Viral Rhodopsins: Structure, Function and Next Generation Optogenetic Tools – Viral_Rhodopsins

Structural characterization:
1. Protein production, in vitro functional characterization by spectroscopic methods including photocycle determination by flash photolysis, and single molecule imaging.
2. Crystallization and structural studies of the proteins in the ground and intermediate states of the proteins. We will use complementary X-ray crystallography, including time-resolved at X-ray synchrotron and XFEL sources and cryo-EM in the case of oligomeric proteins.
Functional characterization:
1. The viral rhodopsins will be expressed in Xenopus oocytes and characterized in terms of light-dependency and selectivity using the single-channel patch clamp technique as well as whole-cell microelectrode recordings. Because bacterial and viral membrane channels often express poorly at the plasma membrane, it will probably be necessary to add signal sequences or coexpress chaperone proteins to promote membrane trafficking.
2. Functional characterization of the newly designed light-driven proteins in vivo including their activity in neuronal cells. The viral proteins will be expressed first in mammalian cells, then in neuronal culture and in hippocampal slices. The electrophysiological properties of each protein, along with its ability to modulate neuronal firing, will be evaluated and compared with existing opto and optopharmacogenetic tools.

The ultrahigh resolution structure of OLPVR1 at 1.4 Å was obtained by X-Ray chrystallography.
Several viral rhodopsins were expressed in xenopus oocytes. The function of OLPVR1 was studied in detail and was found to be unique among all rhodopsins studied to date.
OLPVR1 was expressed in mammalian HEK293 cells and its function was confirmed.
A lentivirus construct incorporating OLPVR1 was designed for expression and characterization in native tissues.

The results should shed light on the role of viral rhodopsins in host infection and could form the basis of new tools for optogenetic applications.

Zabelskii, D., Alekseev, A., Kovalev, K., Rankovic, V., Balandin, T., Soloviov, D., Bratanov, D., Savelyeva, E., Podolyak, E., Volkov, D., Vaganova, S., Astashkin, R., Chizhov, I., Yutin, N., Rulev, M., Popov, A., Eria-Oliveira, A.S., Rokitskaya, T., Mager, T., Antonenko, Y., Rosselli, R., Armeev, G., Shaitan, K., Vivaudou, M., Büldt, G., Rogachev, A., Rodriguez-Valera, F., Kirpichnikov, M., Moser, T., Offenhäusser, A., Willbold, D., Koonin, E., Bamberg, E. and Gordeliy, V., 2020, Viral rhodopsins 1 are an unique family of light-gated cation channels, Nat. Commun. 11: 5707.
Ávalos Prado P, Landra-Willm A, Verkest C, Ribera A Chassot AA, Baron A and Sandoz G. TREK channel activation suppresses migraine pain phenotype. iScience. (in press)
Patent application in preparation.

--- BACKGROUND ---
Light-activated proteins of the rhodopsin family, found in all three domains of life, have also been found to be encoded by viruses, specifically giant viruses. Little is known on these virus-encoded rhodopsins and on their possible role in viral infection. Their structure, their oligomeric organization, and their function in terms of transport and substrates are unknown. In spite of significant amino-acid sequence similarity, we have sufficient preliminary data to show that viral rhodopsins differ greatly from known rhodopsins, and can assemble in complexes resembling human pentameric ligand-gated ion channels, where the agonist is light. These proteins could therefore involve novel biological mechanisms and sustain possible applications in optogenetics.

--- OBJECTIVES ---
Our aims are to decipher the structure and the function of viral rhodopsins, and to examine the possibility of using these proteins as light-sensitive actuators in mammalian tissues. The results should shed light on the role of viral rhodopsins in host infection and could form the basis of new tools for optogenetic applications.

--- CONSORTIUM ---
The consortium assembles three partners with demonstrated, complementary expertise in the fields of structural biology (IBS-Membrane, Institut de Biologie Structurale, Grenoble), electrophysiology in model cells (IBS-Channels, Institut de Biologie Structurale, Grenoble), and electrophysiology and optogenetics in mammalian native cells (iBV, Institut de Biologie Valrose, Nice).

--- WORKFLOW ---
Viral rhodopsins are separated in two phylogenetic groups. We will work on representatives of each group, OLPVRI and OLPVRII.
Partner IBS-Membrane will perform structural characterization of OLPVRI and OLPVRII in their different conformational states by X-ray crystallography, time-resolved serial crystallography, and single-particle cryo-electron microscopy using advanced European instruments (Grenoble synchrotron, Hamburg X-ray free electron laser, and Grenoble Titan Krios). The partner will also perform in vitro functional characterization of the proteins.
In parallel, partner IBS-Channels will characterize the function the viral rhodopsins in model cells and optimize their properties by protein engineering informed by structural data.
Partner iBV will test the optogenetic potential of the proteins in neuronal cells and brain slices.

The proposed tasks are extremely challenging. However, the feasibility of the project is asserted by solid preliminary data and proven successful experience of the partners in the corresponding fields of science and methodologies. In particular, a high-resolution structure of OLPVRII in the ground state has already been obtained and functional characterization of OLPVRI has been started.