following fusion in real time? - (Aug/12/2005 )

Hi everyone,

I'm studying the fusion reaction of HIV-1 with target cells. I'm mostly interested in amino acid mutations that confer resistance to a certain entry (fusion) inhibitor. One of the proposed models for ressistance is that the actual fusion reaction would proceed much faster (thereby reducing the time window the inhibitor can be actively inhibiting fusion).

This leads me to the question if other people think it is technically possible to follow the fusion of 2 membranes in Real Time, by for instance labelling the membranes of virusses (or cells expressing the proteins involved in HIV-1 fusion which renders these cells capable of fusion to other cells) with for instance R18 and target cells with F18 and then measuring FRET between these two. (I know there are other combinations possible or that dequenching of just 1 is possible two, I'm just curious about the principle). I realise I would have to do a lot of background measurements as well, but I am just wondering about the theoretical possibility of doing it this way. I would prefer doing the measurment in a sensitive plate reader (we're buying one soon, the grant is accepted, to the money will roll right in ) instead of confocal microscopy (because this just measures single events...).

anyone with any thoughts on this?

Hi,

can you use a GFP-labelled capside and look for virions entry inside the cells by confocal microscopy? It is possible to put the cells at 37°C in the presence of CO2...

I'm not sure the reader plate could be sensitive for FRET or quenching with such a reaction especially in the early stages of fusion with cell membrane

I think you will learn more with confocal or epifluorescence microscopy since all stages of the reaction will be seen

Seb_

Thanks for your reaction.

I have thought about a gfp-tagged gag protein, but the problem is that the exact mechanism of the inhibitor I'm doing my research on isn't 100% known. Most believe it works before the two membranes actualy have phospholipid-exchange,while others point out that it works after the formation of a small fusion pore, inhibiting expansion of that pore.

In the first model, fusion could be monitored by the spread of "cytoplasmic" (matrix for the virus) dyes or gpf-tagged proteins, while in the other model it couldn't be monitored like that. Also, the viral genome itself being very small, tagging one protein with gfp isn't an easy thing to do, which is why a lot of dye-exchange is carried out with cells labelled with the envelope proteins.

I was thinking of doing these experiments on a "large scale", for instance a 6-well plate instead of a 96-plate, thereby increasing the signal (but sadly enough also the background).

Also, even if the quenching or FRET wouldn't be sensitive enough to really capture the very first fusion events, given that fusion occurs faster for some mutant virusses, it should be still possible to measure FRET sooner for these mutants? (if it begins sooner, the intensity it takes to reach measurable quantities is achieved earlier).