More than 5 years later...

More than 5 years ago, before starting my thesis, and even before my interest in microscopy, I finished my EPHE diploma. This engineer like diploma is specific to one old school, and has the purpose to form scientific through laboratory, instead of university. I had the chance to found a very good laboratory with the Dr Anna SALVETTI, and started to learn molecular and cellular biology as well as virology in the virology laboratory of the ENS of Lyon.

From them, I learnt all the important manual skill to transform me as an engineer. But more importantly, I learnt how to think scientific. Decompose fact, construct hypothesis, test and trial these hypothesis, and the most important point, accepting the failure.

One method to observe a virus, especially its replication, is to observe it by microscopy, or more precisely, its effect on the host cell, like the shape. This heavy use of microscopy make me more and more curious about them, justifying my enrollment in a microscope specialized Master degree and the following PhD.

To come back on the virus, I just realized that it has been more than 5 years since my defense. Putting my work on the public domain. This work, made in French, is partially available on the school website at the following address : https://hal-ephe.archives-ouvertes.fr/hal-01466623, and can be cited like the following:

Simon Leclerc. Association et interactions fonctionnelles entre le génome du parvovirus humain adéno-associé et les domaines nucléaires SC35 . Biologie cellulaire. 2012.

The school version is without images and figures, probably to light off the file. I still have the figures, a total of 34, made with power-point (shame on me).

The virus that I studied is the AAV, or adeno-associated virus, that is a Parvovirus infecting humans but harmless. It is also a good candidate for gene therapy, and a lot of type is actually in clinical trial, with the most famous is probably a treatment for the Leber congenital amaurosis, specifically the RPE65. It is know that this virus replicate in the nucleus of infected cell (with a helper virus), but no work was realized to observe the interaction between the virus and the nuclear bodies composing the nucleus.

At first, be able to detect the nuclear bodies as describe in the literature by immuno-fluorescence (IF). Sc35 si the speckles, coiline the Cajal, PML the PML bodies, and RAP1 the telomere (maybe) and CENPA the centromere of the chromosome.

IF of different nuclear bodies, scale bar 5 microns.

After successful visualization, here the dual view between the nuclear body and the virus, still visualized by IF. The Cajal body and the speckles display the most interesting pattern.

Two nuclear bodies (Cajal in A and speckles in B) change in aspect after viral infection (protein Rep)

At this time, the speckles, shown with the protein SC35, is the most interesting for me. The speckles are known to be a splicing factor storage area. The virus, AAV, make a replication center in the nucleus of the cell, visualized by the protein rep, and where viral DNA replication, RNA transcription and virus encapsulation is realized.

A time-lapse experiment was realized using a Hela cell expressing SC35-GFP and infected by AAV expressing the Rep-Cherry protein.

These experiments showed that SC35, and maybe the speckles, was interacting directly or indirectly with the viral replication center.

This was confirmed by realizing immunoFISH, with the detection of all viral DNA and single strand DNA.

infected cell where the viral DNA (A) and single strand viral DNA (C) is observed with the SC35 protein.

Then by looking at the viral Rep RNA.

Infected cell with viral RNA and SC35 protein detection.

Finally, we wanted to see if the depletion of SC35 (by shRNA) have an impact on the virus. By WB and RT-qPCR, we see no significant differences in the RNA expression and protein production. However, the viral DNA production was 8 times lower than in normal cell. The most interesting result comes from the aspect of the replication center, that loose their integrity.

If these results (with other not shown) were enough for me to graduate. It is not enough for publication. In this case, you want to know if this effect is only caused SC35 or the speckles in general, and then try with other members of the speckles. You want to understand why the DNA replication and not the RNA? What is the mechanism?

Here is the end of the first post. Other will be more about scientific opinion than previous work.

Thanks to have read up to the end.