Proposed model for Tat-mediated splicing in the viral context. Tat binding to TAR aids in the recruitment of the pTEF-b complex that in turn helps the recruitment of Tat-SF1 and CA150, which stimulates the assembly of SF2 onto the GAR enhancer. SF2 interaction with GAR promotes the upstream rev, env specific 3′ss and recruitment of U1 snRNP to the downstream 5′ss, which allows for env mRNA expression.

(A) RNAPII assembly and phosphorylation was analyzed by a ChIP assay using the indicated antibodies. The location of the primers used in the ChIP assay to analyze the promoter (black) or ORF (gray) is schematically indicated in Figure 5A. The reporter plasmids indicated at the left of the panel were transfected in combination or absence of Tat as indicated. Antibodies recognizing RNAPII (RPII) and the phosphorylated form of RNAPII at Ser2 (RP-S2) together with a control anti-IgG are indicated on top. qPCR analysis of the RPII (B) and RP-S2 (C) assays at the promoter and ORF in the presence or absence of Tat. The ChIP values are expressed as percentages of input. Data are represented as means ± SEM.

The structure of the promoter affects Tat-mediated splicing. (A) Schematic representation of the reporter constructs utilized. (B) Quantification of reporter transcripts by RT-qPCR. HEK-293T cells were co-transfected with the indicated reporter construct, and pTat. (C) RT-PCR analysis of alternative spliced mRNAs generated by the indicated reporter construct (top of the panel) transfected in combination with the control (pLuc), splicing factors (pSC35, pSF2) and pTat as indicated. A RNA sample, which was not reverse transcribed, was utilized as negative control (−RT). (D) Unspliced (US) mRNAs and the sum of all mRNA transcripts (TOT) are quantified and their ratio is indicated. (E) The splicing products representing splicing to the tat and env specific 3′ss #3 and 5 were quantified and their ratio is indicated. Data are represented as means ± SEM.

Tat-mediated splicing is dependent on the GAR ESE. (A) Schematic representation of the reporter constructs. Mechanism of ESE/ESS2 and GAR splicing splicing regulation are schematically represented. Wild-type (black bold font) and mutated (red bold font) sequences are indicated below each construct. (B) RT-PCR analysis of alternative spliced mRNAs generated by the indicated reporter construct (top of the panel) transfected in combination with the control (pLuc), splicing factors (pSC35, pSF2) and pTat as indicated, (C) Unspliced (US) mRNAs and the sum of all mRNA transcripts (TOT) are quantified and their ratio is indicated. (D) The splicing products representing splicing to the tat and env specific 3′ss #3 and 5 were quantified and their ratio is indicated on the left. For the pLTR-S1Xm-SS3m-R construct, products representing splicing to the env specific 3′ss #5 and unspliced mRNAs were quantified and their ratio is indicated on the right. Data are represented as means ± SEM.

Tat promotes splicing of a heterologous substrate. (A) Schematic representation of the pLTR-dsx-R minigenes. Dsx exonic sequences (light boxes), LTR promoter and RRE sequences (dark boxes) are shown. GAR splicing enhancer, TAR region and respective mutations are indicated. The GAR wild type and mutated sequences are shown in Figure 3A. (B) Quantification of reporter transcripts. HEK-293T cells were co-transfected with the indicated reporter construct and pTat. Transcripts were quantified by RT-qPCR. A RNA sample, which was not reverse transcribed, was utilized as negative control (−RT). (C) RT-PCR analysis of alternative spliced mRNAs generated by the indicated reporter construct (top of the panel). (D) Spliced and unspliced mRNAs were quantified and their ratio is indicated. Data are represented as means ± SEM.

Tat upregulates env mRNA splicing. (A) Schematic representation of the pMtat(−) proviral construct. The relative position of the viral genes is indicated on the map on top. The Tat start codon (ATG) is mutated. The main mRNAs analyzed by RT-qPCR are indicated with the solid line representing the exons. Location of the primers utilized in the qPCR assays is also indicated (arrows P1 through P10). 5′ and 3′ splice sites are indicated on the unspliced (gag/pol) mRNA. (B–G) HEK-293T cells were transfected with the proviral clone pMTat(−), the control pLuc or pTat. Each graph summarizes the quantification by RT-qPCR of the indicated mRNA species. The amount of each mRNA species generated in the transfection containing the control pLuc was assigned the value 1. (B) The total viral mRNAs (primers P3–P4) data were normalized for the EGFP mRNA content of each sample. (C–G) gag/pol mRNA (primers P1–P2), env1 mRNA (primers P5–P6), tat1 mRNA (primers P7–P8), rev2 mRNA (primers P9–P8) and nef2 mRNA (primers P10–P8) were normalized on the base of the total viral mRNA content of each sample. A RNA sample, which was not reverse transcribed, was utilized as negative control (−RT). (H) Tat expression in 293T cells transfected with the proviral clone pHIV-HXB2 or cotransfected with pMTat(−) and pTat. Data are represented as means ± SEM.

The reporter minigene mimics HIV-1 transcription and splicing regulation. (A) Schematic map of the pLTR-S1Xm-R reporter minigene. Dashed lines indicate the location within the viral genome of the sequences utilized to build the minigene. The size of the different fragments is indicated on top. The Tat start codon (asterisk) is mutated (ATGm). Location of the ESE/ESS2 and GAR splicing regulatory elements are indicated with a schematic representation of their functions. Location of the PCR primers utilized in the RT-PCR (PL1, PL2) and RT-qPCR (PR1, PR2) are indicated. (B) Schematic representation of the main HIV-1 RNA species. Exonic sequences present in each of the main viral mRNAs are drawn in correspondence of their location on schematic map on top. (C) Quantification of reporter transcripts. HEK-293T cells were transfected with pLTR-S1Xm-R, pEGFP-N1 (normalizing control) and the Tat coding plasmid pTAT. mRNAs generated by pLTR-S1Xm-R were quantified by RT-qPCR. The arbitrary value of 1 was assigned to the amount of reporter mRNA generated in the absence of Tat. An RNA sample, which was not reverse transcribed, was utilized as negative control (−RT). (D) Alternative spliced mRNAs generated by pLTR-S1Xm-R. HEK-293T cells were co-transfected with pLTR-S1Xm-R, and the vector indicated on top of the figure in the presence (+) or absence (−) of pTat. Samples were analyzed by RT-PCR, the cDNA input for each sample was normalized on the base of the total amount of transcripts generated by the reporter construct as determined by RT-qPCR. (E) Unspliced (US) mRNAs and the sum of all mRNA transcripts (TOT) are quantified and their ratio is indicated. (F) mRNAs representing splicing to the tat and env specific 3′ss #3 and 5 were quantified and their ratio is indicated. Data are represented as means ± SEM.

Splicing is dependent on Tat-associated cellular factors. (A) Down-regulation by siRNA of cellular transcription-splicing factors. HEK-293T were transfected with the reporter construct pLTR-S1Xm-R, pTat and the indicated a control siRNA (si_Cont) or siRNAs to Tat-SF1 (si_Tat-SF1), CA150 (si_CA150), SKIP (si_SKIP), Tat-CT1 (si_Tat-CT1). Transcripts were quantified by RT-qPCR. A RNA sample, which was not reverse transcribed, was utilized as negative control (−RT). (B) Tat transcription transactivation. Fold activation of transcription in the presence of Tat and the indicated siRNAs. (C) RT-PCR analysis of the alternative spliced mRNAs generated by pLTR-S1Xm-R cotransfected with either the control pLuc or pTat and the indicated siRNA (top of the panel). (D) Unspliced (US) mRNAs and the sum of all mRNA transcripts (TOT) are quantified and their ratio is indicated. (E) The splicing products representing splicing to the tat and env specific 3′ss #3 and 5 were quantified and their ratio is indicated. (F) SF2 and SC35 co-immunoprecipitate with CA150 in HeLa nuclear extracts. CA150 was immunoprecipitated with anti-CA150 antibodies and anti-tubulin antibodies as control (indicated on top). Short RNA transcripts containing TAR or a control sequence were added to the assay mixture as indicated. Recombinant GST-Tat protein was also added to the incubation mixture as indicated. The immunoprecipitates were analyzed by western blotting with the indicated antibodies (panels from top to bottom). Quantification of (G) SF2 and (H) SC35 recovered in each immunoprecipitation assays. The arbitrary value of 1 was assigned to the amount recovered by precipitation with anti-CA150 antibodies in the absence of exogenous RNA and Tat. Data were quantified by optical density analysis. Data are represented as means ± SEM.