I am trying to produce virus for my wild type GOI and a point mutation. For the production I am using a third-generation expression system consisting of RSV, VSV, MDL and my expression plasmid. I have an EF1a promotor and GOI-IRES-GFP sequence.

For the production of the virus I am using HEK 293T cells and MACsfectin. Harvesting the supernatant after 48h, followed by ultracentrifugation. To determine the viral titer, I do the sequential dilution of the concentrated virus, add it to HEK cells and analyse GFP expression via FACS after 48h.

This whole process works fine for the wild type protein I am trying to express. For the mutant however I am running into a strange problem:

In the initial production step I can clearly see the GFP expression in the HEK cells, so I am assuming they should produce my GOI aswell and package it into virus particles.

Now when i put the concentrated virus back onto the HEK cells I barely see any GFP expression even in the lowest 10^-1 dilution. I am talking about 4-5 cells out of 100.000.

The wild type virus that I have produced in parallel works totally fine.

I am assuming that my point mutation is slightly more toxic than the wild type, but I dont see differences in apopotisis (HEK cells) between WT and mutant during the virus production. Both plasmids are sequences and look fine.

Do you have an idea what experiments I could do to see at what step the problems occur?

Thanks for helping!