FUBP3 enhances HIV-1 transcriptional activity and regulates immune response pathways in T cells

Abstract

Far-upstream element-binding protein 3 (FUBP3) was identified at actively transcribing HIV promoters through chromatin affinity purification and mass spectrometry. Known for regulating cellular processes such as transcription and translation by binding to DNAs and RNAs, FUBP3's role in HIV transcriptional regulation was previously unrecognized. This study reveals that FUBP3 enhances HIV-1 transcriptional activation by interacting with Tat and trans-activation response (TAR)-RNA, critical for boosting viral transcription through recruitment of activating factors that promote RNA polymerase II (RNAPII) elongation. Transcriptomic analysis, chromatin immunoprecipitation, and biochemical assays demonstrated that FUBP3 associates with and stabilizes TAR-RNA, in a Tat-dependent manner, and enhances Tat steady-state levels via interaction with Tat's basic domain. Suppressing FUBP3 decreased HIV-1 transcription and altered expression of host genes linked to T cell activation and inflammation, underscoring its broad regulatory impact. Additionally, FUBP3 was enriched at active promoters, confirming its role in transcriptional regulation at specific genomic locations. These findings highlight FUBP3's critical role in the HIV-1 life cycle and suggest its potential as a therapeutic target in HIV-1 infection. Additionally, this study expands our understanding of FUBP3's functions in oncogenic and inflammatory pathways.

Keywords: FUBP3; HIV; MT: Oligonucleotides; RNA stability; T cells; TAR; Tat; Therapies and Applications; immune response; transcription.

Graphical abstract

Figure 1

FUBP3 is important for HIV transcription and replication (A) Knockdown efficiency for FUBP3 and CCNT1 monitored by RT-qPCR (left) and western blot (right) in Jurkat CD4⁺T cells. (B) HIV integration in Jurkat CD4⁺T cells quantified by ALU-PCR assay. (C) Effect of FUBP3 and CCNT1 depletion on Gag-pol abundance in infected Jurkat CD4⁺T cells over time post infection. (D) Viral protein abundance determined by ELISA in infected Jurkat CD4⁺T cell medium. (E) Effect of FUBP3 and CCNT1 depletion on Gag-pol abundance in infected primary CD4⁺T cells over time post infection. (F) Viral protein abundance determined by ELISA in infected primary CD4⁺T cell medium. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, as determined by two-way ANOVA with multiple comparisons. All data are reported as the mean ± SEM.

Figure 2

FUBP3 acts as an HIV transcription activator (A) shRNA depletion of FUBP3 and control CD8B in Jurkat-D6 cells by western blot. (B) Gag-pol transcript abundance upon FUBP3 depletion with or without stimulation (mock, PMA, SAHA) in Jurkat-D6 cells as determined by RT-qPCR. (C) Viral protein abundance determined by ELISA in Jurkat-D6 cell medium. (D) As for (A) but in J-Lat 10.6 cells. (E) Gag-pol transcript abundance upon FUBP3 depletion with or without stimulation (mock, PMA, SAHA) in J-Lat 10.6 cells. (F) Quantification of HIV (%GFP) upon stimulation with PMA. (G) Representative western blot analysis of FUBP3 and GAPDH protein levels in J-Lat 10.6 where FUBP3/CD8B were first depleted before the re-introduction of either FUBP3-WT (WT) or empty-vector (EV) clones. (H) Gag-pol transcript abundance with or without stimulation (mock or PMA) as determined by RT-qPCR. (I) Quantification of HIV (%GFP) upon stimulation with PMA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by two-way ANOVA with multiple comparisons. All data are reported as the mean ± SEM.

Figure 3

FUBP3 regulates pathways involved with inflammation and HIV infection (A) RNA-seq analysis of differentially expressed genes (DEGs) upon FUBP3 knockdown (KD) in J-Lat 10.6 cells (left), uninfected Jurkat cells (middle), and uninfected primary CD4⁺T cells (right). (B) Protein network of downregulated genes in J-Lat 10.6 cells upon FUBP3 depletion grouped by function using STRING db v12. Each node represents a protein, and line thickness indicates confidence in both functional and physical protein associations. (C) Bubble plot of a GSEA of the indicated pathways across the three cell models upon FUBP3 depletion. NES, normalized enrichment score.

Figure 4

FUBP3 acts as a genomic transcription activator (A) Genome tracks of native ChIP-seq to RNAPII, FUBP3, and input in J-Lat 10.6 cells with or without stimulation (mock or TNF-⍺). (B) Scaled average normalized coverage of indicated factors at protein-coding genes >2 kb with expression levels comparable to HIV (1,790 genes) in J-Lat 10.6 cells in non-stimulated conditions (NS) or TNF-α stimulation. The dotted lines represent the coverage at the HIV-1 locus, and the solid lines represent the coverage of the cellular genes. (C) Scaled average normalized coverage of indicated factors at protein-coding genes with an RNAPII (left: 2,952 genes) or FUBP3 (right: 785 genes) peak at −2,000 to +300 of the TSS across indicated regions, binned in 6-kb windows. (D) RNAPII recruitment onto the HIV genome (left) or GAPDH (right) in Jurkat-D6 cells as determined by XChIP qPCR. Results are presented as percentage immunoprecipitated DNA over input, after IgG control background subtraction. All data are reported as the mean ± SEM.

Figure 5

FUBP3 regulates HIV-1 in a Tat-dependent manner (A) HIV mRNA abundance upon FUBP3 or CCNT1 depletion in HIV_GKO/HIV_GKOΔTat clone cells as determined by RT-qPCR. (B) Representative flow cytometry plot showing expression of HIV (MFI GFP+). (C) Tat-FUBPs coIP from HEK293T cells expressing Tat-FLAG and FUBPs-HA. (D) In vitro binding assay of GST-FUBP3 and His-Tat-WT protein. (E) Schematic representation of FLAG-Tat deletion variants and Tat-FUBP3 coIP from HEK293T cells expressing Tat-FLAG deletion variants and FUBP3-HA. (F) Schematic of the mutated Tat basic domain variants and Tat-FUBP3 coIP with Tat-FLAG mutated basic domain variants and FUBP3-HA. (G) Tat-FUBP3 coIP with Tat-FLAG and FUBP3-HA truncations, pretreated or not with RNase before IP. (H) Tat-FLAG degradation assay with FUBP3 or CD8B deletion quantified by western blot and RT-qPCR upon treatment with cycloheximide (CHX) in HEK293T cells. (I) Tat-BRM protein level quantification over time by western blot following CHX treatment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by two-way ANOVA with multiple comparisons. All data are reported as the mean ± SEM.

Figure 6

FUBP3 interacts with and stabilizes TAR-RNA (A) Immunoblotting of the in vitro TAR-RNA pull-down with FUBP3. (B) Immunoblotting of the in vitro TAR-RNA pull-down with GST-FUBP3. (C) Immunoblotting of the competitive in vitro TAR-RNA pull-down with GST-FUBP3 and His-Tat protein. (D) Immunoblotting of the in vitro TAR-RNA pull-down with FUBP3 variants. (E) RIP against control IgG or FUBP3 were subjected to qPCR to measure HIV mRNA level in Jurkat-D6 cells. (F) RIP samples were subjected to RNA-seq to reveal mRNAs bound by FUBP3 transcriptome-wide. (G) Bubble plot of GSEA results from the RIP-seq data. NES: normalized enrichment score. (H) TAR-RNA degradation with FUBP3 or CD8B deletion monitored by RT-qPCR over time upon treatment with flavopiridol in J-Lat 10.6 cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by two-way ANOVA with multiple comparisons. All data are reported as the mean ± SEM.

Figure 7

Model of FUBP3’s role in HIV transcriptional regulation (A) Summary of Tat and FUBP3 interactions with each other and FUBP3 with TAR. (B) FUBP3 binds and stabilizes TAR and Tat, promoting HIV transcription. (a) FUBP3 binds and stabilizes Tat. FUBP3 bound to Tat binds and stabilizes TAR. (b) Tat’s complex binds to TAR and FUBP3, already stabilizing the RNA. FUBP3 also regulates the expression of several T cell receptors as well as CDK1 and RRM2. FUBP3 binds CDK1 and RRM2 mRNAs and stabilizes RRM2 mRNA.