HIV-1 transcription is regulated by splicing factor SRSF1

Abstract

Efficient transcription of the HIV-1 genome is regulated by Tat, which recruits P-TEFb from the 7SK small nuclear ribonucleoprotein (snRNP) and other nucleoplasmic complexes to phosphorylate RNA polymerase II and other factors associated with the transcription complex. Although Tat activity is dependent on its binding to the viral TAR sequence, little is known about the cellular factors that might also assemble onto this region of the viral transcript. Here, we report that the splicing factor SRSF1 (SF2/ASF) and Tat recognize overlapping sequences within TAR and the 7SK RNA. SRSF1 expression can inhibit Tat transactivation by directly competing for its binding to TAR. Additionally, we provide evidence that SRSF1 can increase the basal level of viral transcription in the absence of Tat. We propose that SRSF1 activates transcription in the early stages of viral infection by recruiting P-TEFb to TAR from the 7SK snRNP. Whereas in the later stages, Tat substitutes for SRSF1 by promoting release of the stalled polymerase and more efficient transcriptional elongation.

Figure 1.

Screening of the RBP expression library reveals novel cellular factors regulating Tat transactivation. (A) Schematic map of the pLTR-Xm-LR reporter minigene. Dashed lines indicate the viral sequences utilized to construct the minigenes in relation to the HIV-1 genome (top). (B) Transactivation of the pLTR-Xm-LR reporter. HEK293 cells were transfected with the pLTR-Xm-LR reporter, a Rev expression vector and increasing amounts of Tat expression vector. Expression of the reporter mRNA and gene product were assayed by qPCR (left panel) and an enzymatic luciferase assay (right panel). (C) Screening of the RBP expression library. HEK293 cells were transfected with each RBP and the control EGFP expression clones in the presence or absence of Tat. The TrC measures the change in transactivation efficiency and is defined as log2[(LTBP/LBP)/(LTE/LE)], where LTBP and LBP are luciferase expression values in cells transfected with a given RBP in the presence or absence of Tat, respectively, whereas LTE and LE are luciferase expression values in cells transfected with the EGFP control in the presence or absence of Tat, respectively. SRSF1 inhibits Tat-dependent transactivation of the HIV-1 promoter. HEK293 cells were transfected with either pLTR-Xm-LR (D) or pLTR-Luc (E) and the expression clones for Tat and SRSF1, as indicated. (F) Tat and SRSF1 expression. The SRSF1 expressed from the pSRSF1 vector is tagged with a T7 epitope (T7-SRSF1) and migrates slower than the endogenous protein (endo-SRSF1).

Figure 2.

Tat transactivation is a primary target for SRSF1. (A) HEK293 cells were co-transfected with the pLTR-Xm-LR reporter, Tat alone or in combination with SRSF1. Cells were harvested during a 48-h time course. Transcripts generated by the reporter construct were quantified by qPCR. (B) Luciferase activity of the cells transfected during the time course. (C) Tat and SRSF1 expression during the time course; expression of SRSF1 does not interfere with expression of Tat (Figure 1F). Dose-response curves in HEK293 cells transfected with the pLTR-Xm-LR reporter, increasing amounts of the Tat expression construct and either a control (pEGFP), the SRSF1 expression construct or control (D) or SRSF1-specific siRNAs (E). (F) SRSF1 expression following transfection of the SRSF1 expression clone (left panel) or the anti SRSF1 siRNAs (right panel).

Figure 3.

The SRSF1 RRM2 is required and sufficient to abolish Tat transactivation. (A) Schematic representation of the SRSF1 deletion clones. (B) Luciferase activity in HEK293 cells transfected with the pLTR-Xm-LR reporter, the Tat expression vector, the control pEGFP and the SRSF1 expression constructs. (C) Relative expression level of the T7-tagged SRSF1 deletion clones.

Figure 4.

SRSF1 and Tat bind overlapping sequences within TAR. (A) RAC assays were set up with bait RNAs containing the wild-type (TARWT) and mutated (TARM) TAR sequences. The RNA substrates were incubated with 100 ng of recombinant Tat or purified SRSF1 in separate reactions. (B) Tat and SRSF1 compete for binding onto TAR. RAC assays were set up with the wild-type TAR sequence as bait, and either 100 ng of recombinant Tat and increasing amounts of purified SRSF1 (upper panel) or increasing amounts of Tat and 100 ng of SRSF1 (lower panel).

Figure 5.

ChIP analysis of transcription and splicing factors at the HIV-1 promoter of transiently transfected and integrated proviruses. (A) The schematic diagram shows the HIV-1 promoter and downstream sequences, indicating the location of the primer sets used for ChIP of the TAR and ORF. (B and C) HEK293 cells were transfected with the viral clone pMtat(–), a control plasmid, the Tat expression vector alone or in combination with SRSF1. (D and E) HLM1 cells were transfected with a control plasmid, the Tat expression vector alone or in combination with SRSF1. ChIP assays were performed 48 h after transfection, with antibodies for the indicated proteins and the GFP-tagged Tat. Values represent the relative enrichment in ChIP signal to the TAR (B and D) and ORF (C and E) relative to the control IgG, and are the average of three independent PCRs from two independent ChIP experiments.

Figure 6.

SRSF1 binds to the 7SK RNA and activates transcription of the viral promoter. (A) SRSF1 transcription activation is dependent on TAR. HEK293 cells were transfected with the reporter constructs pLTR-Xm-LR and pLTR-XTm-LR, which carries a deletion of the TAR sequence, the control pEGFP, the SRSF1 expression constructs, control or SRSF1-specific siRNAs I the absence of Tat. Luciferase activity and mRNA expression were assayed 48 h after transfection. (B) SRSF1 and Tat bind overlapping sequences within the 5′ hairpin of the 7SK RNA. RAC assays were set up with bait RNAs containing the wild-type (5hpWT) and mutated (5hpM) sequences of the 5′ hairpin of the 7SK RNA. The RNA substrates were then incubated with 100 ng of recombinant Tat or purified SRSF1 in separate reactions. (C) HEK293 cells were transfected with the viral clone pMtat(–), a control plasmid, the SRSF1 expression clone or SRSF1 siRNA. ChIP assays were performed 48 h after transfection, with antibodies for the indicated proteins and the GFP-tagged Tat. Values represent the relative enrichment in ChIP signal to the TAR and ORF relative to the control IgG, and are the average of three independent PCRs from two independent ChIP experiments.

Figure 7.

SRSF1 regulates HIV-1 transcription. (A) In the early stage of viral infection, SRSF1 associates with gene promoters, as part of the 7SK snRNP. Synthesis of TAR triggers the switch of SRSF1 from the 7SK RNA to the viral transcript, and the simultaneous release of P-TEFb from the 7SK complex and its recruitment onto the nascent RNA. The activated P-TEFb phosphorylates RNAPII and the components of the pausing complex NELF and DSIF, which result in transcription-pause release. (B) During the later stages of the viral infection, Tat is present at higher concentrations and is found in a complex with P-TEFb within the 7SK particle. Tat binding to TAR induces relocation of P-TEFb from the 7SK complex and other nucleoplasmic complexes to the nascent transcript within the paused RNAPII complex. This triggers activation of the P-TEFb kinase and hyper-phosphorylation of the RNAPII CTD and the NELF/DSIF complex. The difference in the efficiency of transactivation between Tat and SRSF1 might be due to the ability of Tat to recruit P-TEFb from nucleoplasmic complexes other than 7SK (dashed arrows).