver or gold nanoparticles. Our first efforts were focused on fabricating a virus-gold hybrid to exploit the SERS signal enhancement.
We then obtained a 3D representation of our hybrid virus-like particle by taking a series of tilt images with an electron microscope followed by computer reconstruction as shown in Figure 1(a,b).10 The imaging and subsequent biochemical analysis revealed that the incorporated AuNPs replaced one viral subunit, with SH-bond formation between two cysteine residues and molecular gold. Before incorporation, the cysteines formed disulfide bonds between the viral subunits.6 The size of the incorporated AuNPs was in the 5–7nm range, similar to that of the viral subunits. We also demonstrated that the incorporation of AuNPs into the capsids did not interfere with the ability of the virus to enter susceptible cells, as shown in Figure (1c).
We also recorded SERS spectra of assembled and disassembled viruses mixed with gold nanoparticles in vitro, which yielded distinct spectral signatures, as shown in Figure 2. Overall, the spectra were reproducible with high accuracy. The spectra of both forms also had the same general features, indicative of minor conformational changes during disassembly.
Figure 2. Assembled (a) and disassembled (c) VLPs show distinct Raman spectra in vitro. EGTA and dithiothreitol, the agents used for disassembly, are Raman-insensitive at the concentrations used. A disassembly intermediate is shown in (b), in which some capsids are still (partially) assembled, while others have already dissolved into capsomeres. This intermediate only appears transiently, and no Raman spectrum could be recorded. White arrows point to capsids and black arrow to capsomeres.
Our work thus yielded a multimodal viral vector for imaging and therapy. An AuNP incorporated into a capsid shell leaves the inner cavity free to transport a potential payload for therapy. In this context, the imaging could be done by diffraction or electron microscopy, or by magnetic resonance imaging. In addition, the gold itself could be used therapeutically. For instance, in hyperthermal therapy, laser or microwave radiation could be focused on the metal particles to heat them up and kill the surrounding tissue.
We showed that SERS can be used to differentiate between assembled and disassembled capsids in vitro. In future experiments, we intend to combine the techniques which we have pioneered to obtain resolved SERS spectra of (dis-)assembly intermediates from internalized viruses in vivo. It was recently shown that SERS spectra can be obtained from intracellular AuNPs.11 Having demonstrated the ability of hybrid particles to enter living cells, we hope to use them to track virus movement from the cell surface into the cytoplasm during viral infection. Obtaining time-resolved SERS spectra from internalized viruses would undoubtedly provide new insights into how conformational changes affect disassembly and therefore trafficking in vivo.
Australian Institute for Bioengineering and Nanotechnology
University of Queensland
Marcus Niebert is the coordinator of the challenge project ‘Cellular Delivery of Nanoparticles’ at the Australian Institute for Bioengineering and Nanotechnology of the University of Queensland. He obtained his PhD at Johann Wolfgang Goethe University in Frankfurt, Germany.
2. J. Forstova, N. Krauzewicz, V. Sandig, J. Elliott, Z. Palkova, M. Strauss, B. E. Griffin, Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells, Hum. Gene Ther. 6, no. 3, pp. 297-306, 1995.
3. K. T. Nam, D.-W. Kim, P. J. Yoo, C.-Y. Chiang, N. Meethong, P. T. Hammond, Y.-M. Chiang, A. M. Belcher, Virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes, Science 312, no. 5775, pp. 885-888, 2006.
4. C. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, A. M. Belcher, Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires, Science 303, no. 5655, pp. 213-217, 2004.