How you can distinguish between the mutation effect and the insertion effect, if there is any? I think you have to do the mutation part and see the results, then you may do the insertion step which is another kind of mutation and both unpredictable!. In this case you will be able to track the mutation effects!

Best luck.

Hi Mushtak,

Thank you for your response. I already did the clonings and the experiments. It is only afterwards that I am realizing that I have this 8 bp site in between the U3 and R regions of my LTR and am wondering if it would have any effect of its own? I do not want it to have any effect, which is my concern.

The only way to tell for sure is to try it and see. Test a virus with and without this site, but no other differences, and see if the results are different for whatever factor it is you are interested in (replication rate, time between infection and first round of replication, etc).

The "R" is the beginning of the viral transcript so that region is involved in a key part of HIV replication. The human RNA polymerase (transcriptase) and various other human proteins bind to the HIV proviral DNA integrated into the human genome nearby the R site, and direct the transcription to begin at a specific site in the proviral genome. In theory 8 bases might make a difference, but it would depend on which 8 bases and real life is more complicated than our theories can always predict, so you should just test it and see.

https://www.hiv.lanl.gov/content/sequence/jbrowse/?loc=HXB2%3A416..467&tracks=DNA%2CHXB2_LTR_sites_of_interest&data=hivdata%2FEpitope-nucleic&highlight=

https://www.ncbi.nlm.nih.gov/pubmed/?term=immunodeficiency+transcription+initiation

Hi Brian,

Thank you so much. You are right. In hindsight, I should have also cloned a control where I performed the exact cloning but with a U3 region that had no mutations. And then compare it to a wildtype virus to see the effects of just the 8 bp insertion and cloning itself.