Figure 1.
ZASC1 binds to specific DNA elements in the HIV-1 promoter and regulates viral transcription.
(A) Sequence of the HIV U3 region from −150 to +1 of HIV-1 isolate NL43 showing the relative location of the four putative ZBS. Known transcription factor binding sites are indicated as follows: NFκB (orange), SP1 (purple), TBP (blue) and the Ras-responsive binding elements (RBE) RBEI and RBEIII (underlined). Mutations that were introduced in the ZBS are indicated in red. A control downstream mutation (mDS) is indicated in purple. Alignment of ZBS 1 and 2 indicating the 2 bp offset palindromic structure and the GC to TA mutation introduced into the site that alters the same bases on both strands is shown below the promoter sequence. (B) EMSA of the WT HIV U3 DNA probe (nucleotides −454 to +66) alone (P) or incubated with in vitro transcribed/translated luciferase (L) or ZASC1 (Z) proteins. (C) Total ZASC1 bound to the HIV promoter was measured by chromatin immunoprecipitation with an anti-ZASC1 antibody on HEK293 or Jurkat cells transduced with NL43E-R-Luc using a primer set that spans the ZBS at the U3/R boundary (−116 to +25) or a primer that amplifies the luciferase gene in the nef locus located approximately 9 kBp downstream from the transcription start site. Real-time PCR analysis was performed in triplicate and normalized to input controls. (D) ChIP assays of ZASC1 recruitment to the HIV-1 promoter in Jurkat cells transduced with similar levels of NL43E-R-Luc or modified viral constructs containing the TA mutation or the mutation of all four ZBS (Fig. 1A). Immunoprecipitation and Real-time PCR analysis was performed in triplicate and normalized to input controls and is reported as fold-enrichment relative to control pull-downs with non-specific rabbit IgG. The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and each is representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student's t-test and significant changes relative to WT or relevant bracketed comparisons indicated.
Figure 2.
ZASC1 contributes to HIV-1 vector gene expression.
(A) Jurkat cells were challenged in quadruplicate with NL43E-R-Luc derivatives containing mutations in the indicated ZBS as described in Fig. 1A. Infections were monitored by chemiluminescent assays as described in materials and methods and normalized to input virus by anti-capsid (p24) western blots and reported as the ratio of [reporter gene activity∶input capsid] observed. (B) HEK293T (2×105 cell/well of 12 well plate) cells were transiently transfected with 1 µg of plasmids expressing GFP and either one of four shRNAs targeting the open reading frame of ZASC1 or the empty shRNA vector. Three days post transfection, replicate wells were harvested for either total RNA or protein. Quantitative RT-PCR analysis of ZASC1 and actin RNA was performed in triplicate (Bar graph), representative western blots with the indicated antibody are shown below. (C) HEK293T cells (1×104 cells/well) in a 96 well plate were transiently transfected with 80 ng of plasmids expressing either one of four shRNAs targeting the open reading frame of ZASC1 or the empty shRNA vector along with an 20 ng of an expression vector encoding the ASLV receptor TVA800 [35], [36]. Three days post transfection, cells were challenged with EnvA pseudotyped HIV vectors, either NL43E-R-Luc, which directs HIV LTR-driven firefly luciferase gene expression, or the mTA mutant (Fig. 1A). (D) HeLa cells expressing the Tet repressor were stably transduced with a lentivirus construct encoding a Tet-inducible dominant negative GFP-ZASC1 fusion protein. GFP-ZASC1 expression was induced with 1 µg/ml doxycycline, 24 hours post induction the cells were challenged with the VSV-G pseudotyped NL43E-R-Luc or a derivative with all 4 ZBS mutated (Fig. 1A). Infection was monitored using chemiluminescent assays as described in materials and methods. The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and each is representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student's t-test and significant changes relative to WT indicated.
Figure 3.
ZASC1 promotes HIV-1 transcription elongation.
(A) Schematic of the HIV-1 vector NL43E-R-Luc indicating the two types of primary transcripts and the PCR primers utilized to differentiate them. (B) The amount of initiated and extended transcripts was assayed in cells challenge with NL43E-R-Luc or ZBS variants by isolating total RNA 48 hpi and performing real-time reverse transcription PCR with primers that amplify all initiated transcripts (+1 to +58) or transcripts that have been initiated and extended (+9961 to +10,191). Transcript values were normalized for the amount of input virion RNA and total cellular actin mRNA. Real-time PCR was performed in triplicate. Elongation efficiency of panel B was determined by dividing the extended transcripts by the total initiated transcripts with WT efficiency set at 100%. (C) HEK293 cells (1×105) were transfected with an shRNA vector plasmid or an shRNA targeting ZASC1 (shRNA ZASC1#4, see Fig. 2B), 72 hrs post transfection, the cells were challenged with WT NL43-luc. 48 hrs post infection, total RNA was harvested and initiated and extended transcripts were analyzed and elongation efficiency determined as in B. ZASC1 RNA was analyzed as in Fig. 2B. (D) Total RNA polymerase II bound to either NL43E-R-Luc or a derivative with all ZBS mutated (Fig. 1A) was measured by chromatin immunoprecipitation assays using a primer set that spans the transcription initiation site (−116 to +25) or (E) a primer set that amplifies the luciferase gene in the nef locus approximately 9 kBp downstream from the transcription start site. Error bars indicate the standard deviation of the data and are representative of three independent experiments. P-values were calculated using a standard Student's t-test and significant changes relative to WT or relevant bracketed comparisons indicated.
Table 1.
Real-time PCR primers.
Figure 4.
ZASC1 stimulates transcription elongation from the HIV-1 promoter in primary T-cells.
(A) Isolated CD4+ were placed in culture and stimulated for 24 hrs with CD3/CD28 beads, total RNA was isolated from cells (1×105) and expression of ZASC1 and actin was monitored by quantitative real-time RT-PCR. (B) Unstimulated or CD3/CD28 stimulated primary T-cells (1×106) were harvested and analyzed by immunoblotting with anti-ZASC1 and anti-Histone H3 antibodies. (C) Cells (1×106) from CD3/CD28 stimulated primary T-cells or the indicated cell lines were harvested and analyzed by immunoblotting with anti-ZASC1 and anti-Histone H3 antibodies. A representative blot of three independent experiments is shown (D) Stimulated primary T cells were challenged with either WT NL43-luc or mZBS mutant and 2 dpi, total ZASC1 bound to the HIV promoter was measured by chromatin immunoprecipitation with an anti-ZASC1 antibody using a primer set that spans the ZBS at the U3/R boundary (−116 to +25) or a primer that amplifies the vif locus located approximately 5 kBp downstream from the transcription start site. Real-time PCR analysis was performed in triplicate and normalized to input controls and fold enrichment relative to rabbit IgG controls reported. (E) The amount of initiated and extended transcripts was assayed in stimulated primary T-cells challenged with NL43E-R-Luc or ZBS variants by isolating total RNA 48 hpi and performing real-time reverse transcription as described in Fig. 3B. Transcript values were normalized for the amount of input virion RNA and total cellular actin mRNA. Real-time PCR was performed in triplicate. Elongation efficiency was determined by dividing the extended transcripts by the total initiated transcripts with WT efficiency set at 100%. (F) Stimulated primary T-cells were challenged with the indicated NL43-Luc variants and 2 dpi total ZASC1 bound to the HIV-1 promoter was determined by ChIP analysis as in C. Error bars indicate the standard deviation of the data and are representative of three independent experiments. P-values were calculated using a standard Student's t-test.
Figure 5.
ZASC1 stimulates TAT-activation of the HIV-1 promoter.
(A) HEK293 cells transiently transfected with a plasmid that expresses Guassia luciferase under the regulation of either the WT or mZBS 1–4 HIV promoter (all 4 putative ZBS mutated as in Fig. 1A) transfected in the presence of a ZASC1 expression plasmid. The basal activities of the WT and mZBS HIV promoters in the absence of ZASC1 were set to 100%. (B) Transient transfection of WT or mZBS HIV-1 promoters in the presence of plasmids that express TAT, ZASC1, or both TAT and ZASC1. (C) Transient transfection of WT or mZBS HIV-1 promoters in the presence of plasmids that express TAT, dominant negative GFP-ZASC1, or both TAT and GFP-ZASC1. (D) Transient transfection of WT or mZBS HIV-1 promoters in the presence of plasmids that express TAT, an shRNA plasmid that targets ZASC1 (shZASC1#4 see Fig. 2B), or both TAT and shZASC1#4. Fold activation by TAT and ZASC1 on the WT and mZBS promoters was determined relative to promoter expression in the presence of an empty expression plasmid. The transfection data shown are the average mean values obtained in an experiment performed with quadruplicate samples and each is representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student's t-test and significant changes relative to WT or relevant bracketed comparisons indicated. Western blot of ZASC1, TAT and Histone H3 levels in HEK293 cells transiently transfected with plasmids that express (E) express TAT, ZASC1, or both TAT and ZASC1 or (F) TAT, dominant negative GFP-ZASC1, or both TAT and GFP-ZASC1.
Figure 6.
HEK293 cells (1×107) were transfected with expression plasmids encoding the epitope tagged forms of the indicated proteins. 48 h post-transfection, cells were lysed and epitope tagged proteins were immunoprecipitated (IP), separated by SDS-PAGE and analyzed by western blotting (WB) using the indicated antibodies as described in materials and methods. (A) Co-immunoprecipitation of Flag-ZASC1, and HA -TAT following IP with anti-HA beads.(B) Co-immunoprecipitation of HA-TAT, Flag- ZASC1 and Myc-tagged CDK9 following IP with anti-Myc beads. (C) Co-immunoprecipitation myc-Hexim1 and Flag-tagged ZASC1 following IP with anti-Flag beads. (D) Western blots of HEK293 cell fractionation into nuclear pellet and soluble fractions before glycerol gradient analysis. (E) Western blot of fractions from a 5% to 45% glycerol gradient of HEK293 cell lysates. All blots are representative of at least three independent experiments.
Figure 7.
ZASC1 binds to active and inactive forms of P-TEFb.
HEK293 cells (1×108) were transfected with expression plasmids encoding Myc-tagged ZASC1 and eGFP. 48 h post-transfection, cells were treated with 10 µM DRB or 1 µg/ml actinomycin D for one h, lysed, fractionated on a 5% to 45% glycerol gradient and (A) analyzed by western blot with the indicated antibodies. (B) The indicated gradient fractions were pooled and immunoprecipitated with either a non-specific mouse IgG or mouse anti-Myc IgG. Input (I) non-specific (N) and myc-ZASC1 (Z) immunoprecipitations were analyzed by western blot with the indicated antibodies. All blots are representative of at least three independent experiments.
Figure 8.
ZASC1 recruits P-TEFb and TAT to the HIV-1 promoter in the absence of TAR.
The ability of ZASC1 to recruit P-TEFb and TAT to the HIV promoter was assessed by stably transfecting HeLa cells with a reporter plasmid containing a HIV promoter lacking a TAR element (WT or a variant with all ZBS mutated) and driving expression of the gLUC reporter enzyme. For TAT ChIP, the cells were transfected with HA-tagged TAT. ChIP experiments against endogenous proteins or TAT were performed using antibodies against (A) ZASC1 (B) CycT1 (C) CDK9 (D) Hexim1 (E) HA epitope (TAT) and (F) SP1. Real-time PCR was performed in triplicate using a primer set that spans the ZBS at the U3/R boundary (−116 to +25). Error bars indicate the standard deviation of the data and are representative of three independent experiments. P-values were calculated using a standard Student's t-test.
Figure 9.
Alignment of ZBS1,2 binding sites.
HIV-1 sequence from −7 to −34 from circulating HIV-1 strains from major and outlier clades, simian immunodeficiency viruses from Chimpanzee, African green monkey, Mandrill, Macaque, HIV-2, feline immunodeficiency virus, equine infectious anemia virus and bovine immunodeficiency virus were aligned to the HIV-1 NL43 strain used in this study as described in materials and methods. Conserved sequence shown as dashes, changes from NL43 are indicated.
Figure 10.
Model for ZASC1 function during HIV-1 transcription.
Transcription efficiently initiates but the RNA pol II is non-processive. 7SK inhibited P-TEFb and TAT are recruited to the promoter by ZASC1. The complex undergoes a conformational change when TAT and TAR disassociate the inhibitory 7SK snRNA complex, TAT and P-TEFb transfers to TAR facilitating phosphorylation of the CTD of pol II and stimulating transcription elongation.