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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: FEBS J. 2017 Nov 8;284(23):4096–4114. doi: 10.1111/febs.14293

Figure 4. Enhancing proviral activity in HIV-NanoLuc clone E9.

Figure 4

A series of experiments were conducted to test the activity of the integrated provirus in clone E9. (A) Cells were plated in a 96 well plate and stimulated one time with either LPS (100ng/ml) or TNF-α (50ng/ml) for 24, 48, or 72 hrs (timeline in A.1). We observed significant increases in luciferase activity after simulation with both LPS (A.2) and TNF-α (A.3). Significant induction persists up to the 72 hr. time point. LPS: **p<0.0001, *p<0.0002; TNF-α: ****p<0.0001. (B) Cells were treated 1x, 2x or 3x with LPS or TNF-α to determine if proviral activity could be increased by multiple treatments (timeline in B.1). There is a significant effect of proviral induction with multiple treatments of pro-inflammatory factors; LPS (B.2), TNF-α (B.3). LPS: *p<0.0001 vs Untreated, #p<0.001 vs 1x treatment; TNF-α: *p<0.0001 vs Untreated, #p<0.0001 vs 1x treatment, (x0005E)p<0.0004 vs 2x treatment. (C) Myc-tagged HIV protein expression plasmids were developed from their corresponding coding sequence in the provirus. Plasmids were transfected into HEK293 cells for expression testing and Wes analysis was performed to confirm protein expression. (D) E9 cells were transfected with pTat-myc and pRev-myc to determine the effect of HIV regulator proteins on proviral activity. LPS was used as a positive control for induction and pBSII plasmid was used as a transfection control. pRev-myc transfection produced the same relative NanoLuc levels as pBSII transfection. pTat-myc significantly increased NanoLuc activity similar to the levels of LPS. *p<0.05 vs. untreated, **p<0.0001 vs. untreated. Data are expressed as the mean ± SEM. N=3 independent cell passages.