Advances in structural studies on TrV: Capsid Disassembly and RNA Release.

AGIRRE, J; SANCHEZ- EUGENIA, R; MARTI G. A; GAËL GORET; NEUMANN, E; LEGOFF, M; NAVAZA J; SNIJDER, J; UETRECHT, C; ROSE, RJ; WUITE, G; ROOS, W; HECK, A; GUERIN, D.M.A.

Cochabamba

Workshop; II International Workshop on Chagas Disease, triatomine vectors, Trypanosoma cruzi, and Triatoma virus.; 2012

Facultad de Medicina. Universidad Mayor de San Simón

Triatoma Virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid (T=1, p=3) that encloses a +ssRNA genome. Here we present results on the externalisation of the viral RNA and the genome influence on the stability of capsids as a function of the pH of the medium. Firstly, we employed electron microscopy (EM), intrinsic fluorescence, and light dispersion to observe that the virion is stable at acidic pHs. This result is in line with the acidic blood digestion of triatomines. Secondly, we did an analysis at neutral pH by using 3D cryo-EM reconstruction combined with the crystallographic atomic model. We present three reconstructions of the TrV capsid in different states. Along with other biophysical results, these reconstructions show that upon genome release, no striking structural changes are observed on the empty capsid proteins to respect their initial position in full particles. Finally, we analysed in vitro by atomic force microscopy nano indentation and native mass spectrometry, and from mild acidic to basic pHs, the capsid assembly/disassembly, stability, and genome uncoating. We show that at neutral pH the protein shell is stabilised but under increasingly alkaline conditions an opposite effect occurs: the RNA starts to destabilise the capsid. The RNA containing particles exhibit pronounced different mechanical characteristics as compared to the empty capsids, even though no major structural changes are observed. Therefore, these differences can be assigned to the presence/absence of the genome, revealing the intricate interplay between genome encapsidation and capsid stability. In summary, our results support in TrV a genome delivery mechanism with strong differences to respect the processes described for picornaviruses Human Rhino Virus 2 and Polio Virus. Acknowledgments: EN thanks the FBB and JN an Ikerbasque fellowship to support their visit to DMAG?s laboratory. JA was awarded by EMBO (ASTF 309-2008) to perform a stay at the IBS, Grenoble. RSE acknowledges a predoctoral grant from the Basque Government (BG). GAM is a researcher of the CONICET, Argentina, and is partially supported by ANPC-PICT Nº 32618/05, Argentina. DMAG received support from Bizkaia:Xede, UPV/EHU (IT-461-07), MV-2012-2-41 from BG, and MICINN (BFU2007-62062), Spain. This work was partially supported by Fundamenteel Onderzoek der Materie through the ?Physics of the genome? programme (to GJLW), by the Netherlands Proteomics Centre and by the Netherlands Organisation for Scientific Research. ALW-ECHO (819.02.10) to AJRH. DMAG thanks the Ibero-American Programme for Science, Technology and Development (CYTED, 209RT0364)