Gag-VLP are empty HIV capsids made solely from the Gag protein. They can be fused with fluorescent molecules, are devoid of pathogenicity and still retain some of their viral properties namely: same budding mechanism, same size distribution.

Authors: Charlotte Floderer, Jean-Baptiste Masson, Elise Boilley, Sonia Georgeault, Peggy Merida, Mohamed El Beheiry, Maxime Dahan, Philippe Roingeard, Jean-Baptiste Sibarita, Cyril Favard & Delphine Muriaux 

#nanoparticle #SuperResolution #VirusLike #biomimetic #nanoscopy

How I met with Gag-VLP

Super-resolution microscopy often sheds new (and always beautiful) light on existing biological mechanisms. 
When I read the recent paper in Nature Scientific Reports Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells I was thrilled to know more and got in touch with Delphine.

In case you want a quick overview of the paper, please follow me below.

Odoo • Image and Text credit: (c)

First, why are VLP important?

When I discover a new research tool, I can't help wondering to myself: what challenge is awaking these scientists every morning? What drives them to innovate?  

In the case of VLP, the challenge is having something as much virus-like as possible while still being safe to handle.

Research is often about frequent monitoring, quick iterations and swift adjustments. 

Now imagine having to wear/unwear a complete overall with blouse, gloves, facemask and going through a decontamination process just to look at something under a microscope… 

Respecting P3 Biosafety conditions means that even simple actions will necessitate some degree of planning and organization to save time and more importantly spare risks.

Hence, scientists are always seeking for alternatives: models that are safer/simpler to use while retaining most or some of the properties of the original. Sometimes, bacteria and viruses are used as surrogates from their more virulent counterparts. Sometimes sub-components of the pathogens are used to reproduce a given aspect of the infection process as it is the case for Gag-VLPs.

How do Gag-VLP work?

Gag-VLP were created to study the viral assembly and budding of HIV.

This mechanism occurs at the end of the infection cycle when newly synthesized viruses exit the cell to spread the infection.

Because studying the pathogen is difficult, there are many unknowns concerning the budding process notably the amount of time and energy it takes.

Fortunately, transfecting cells with the capsid protein Gag is sufficient for the cells to produce buds of virus-like particles, aka VLP.

Odoo • A picture with a caption
Schematic of the late phases of HIV infection. The GAG protein is represented in green and red. Image Credits © : Nature Scientific Reports

1. Playing with the Gag protein

Jurkat Cells ( immune cells surrogates) were transfected with a Gag(i)mEOS2 DNA construct to mimic the budding of HIV as it occus during infection.

With this much simpler/safer model, the scientist studied mutants of the GAG protein to identify which subsections were most crucial for the budding mechanism.

In addition the fused mEOS protein  enabled the detection of VLP in super-resolution microscopy from live samples which was something never done before.

Odoo • Text and Image
Odoo • Text and Image   Image Credits © : Nature Scientific Reports

2. Imaging the budding using advanced optical microscopy

In super-resolution microscopy the operator controls the fluorescence emission (through blinking or selective deactivation) to bypass the diffraction limit of optical imaging. This enables the detection of nanometer sized objects such as the VLP. 

TIRF microscopy, on the other hand, uses the total internal reflection to selectively illuminate a thin (200 nm) layer of sample close to the glass slide. As the cell’s membrane is usually located within this area, TIRF is the tool of choice to observe membrane dynamics without disruption from the inner parts of the cell. 

Combine those two and you have a great tool to study nanosized objects moving close to a cell's membrane such as VLP budding!

Odoo • Text and Image Image Credits © : Nature Scientific Reports

3. DATA Analysis

See the figure on the right where you may recognize a budding event in super resolution microscopy.

As the Gag proteins assemble into a budding site, their density increases while their diffusivity decreases. 

Concomitantly the number of fluorescence events in the field of view is decreasing as the VLP is progressively exiting the observation area. 

Yet, extracting the mean budding time and trapping energy from those image acquisitions required a lot of data analysis  and mathematical models.

I will try to explain it with my own words below.

Odoo • Text and Image
 Image Credits © : Nature Scientific Reports
Odoo - Sample 1 for three columns

 Image Credits © : Nature Scientific Reports

They tracked the Gag proteins over short (0,2s) time frames using live PALM so they could tag budding events (white circles).

Odoo - Sample 2 for three columns

  Image Credits © : Nature Scientific Reports

In the vicinity of those tagged budding areas, they measured the movement of those single Gag proteins using Bayesian inference and their aggregation using Voronoi tessellation.

Odoo - Sample 3 for three columns

 Image Credits © : Nature Scientific Reports

Finally from those measurements they extracted the budding time and energy maps of single VLP formations.

Closing: models in biology 

Biology is always looking for more accurate, more ethical and safer models.

Here fusing Gag with mEOS and transfecting it into cells not only made for a safe budding model but it also opened the road for the high tech microscopy and the cutting edge data analysis. 

And there are plenty of opportunities to find beyond the study of budding...

Come think of it: a safe, biological, nano-object of standard size and tunable fluorescent composition? This might make one heck of a calibration tool!


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