Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation

Abstract

BackgroundHIV-1 replication is critically dependent upon controlled processing of its RNA and the activities provided by its encoded regulatory factors Tat and Rev. A screen of small molecule modulators of RNA processing identified several which inhibited virus gene expression, affecting both relative abundance of specific HIV-1 RNAs and the levels of Tat and Rev proteins.ResultsThe screen for small molecules modulators of HIV-1 gene expression at the post-transcriptional level identified three (a pyrimidin-7-amine, biphenylcarboxamide, and benzohydrazide, designated 791, 833, and 892, respectively) that not only reduce expression of HIV-1 Gag and Env and alter the accumulation of viral RNAs, but also dramatically decrease Tat and Rev levels. Analyses of viral RNA levels by qRTPCR and RTPCR indicated that the loss of either protein could not be attributed to changes in abundance of the mRNAs encoding these factors. However, addition of the proteasome inhibitor MG132 did result in significant restoration of Tat expression, indicating that the compounds are affecting Tat synthesis and/or degradation. Tests in the context of replicating HIV-1 in PBMCs confirmed that 791 significantly reduced virus replication. Parallel analyses of the effect of the compounds on host gene expression revealed only minor changes in either mRNA abundance or alternative splicing. Subsequent tests suggest that 791 may function by reducing levels of the Tat/Rev chaperone Nap1.ConclusionsThe three compounds examined (791, 833, 892), despite their lack of structural similarity, all suppressed HIV-1 gene expression by preventing accumulation of two key HIV-1 regulatory factors, Tat and Rev. These findings demonstrate that selective disruption of HIV-1 gene expression can be achieved.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-017-0330-0) contains supplementary material, which is available to authorized users.

An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing

“…Rev is critical in mediating nuclear export of incompletely-spliced (US/SS) HIV-1 RNAs to the cytoplasm while Tat is essential as a transactivator of viral transcription [35][36][37][38]. These results (and data on HIV-1 RNAs and SR proteins described below) are in mark contrast to previous HIV-1 RNA-processing inhibitors reported, suggesting a novel mechanism of action [27,28,33,[39][40][41][42][43].…”

“…In comparison with other HIV-1 RNA processing inhibitors, 5342191 induces fewer alterations to host AS events (0.25-0.67% change and r = 0.97 compared to DMSO, resp., in Fig 2E and 2F) than those detected for the CS digitoxin (20.6%) and ABX464 (r = 0.89) by RNA-Seq or chlorhexidine (8.1%) by exon microarray [41,72,73]. 5342191-induced changes in AS convert to only 0.46% of host genes becoming DE by � 2 fold in cells ( Fig 2G) which is lower than those reported for other HIV-1 RNA processing inhibitors at this threshold (and same cell type): 1C8 (0.95%) and 9147791 (0.75%) [40,42]. Although not previously reported for any other HIV-1 RNA processing inhibitors, 5342191 perturbs the abundance of only 0.02-0.34% of host proteins by � 1.5-2.0 fold ( Fig 3D) which is over 2.2-6.5-fold lower than the changes observed in 9147791-treated cells at this cut-off (0.13-0.75%, S9C Fig).…”

“…The possibility that 5342191 may be affecting Tat expression by altering its synthesis/degradation ( Fig 3A and 3B) raised the question of whether host proteins were similarly affected. To test this possibility, HeLa rtTA-HIV-ΔMls cells were treated with 5342191, 9147791 (a previously identified HIV-1 RNA processing inhibitor) [40], or DMSO (+/-Dox) and abundance of proteins analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using tandem mass tags (TMT) [57]. From volcano plots (S8A-S8C Fig) of the abundance and statistical significances of 5,326 proteins identified by TMT LC-MS/MS (S4 Table), a cut-off of � 1.5-fold change was selected to identify the proteins which were substantially affected above the normal ranges of each treatment ( Fig 3C-3E and S9A-S9E Fig).…”

Inhibition of HIV-1 gene transcription by KAP1 in myeloid lineage

“…Thereby, inhibiting Tat activity results in hindering the positive feedback loop required for efficient HIV-1 transcription. Interestingly, Tat has been shown to be sensitive to proteasome degradation (39)(40)(41)(42)(43)(44)52). We observed that Tat expression was inversely proportional to KAP1 expression level suggesting that KAP1-mediated repression of Tat function results from Tat degradation.…”

“…Among the mechanisms controlling protein half-life in eukaryotic cells, the degradation via the proteasome pathway has been long studied. Interestingly, Tat has been described to be directed towards proteasome degradation (39)(40)(41)(42)(43)(44). To determine whether the reduced expression of Tat in the presence of KAP1 is related to proteasome degradation, we quantified Tat expression levels in the presence of KAP1 and the proteasome inhibitor MG132, (Figure 8 D).…”