Structural elements of RABV nucleoprotein required for the formation of biomolecular condensates in a cellular minimal system.

A) Ribbon representation of the crystal structure of RABV nucleoprotein (PDB 2GTT). Three protomers are shown in complex with RNA (in magenta). The RNA molecule is located in a groove between N amino-terminal domain (NTD) and carboxyterminal domain (CTD). Both domains are extended by two elongated arms (NTarm and CTarm). The central protomer is rainbow colored from N (blue) to C (red) terminus. The loops L1 and L2, absent in the crystal structure, are represented by dotted lines and their position indicated by arrows. The insertion sites between residues 128–129 and 130–131 are also indicated by arrows. B) BSR-T7/5 cells were co-transfected with pTit plasmids encoding P protein and the indicated fluorescent (GFP-N, N-106GFP107, N-112GFP113, N-128GFP129 and N-130GFP131) or non fluorescent N constructions. Cells were fixed 24 h post transfection and the formation of N-P inclusions was assessed by fluorescence confocal microscopy. P was revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 goat anti-rabbit IgG. Scale bar: 10 μm. C) N/P-mCherry inclusions as well as GFP-N/P, and N-128GFP129/P inclusions (observed in BSR-T7/5 cells co-transfected as in B) were photobleached and the recovery of fluorescence was imaged with a Spinning-Disk microscope. Scale bars: 5 μm. For the plots on the right, FRAP data were corrected for background, normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD. D) Sequence of N mutants in which one or several stretches of amino acids in loops L1 and L2 have been replaced by one or several GGGS linkers (L). E) BSR-T7/5 cells were co-transfected with pTit plasmids encoding the indicated N mutant (mutated in L1 and L2) and the P protein. Cells were fixed 24 h post transfection and the formation of N-P inclusions was assessed by fluorescence confocal microscopy. N was revealed with a mouse monoconal anti-N antibody followed by incubation with Alexa-488 goat anti-mouse IgG antibody and P was revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 donkey anti-rabbit IgG. Scale bar: 10 μm. F) View of the RNA binding groove of the crystal structure of RABV N. Arginines pointing toward the phosphodiester backbone (in sticks representation) are indicated. G) BSR-T7/5 cells were co-transfected with pTit plasmids encoding the indicated N mutant and the P protein. Cells were fixed 24 h post transfection and the formation of N-P inclusions was assessed by fluorescence confocal microscopy. N was revealed with a mouse monoclonal anti-N antibody followed by incubation with Alexa-488 goat anti-mouse IgG and P was revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 donkey anti-rabbit IgG. Scale bar: 10 μm.