How do icosahedral viruses package their RNA genome?
The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified some assembly pathways, few experimental measurements are available to date due to the difficulty of detecting biological molecules in water over a wide range of timescales.
Cowpea chlorotic mottle virus (CCMV) is an icosahedral RNA virus infecting plants. The virus was spontaneously reconstituted from purified proteins and genomic RNA, and its assembly was probed by using time-resolved small-angle X-ray scattering with a synchrotron source. Measurements revealed that the adsorption of proteins on RNA occurs in a few tens of milliseconds, while the structural reorganization of the formed species can take several hours. Before the completion of virus, the proteins are loosely bound on the RNA, which may ensure a good selectivity for the viral genome during assembly in host cell. The structural reorganization is limited by an energy barrier, thus minimizing the misassembly of the protein shell. Quite unexpectedly, viruses packaging synthetic polyelectrolytes are reconstituted more easily than viruses packaging genomic RNA, which should promote further studies on the role of genome in the self-assembly dynamics.
(Top, from left to right) Cryo-electron microscopy images of partially reconstituted viruses, completely reconstituted viruses and reconstitued viruses packaging polyelectrolytes. Scale bar is 30 nm. (Bottom) Schematical representation of a self-assembling virus superimposed on experimental X-ray scattering intensities. [Nat. Commun. 9 (2018) 3071]
A viral nanocage assembled within an X-ray beam
We have investigated the self-assembly of capsid proteins derived from a norovirus by time-resolved small-angle X-ray scattering (TR-SAXS) with a synchrotron source. The three-dimensional structure, at nanometre resolution, of an intermediate species that plays a pivotal role in the self-assembly was extracted through an original global fitting algorithm applied to the time-resolved data. We found that in the first step, some ten dimers combine to form this stave-shaped intermediate possibly made of two pentamers of dimers connected by an interstitial dimer. In the subsequent, slower step, which takes hours, these intermediates interlock into a capsid. In contrast, capsids form by sequential addition of dimers in many other viruses such as the hepatitis B virus.
By clarifying the kinetics involved in norovirus assembly, this study provides a better understanding of the physical processes at work in the self-assembly of a viral capsid. It could also advance efforts to treat or prevent these infections, and it could be applied to engineer viral nanocages to make diagnostic agents and tailored therapeutics.
Kinetic scheme of norovirus capsid assembly. Free dimers are represented in magenta, dimers related by five-fold symmetry in the final capsid in blue, and interstitial dimers in red. Above the last assembly step, a representation of interlocking intermediates is given as a possible mechanism. Six intermediates, each in a different color, have been positioned above the six contiguous fragments made of two pentamers of dimers connected by an interstitial dimer. [J. Am. Chem. Soc. 135 (2013) 15373]
Related publications
- M. CHEVREUIL, D. LAW-HINE, J. CHEN, S. BRESSANELLI, S. COMBET, D. CONSTANTIN, J. DEGROUARD, J. MÖLLER, M. ZEGHAL, G. TRESSET (2018) Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte. Nat. Commun. 9 3071.
- G. TRESSET, M. CASTELNOVO, A. LEFORESTIER (2017) Assemblage et désassemblage des virus : Mode d’emploi. Reflets de la Physique 52 22-26.
- D. LAW-HINE, M. ZEGHAL, S. BRESSANELLI, D. CONSTANTIN, G. TRESSET (2016) Identification of a major intermediate along the self-assembly pathway of an icosahedral viral capsid by using an analytical model of a spherical patch. Soft Matter 12 6728-6736.
- D. LAW-HINE, A. K. SAHOO, V. BAILLEUX, M. ZEGHAL, S. PREVOST, P. K. MAITI, S. BRESSANELLI, D. CONSTANTIN, G. TRESSET (2015) Reconstruction of the disassembly pathway of an icosahedral viral capsid and shape determination of two successive intermediates. J. Phys. Chem. Lett. 6 3471-3476.
- G. TRESSET, C. LE COEUR, J.-F. BRYCHE, M. TATOU, M. ZEGHAL, A. CHARPILIENNE, D. PONCET, D. CONSTANTIN, S. BRESSANELLI (2013) Norovirus capsid proteins self-assemble through biphasic kinetics via long-lived stave-like intermediates. J. Am. Chem. Soc. 135 15373-15381.