Hi,
I am trying to mutate HIV-1 reverse transcriptase and integrase to make dysfunction phenotypes. I am wondering are they necessary for TZM-bl cell infection assay (X-gal or luc)?
Another question: When 293T cell assembles HIV-1 virus, does it package intact mRNA transcripts coming from plasmid transcription? Or some virus can integrate into 293T cell first, and produce more RNA genome?
Thanks a lot!
Hi Nanxi
1. Yes, you need both RT and IN to make virus. Without these, the virus that gets produced from 293T cells will be incapable of replicating and integrating into the cell line used for the infection assay which measures either production from cell death or direct luciferase-based integration
2. The virus amde frmo 293T cells packages full-length intact viral RNA that are generated by transcription off of your plasmid. This is the main method by which the virus is produced. However, if your virus is pseudotyped with VSV-G then during the production phase some of this virus will actually infect the 293T cells, integrate, and produce even more virus. This is what results is such highly produced levels of virus. You can compare the difference in virtus produced by using a virus that is incapable of integration.
Dear Nanxi,
I was wondering if you were able to produce the viral particles. I am trying to express the RT and IN protein separately, but there is no protein expression. Do I need to express them in the same transcript design. Currently, I am trying to infect them separately.
Thanks!
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