Figure 6.

SEC promotes both initiation and elongation of transcription and is recruited to the HIV promoter via RNA. (A) Scheme of the HIV-Luc cassette and location of primers used for qPCR analysis and RNA IP. (B and C) In the absence of Tat, AFF1 and AFF4 promote initiation rather than elongation of transcription. RNA was isolated from cells over-expressing either HA-AFF1 or HA-AFF4 (panel B), or from cells where the corresponding protein expression was KO (C). Following cDNA synthesis, mRNA was amplified with the designated primers corresponding to either short (core promoter sites) or long transcripts, (luciferase coding genes). Results are shown as fold of mean enrichment relatively to data obtained in cells expressing LTR-Luciferase alone—set to 1. (D) P-TEFb modulates the association of SEC with HIV RNA. Cells expressing wild type or cyclin T1 KO cells were transfected with HIV LTR-Luciferase and subjected to RNA-immuno-precipitation (IP) with either αHA IgG, corresponding to HA-AFF1 and HA-AFF4. Non-immune human IgG served as control. RNA was extracted from immuno-precipitated and input samples (1%) and was then subjected to cDNA synthesis, which was further analyzed by qPCR using the indicated primers, located on either, short (core promoter sites) or TAR-Luc, located on both the TAR and N-terminal luciferase sequences. Primers that measured association to 7SK snRNA were also tested. qPCR reactions were performed in triplicates and presented as percentage of input over non-specific IgG.