Cryo-electron Microscopy of Rhinovirus Uncoating Intermediates: Geometry of Membrane Attachment and Conformation of the RNA Genome
Mohit Kumar, Shushan Harutyunyan, Heinrich Kowalski and Dieter Blaas
Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Austria.
Human rhinoviruses (HRVs) are the main cause of the common cold. During uncoating they undergo conformational changes, first converting into the subviral A-particle and finally, on releasing the RNA genome, into the (empty) B-particle. Mimicking in vivo conditions of uncoating in the late endosome we attached the A-particle to the liposomes and demonstrated that the RNA was transferred into the liposomal lumen upon acidification; arrival of the RNA was shown with RT/PCR (1) and fluorescence correlation spectroscopy. Single particle 3D cryo-electron microscopy reconstruction (cryo-EM 3DR) of membrane bound A-particles showed that they are attached via one of the 30 two-fold icosahedral axes (2). In Enteroviruses, such as poliovirus, it has been shown that the RNA was released close to a 2-fold axis when native virus was heated to 56°C. When the RNA was crosslinked with psoralen within native HRV-A2 followed by exposure to 56°C, particles representing an intermediate stage different from the classical A- and B- subviral particles accumulated. Cryo-EM 3DR revealed a rod-like internal density, which presumably represents the condensed form of the viral RNA. One end of this ‘rod’ was near a viral icosahedral 2-fold axis (4). This suggests that the rhinoviral RNA was in the process of being released but got stuck because double stranded regions could not be unfolded as a consequence of the crosslinking. It remains to be seen whether such condensed RNA plays any role during uncoating under more physiologic conditions. A mechanism imparting directionality to the genome release process might be common to many icosahedral non-enveloped single stranded RNA viruses.
References:
1. Gerhard Bilek, Nena M. Matscheko, Angela Pickl-Herk, Victor U. Weiss, Xavier Subirats, Ernst Kenndler, and Dieter Blaas (2011). Liposomal Nanocontainers as Models for Viral Infection: Monitoring Viral Genomic RNA Transfer through Lipid Membranes. J. Virol; 8368–8375.
2. Mohit Kumar, Dieter Blaas (2013). Human Rhinovirus Subviral A Particle Binds to Lipid Membranes over a Twofold Axis of Icosahedral Symmetry. J. Virol .87; 1309–11312.
3. Shushan Harutyunyan, Mohit Kumar, Arthur Sedivy, Xavier Subirats, Heinrich Kowalski, Gottfried Köhler, Dieter Blaas (2013). Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3’-End. PLOS Pathogens; 9.
I would like to thank my professor Dr. Dieter Blaas for his continuous support during my PhD. My PhD committee, Dr. Thomas Marlovits and Prof. Kristina Djinovic-cargo for their important inputs and discussion. I would also like to thank Prof. Heinrich Kowalski and Prof. Holland Cheng for their valuable suggestions and support, and finally my colleagues from the lab.