Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

Emmanuel T Agbottah,Georges C St Laurent,James Mcardle,Sambhav Karki,Christine Traviss,Ajit Kumar

doi:10.1186/1742-4690-4-41

Abstract

BackgroundExamination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.ResultsUtilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.ConclusionStructural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.

Highlights

Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication
The nascent transcripts from HIV-1 Long Terminal Repeat (LTR) contain a unique structured RNA domain within the 5'-nontranslated region known as the transactivation response (TAR) element which is critical for efficient transcription of viral promoter in response to Tat [3,4]
Isolation and identification of HIV-1 TAR binding proteins To detect proteins that interact with the TAR region of HIV-1 LTR, a three-step purification process was devised to fractionate nuclear proteins from HeLa cells, including a Sulpho-phosphate Sepharose (SP Sepharose) chromatography step followed by TAR RNA affinity chromatography

Summary

Results

Isolation and identification of HIV-1 TAR binding proteins To detect proteins that interact with the TAR region of HIV-1 LTR, a three-step purification process was devised to fractionate nuclear proteins from HeLa cells, including a Sulpho-phosphate Sepharose (SP Sepharose) chromatography step followed by TAR RNA affinity chromatography. The resulting complex was washed and bound proteins were resolved by 4–20% SDS/PAGE, and Western blotted with either anti-Tat or anti-NF90ctv antibody Results of such an experiment is shown, where wild type Tat bound to TAR RNA and the binding could be competed with excess wild type but not mutant TAR (TM26; compare lanes 4, 5, upper panel). Interference of Tat-transactivation by TAR RNA-NF90ctv binding results in histone methylation of HIV chromatin We previously reported that transcriptional activation of latent proviral DNA by TNF-α induction of OM10.1 cells, a promyelocytic cell line that contains single copy integrated proviral DNA, is mediated by Tat [12]. The data suggest an indirect role of NF90ctvmediated interference of Tat/TAR interaction that leads to the transcriptional repression

Background

Discussion

Siliciano RF