Thank you.

So today I'm going to talk about the characterization of rabies virus phosphoprotein and its thermoresponsive

liquid-liquid phase separation.

So just to give you a brief introduction on rabies and rabies virus.

Rabies virus causes rabies.

And the estimated population death per year is about 60,000 people

according to World Health Organization, but there is probably more than that. It's probably underestimated.

99% of the human cases of rabies are caused by dog bites and scratches

from dogs and it's mainly present in Asia, Africa and a little bit in South America.

Once you start to develop the symptoms of rabies, the fatality rate is 100%.

So you are sure you're gonna die from this.

But there is a vaccine that you can get before exposure or even after exposure

before you start to get symptoms.

So the rabies virus has a bellet shape

like this.

Its genome is composed of a negative sense RNA. It encodes for five proteins.

And we are in the lab, we are interested in the phosphoprotein, mainly

nuclear protein and how they assemble to form the ribonucleotide capsid that is forming with a nuclear protein and a phosphoprotein.

So the genome is encapsidated with those two proteins.

All right.

So if we look at the phosphoprotein and its structure, I'm sorry, my slides are a bit funny.

So the phosphoprotein is composed of different domains. So there is a nuclear,

binds the nuclear protein, this domain, the C-terminal domain. There is a dimerization domain and there is

intrinsically disordered regions. So this region here, the N-terminal domain, is known to bind the free

nuclear protein. There is an all region domain and another domain here that is known to bind other proteins.

So the B-protein is a multitasking

protein that orchestrates interplay between different components of the replication and translate transcription machinery.

So it's known to interact, as I mentioned, with a free nuclear protein with a nuclear protein bound on RNA,

but also known to interact and be a cofactor of the polymerase.

So more than 100 years ago,

Aldo

Negri, sorry,

which was an Italian scientist,

identified membrane-less organelle present in the neurons of patients who died from rabies and

they are called Negri bodies since then. So here you can see a picture of in green Negri bodies and in blue,

this is the bullet-shaped rabies viruses.

And basically what we found out in other labs

identified is that those Negri bodies are viral factories.

So basically inside these, a reaction transcription of the genomic RNA replication and assembly of the

ribonucleocapsids takes place.

While working on those

interactions in the lab, we found out that the phosphor protein by itself is able to induce phase separation.

So here I'll show you just a picture of the protein at 4 degrees and 20 degrees and here you can see droplets formation.

And so we started to look at the

biophysics behind the formation of those liquid droplets.

It's all shifting, that's okay. So here I present to you the experiments we've used to look at those

investigate

this formation of liquid-liquid phase separation.

So we use first a bright-field microscopy just to look at the droplets.

We use turbidity, optical density measurement at 350 nanometers just to look at the formation of the droplets.