Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

HIV-1 provirus, either as a chromosomal integrant or as an episomal plasmid in HeLa cells, forms a transcription-dependent gene loop structure between the 50LTR promoter and 30LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocks 50 to 30LTR juxtaposition, indicating that this structure is maintained during transcription. Analysis of mutant or hybrid HIV-1 plasmids demonstrates that replacement of the 50LTR promoter withCMVor the 30LTR poly(A) signal with a synthetic element (SPA) permits gene loop formation, suggesting that these interactions are not retroviral specific. In addition, activation of the 50LTR poly(A) signal or inactivation of the 30LTR poly(A) signal abolishes gene loop formation. Overall, we demonstrate that both ongoing transcription and pre-mRNA processing are essential for gene loop formation, and predict that these structures represent a defining feature of active gene transcription.