Overexpression of CA150 inhibits basal and Tat-activated transcription from the HIV-1 LTR. (A) Analysis of CA150 protein expression. Cells were transfected with 1, 2, and 3 μg of vector alone (pEFBOST7) (lanes 1 to 3) and the same amounts of the CA150-expressing construct (pEFBOST7-CA150) (lanes 4 to 6). Whole-cell lysates were prepared, and proteins were resolved by SDS-PAGE and transferred to a membrane. Antibodies against the protein were used to localize CA150. (B) Effect of CA150 overexpression on basal and Tat-activated transcription from the HIV-1 promoter. The levels of CAT activity in extracts from cells that were cotransfected with 0.1 μg of HIV-1 LTR reporter vector and the indicated amounts of Tat in the presence of 3 μg of vector alone or the CA150-expressing construct were measured. Transfection and activity assays were done as described in Materials and Methods.

Repression mediated by overexpression of CA150 requires specific promoter sequences. (A) Effect of CA150 overexpression on the activity of HIV-1 LTR constructs: CA150 overexpression inhibits HIV-1 promoter activity in a TATA-box-dependent manner. A schematic representation of the HIV-1 promoter constructs is shown. Numbers on the constructs indicate the amount of viral sequences present. NF-R has both NF-κB sites in a reverse orientation (arrow on top). The indicated HIV-1 LTR constructs (0.1 μg) were cotransfected into 293T cells in the presence of 3 μg of vector alone (−CA150) or CA150-expressing vector (+CA150). Numbers indicate CAT activity from transfected-cell extracts (see Materials and Methods). The transcription activity from HIV-CAT, NF/SP (wild-type), and NF-R constructs was repressed by overexpressing CA150. Changing the TATA box sequence to TATTTAT (SV40-TATA) or to the random sequence GTCAC (m-TATA) or using a construct where the TATA box was deleted (d-TATA) abrogated the repression mediated by overexpression of CA150. The abilities of these constructs to respond to Tat are shown in the last column. 293T cells were cotransfected with 0.1 μg of the indicated HIV-1 reporter constructs, 25 ng of pcTat, and 10 ng of HSV-TK-LUC expression plasmid. The level of Tat activation of NF/SP (wild-type) plasmid was set at 100. (B) Effect of CA150 overexpression on the activity of the α4 integrin promoter. A schematic representation of the 5′ α4 integrin reporter constructs is shown. Numbers in the construct name indicate the amount of 5′ flanking region present. The 5′ α4 integrin reporter constructs were cotransfected into 293T cells in the presence of 3 μg of vector alone or the CA150-expressing construct. The approximately threefold reduction in transcription activity of the constructs containing sequences upstream to bp +600 has been described previously (45). Transfection and activity assays were done as described in Materials and Methods.

Overexpression of CA150 reduces transcriptional elongation from the HIV-1 LTR. RNA from transfected 293T cells was isolated and subjected to RT-PCR analysis. (A) Representation of the RT-PCR assay used. A schematic map of the relevant regions in the HIV-CAT reporter construct is shown. The start site of transcription (+1) is indicated with an arrow. The relative positions of the specific primers used in the RT reaction mixture are shown. These RT specific primers measured transcription complexes that reached nucleotides +82 (Short) and +542 (Long) relative to the +1 start site of transcription. For PCR, the RT reaction mixtures were subsequently amplified with the same set of primers (F and Short) to compare directly the radioactive signals (see Materials and Methods). (B) RT-PCR assay. PCR products were linear over the range of concentrations used. RNA from cells transfected with the control plasmid was isolated and reverse transcribed in the presence (+RT) or absence (−RT) of reverse transcriptase enzyme. One microliter of the RT reaction mixture and subsequent fivefold dilutions were subjected to amplification by PCR and visualized as described in Materials and Methods. Molecular size markers (in base pairs) are indicated on the left. The position of the labeled PCR product is also indicated. (C) Inhibition of HIV-1 transcriptional elongation by overexpressed CA150. RNA from cells transfected with the control plasmid or CA150 expression construct (CA150) was isolated and reverse transcribed by using the short and long RT specific primers. RT reaction mixtures were subsequently amplified by the PCR approach described for panel A. Molecular size markers (in base pairs) are indicated on the left. The position of the labeled PCR product is also indicated. Quantification was performed to yield values for the radioactive signals from PCR products (see Materials and Methods and Results).

Overexpression of CA150 inhibits the activity of the α4 integrin promoter. (A) The indicated amounts of the α4 integrin reporter vector were cotransfected into 293T cells in the presence of 3 μg of vector alone or the CA150-expressing construct. (B) The same cotransfection experiment was carried out with a fixed amount of α4 integrin reporter vector (1.6 μg) and the indicated concentrations of the CA150-expressing plasmid. Empty vector was used to keep the total amount of DNA constant. Transfection and activity assays were done as described in Materials and Methods.

TBP can alleviate CA150-mediated repression of the HIV-1 promoter. (A) Analysis of TBP expression. Fifty micrograms of extracts from 293T cells transfected with 1, 10, 50, 100, and 500 ng of pCGNTBP (lanes 1 to 5) or mock transfected (lane 6) was subjected to SDS-PAGE and Western blot analysis. Antibodies against TBP (α-TBP) and the HA epitope tag (α-HA) were used to visualize the proteins. (B) HIV-CAT reporter plasmid (0.1 μg) was cotransfected into 293T cells with 0.1 μg of pCGN (empty vector) (−TBP) or pCGNTBP (+TBP) in the presence of 3 μg of vector alone (black bars) or CA150-expressing construct (hatched bars). Transfection and activity assays were done as described in Materials and Methods. (C) Analysis of CA150 and TBP protein expression in the transfected cells. Twenty micrograms of transfected-cell extracts was used in SDS-PAGE and Western blot analysis to analyze protein expression. Antibodies against the T7 and HA epitope tags were used to visualize CA150 and TBP, respectively.