Green fluorescence protein as a transcriptional reporter for the long terminal repeats of the human immunodeficiency virus type 1

Journal of Virological Methods 84 (2000) 127 – 138 www.elsevier.com/locate/jviromet

Green fluorescence protein as a transcriptional reporter for the long terminal repeats of the human immunodeficiency virus type 1 Anindita Kar-Roy a, Wei Dong a, Nelson Michael b, Yen Li a,* a

Department of Microbiology and Immunology, Uni6ersity of Maryland School of Medicine, Baltimore, MD 21201, USA b Di6ision of Retro6irology, Walter Reed Army Institute of Research, Rock6ille, MD 20850, USA Received 8 April 1999; received in revised form 26 August 1999; accepted 30 August 1999

Abstract Using the enhanced green fluorescence protein (EGFP), a transient reporter expression system was established to assess the transcriptional activity of the long terminal repeats (LTR) of primary isolates of the human immunodeficiency virus type 1 (HIV-1). Consistent with the conventional chloramphenicol acetyl transferase (CAT) reporter, EGFP expression, under the direction of HIV-1 LTR, was readily detected in the transient transfection and was elevated by co-transfection of HIV-1 tat-expression vector. Comparing to CAT, however, EGFP expression system has two advantages: (i) Using a fluorescence activated cell sorter (FACS), it was possible to simultaneously measure transfection efficiency and fluorescence intensity of the transfected live cells without the necessity of co-transfection of a reference plasmid for comparing the transcriptional activity of two promoters; and (ii) EGFP expression was readily detected at a DNA concentration where CAT activity was not detectable possibly because the transfectants could be ‘gated’. On the other hand, at a higher concentration of DNA, CAT signal became more prominent than that of EGFP, possibly because the enzymatic activity of CAT ‘amplified’ the signal. EGFP fluorescence detected by FACS was a direct measurement of the expressed chromophore. It is concluded that the system is rapid, reproducible, convenient and useful for quantitative analysis of transcription. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Green fluorescence protein; Transcriptional reporter; Human immunodeficiency virus type 1

1. Introduction One technique used widely to evaluate promoter and enhancer activity is to transfect recom-

* Corresponding author. Present address: SRP/DEA/NIAID/NIH, 6700B Rockledge Drive, Bethesda, MD 208927610, USA. Tel.: +1-301-4962550; fax: +1-301-4022638.

binant plasmids containing transcriptional regulatory elements linked to a reporter gene into cells of interest. The reporter genes used commonly include chloramphenicol acetyl transferase (CAT), b-galactosidase (b-gal), alkaline phosphatase, and luciferase. Their enzymatic activities make them easily detectable both in vitro and in vivo (Gorman et al., 1982; Gould and Subramani, 1988; Urdea et al., 1988; Alam and Cook, 1990).

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However, the disadvantages of these reporters include the need for additional substrates or cofactors and the requirement of lysis or fixation of cells to detect their respective activities. Furthermore, with the enzymatic reporter assays, cell extract of the entire population is used instead of only the transfected population, since there is no easy method to separate the transfected cells from the untransfected cells. In addition, plasmid controls such as the b-gal-encoding vector is necessary for optimizing and normalizing transfection. The green fluorescent protein (GFP) gene of the bioluminescent jelly fish Aequorea 6ictoria, has been introduced recently as a novel reporter molecule for analyzing gene expression, protein trafficking and cellular localization (Chalfie et al., 1994; Ogawa et al., 1995; Reilander et al., 1996). The use of GFP as a reporter molecule may alleviate some of the problems encountered by the previously mentioned reporters. GFP expression is species independent and does not require additional co-factors, substrates, or other gene products from the jelly fish (Chalfie et al., 1994; Inouye and Tsuji, 1994; Barthmaier and Fryberg, 1995; Ogawa et al., 1995; Schlenstedt et al., 1995; Reilander et al., 1996). The fluorescence intensity of GFP is a direct measurement of the transcriptional activity of the promoter that directs the expression of the protein. The wild type protein, contains 238 amino acids and emits bright green fluorescence (emission lmax =509 nm) when illuminated with a blue light (excitation lmax =395 nm) (Chalfie et al., 1994). The chromophore of GFP is derived from the primary amino acid sequence through cyclization of serine-dehydrotyrosine-glycine within a hexapeptide (Cody et al., 1993). In this study, a red shifted variant of the wild type GFP was used that contained a double amino acid substitutions, F-64-L and S-65-T (EGFP-1, excitation lmax =488 nm; emission lmax =507 nm) (Cubbit et al., 1995; Heim et al., 1995; Cormack et al., 1996). Furthermore, the coding sequence of the EGFP-1 gene also contained more than 190 preferred human codon usage silent base changes for optimized expression in human cells (Haas et al., 1996). The human immunodeficiency virus type 1 (HIV-1) is a member of the retrovirus family. A

hallmark structure of retroviruses is the long terminal repeats (LTR) which contain several transcriptional regulatory motifs including Sp1 binding site and TATA box that are critical for basal level HIV-1 transcription (Jones et al., 1986; Garcia et al., 1987; Hauber and Cullen, 1988; Jones et al., 1988; Parrott et al., 1991; Berkhout and Jeang, 1992; Kashanchi et al., 1994). Other regulatory elements in the LTR include NF-kB and NFAT-1 binding sites that interact with factors present in the activated T cells, allowing enhanced viral transcription and replication (Rosen et al., 1986; Kaufman et al., 1987; Nabel and Baltimore, 1987; Tong-Starksen et al., 1987; Kawakami et al., 1988; Wu et al., 1988a,b; Ross et al., 1991; Perkins et al., 1993). A regulatory element known as TAR is located within the R region of the LTR which is critical for Tat transactivation (Feng and Holland, 1988; Garcia et al., 1988). Tat protein, encoded by the virus is a potent transactivator for HIV gene expression and viral replication, binds to the TAR region within the LTR (Cullen, 1986; Dayton et al., 1986; Fisher et al., 1986; Peterlin et al., 1986; Wright et al., 1986; Hauber et al., 1987; Sakai et al., 1988; Golub et al., 1990; Jeang et al., 1993; Michael et al., 1994). The usefulness of GFP was studied as a reporter for measuring transcriptional activity of HIV-1 LTR. To compare transcription from the LTRs of different primary isolates of HIV-1, various pLTR-EGFP-1 plasmids were constructed. Upon transfection, fluorescence activated cell sorter (FACS) was used to distinguish the transfected population from the dead and untransfected population. The transfected live cells emitted bright fluorescent signal which was distinguishable from the dull background auto-fluorescence emitted by the untransfected population and from the dead cells. FACS analysis was used to quantitatively determine transcriptional activity of the variant LTRs by fluorescence intensity of GFP. Tat-independent transcription, which had previously been described as undetectable (Arya et al., 1985; Sodroski et al., 1985), was detected using this system, suggesting that GFP is a sensitive reporter. Co-expression of HIV-1 Tat in trans, greatly enhanced the levels of GFP expres-

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sion. LTRs that responded to Tat was distinguishable from those that did not. It is shown that GFP allowed accurate, quantitative and sensitive evaluation of promoter activity. The method is rapid, reproducible, convenient and may prove useful as a quantitative assay to study transcription, which was confirmed by other investigators recently (Dorsky et al., 1996; Gervaix et al., 1997; Page et al., 1997).

2. Materials and methods

2.1. Construction of LTR-EGFP-1 6ectors and LTR-CAT constructs The HIV-1 LTRs 1E6, 1E17, 4A8, 4A23 were obtained as described (Michael et al., 1994) and LTR 601 (pEG601) was kindly provided by Volsky (Sakai et al., 1988; Golub et al., 1990). LTRs 1E6 and 1E17 were digested with XbaI and XhoI, LTRs 4A8, and 4A23 were digested with XbaI and HindIII, and LTR 601 (pEG601) was digested with XhoI and HindIII. The released fragments were rendered blunt by treatment with the Klenow fragment of the DNA polymerase I and cloned in the SmaI site of pEGFP-1 (Clontech Laboratories, CA) to generate LTR-EGFP-1 clones. The cytomegalovirus (CMV) immediate early (IE) promoter was isolated from pEGFP-N1 (Clontech) by digestion with AseI and BglII. BamHI linkers were added to the CMV IE promoter and the construct was subsequently cloned into the BamHI site of pEGFP-1 to generate pCMV-EGFP-1. To generate the LTR-CAT constructs, p1E6-EGFP-1 and p1E17-EGFP-1 were first digested with BamHI, and XbaI linker was then added. The linearized vector was then digested with PstI and XbaI to release the LTR fragments which were subsequently cloned in the PstI and XbaI site of pCAT-Basic vector (Promega). The pCMV-CAT construct was generated by first digesting pCMV-EGFP-1 with AgeI, and XbaI linkers were then added. The construct was then double digested with PstI and XbaI and cloned in the PstI and XbaI site of pCAT-Basic vector.

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2.2. Screening of HIV-1 molecular clones PCR was used for identifying clones with the proper insert without isolating plasmid DNA (Dong et al., 1999). It allowed rapid screening of a large number of colonies. After the cloning of the LTR fragments into pEGFP-1 or pCAT-Basic vector, these constructs were used to transform E. coli DH5a. Colonies were screened by PCR using lysates directly. Briefly, colonies were picked and streaked onto a master plate, and the remaining bacteria were suspended in 25 ml of 1 × PCR buffer. Mineral oil (70 ml) was added to each tube and the bacterial lysate was heat denatured at 94°C for 15 min. Added to it, beneath the oil layer, was the PCR master mix containing 1× PCR reaction buffer, 200 mM dNTP’s, 1 mM of the primers, 2 mM MgCl2 and 0.5 units of Taq DNA polymerase (Perkin-Elmer). The PCR reaction was carried out under the following cycling conditions: denaturing at 94°C (1 min.), annealing at 55°C (2 min), and extension at 72°C (2 min) for 30 cycles followed by a 10 min extension at 72°C. The primers were designed to allow amplification of clones that had the correct orientation. The primers used for identifying p1E6/1E17-EGFP-1 clones were 5%-TAGCGCTACCGGACTCAGAT3% (sense primer) and 5%-GGACGGCGCCTGCTAGAGATTTTCCACACTGACTAA-3% (antisense primer). Primers used for identifying p4A8/4A23-EGFP-1 clones were 5%-TAGCGCTACCGGACTCAGAT-3% (sense primer) and 5% - GGACAAGCTTTATTGAGGCTTAAGCAGTGG-3% (antisense primer). Primers used to identify p1E6/1E17pCAT clones were 5%-TGGATGGTGCTACAAGCTAGT-3% (sense primer) and 5% - GGACGGCGCCTGCTAGAGATTTTCCACACTGACTAA-3% (antisense primer). Primers used to identify pCMV-CAT clones were 5%-GTCCTCGAGTAGTTATTAATAGTAATCAAT - 3% (sense primer) and 5%-GTCAAGCTTGATCTGACGGTTCACTAAACC-3% (antisense primer).

2.3. Cell culture and transfections HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL) supplemented with 10% heat inactivated fetal

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bovine serum (Gibco-BRL), penicillin G (50 U/ ml) and streptomycin (50 mg/ml). 6 × 105 cells were plated in a 60 mm culture dish (Corning) and incubated in a humidified incubator containing 5% CO2 at 37°C for 12 – 18 h to obtain 70 – 80% confluence. DNA-lipofectamine complexes were formulated by mixing various amounts of LTR-EGFP-1 and LTR-CAT DNA (10 – 1500 ng), in the presence or in the absence of Tat (1–500 ng) pSV2tat72), obtained from AIDS Research and Reference Reagent program, with 8 ml of lipofectamine (Gibco-BRL), in a total volume of 200 ml of incomplete (without fetal calf serum and without penicillin/streptomycin) DMEM. Total DNA amount was kept constant by adding carrier plasmid DNA (pBS from Stratagene). The complexes were incubated for 45 min at room temperature. HeLa cells were rinsed with 1 × phosphate buffered saline (PBS) and 800 ml of incomplete medium was added. The DNA – lipofectamine complexes were added on top of the cells, and incubated in the absence of serum for 8 h at 37°C and 5% CO2. Cells were then supplemented with 0.5 volume of complete DMEM to obtain final serum concentration of 10% and incubated for an additional 48 h. The DNA for transfection was prepared using endotoxin free column chromatography (Qiagen, Chatsworth, CA).

2.4. FACS analysis Flow cytometric analysis was undertaken using a FACSORT (Becton Dickinson) equipped with a 488 nm argon laser. The FL-1 emission channel normally used to detect fluorescein isothiocyanate was used to monitor GFP expression and the FL-3 channel was used to identify 7-amino actinomycin D (7-A.A.D)-stained red fluorescence of dead cells. 48 h after transfection, the cells were trypsinized and washed twice with PBS containing 2% fetal bovine serum and stained with 7-A.A.D (7-A.A.D is a vital dye that allows the gating of living cells) at a 1: 50 dilution (stock solution is 1 mg/ml), for 1 h. Cells were then washed twice with 1×PBS containing 2% serum, to remove the excess stain. After the final wash, cells were resuspended in 300–500 ml of 1× PBS containing 2% bovine serum, at a concentration of 1× 106

cells/ml. Cells were kept on ice at all times. Gating for living cells was achieved either by the exclusion of the 7-A.A.D (high) stained cells, or by the use of light scattering measurements of the FACS. Using the parameter forward scatter (FSC) and side scatter (SSC) in a dot plot it was possible to estimate cell size and granularity, which helped to distinguish living cells from damaged or dead cells, since damaged or dead cells have a lower refractive index and thus produce a smaller forward scatter index. The histogram of the fluorescence intensity of the living cells was used to further distinguish between the transfected population and the untransfected population, since the transfected population of cells emitted a fluorescent signal above background. Marker bounds were set in the histogram which defined the population of transfected cells, and also determined the percentage of transfected cells. Quantitation of promoter activity was accomplished by measuring the median of the histogram of the fluorescence intensity of the transfected population. Populations expressing different amounts of GFP (measured by the median fluorescence intensity) indicated differences in the transcriptional efficiency of the variant LTRs. Since this system was based on a transient transfection assay, it was observed that the population of transfected cells were extremely heterogeneous in the level of GFP expression. The distribution of the fluorescent signal emitted from the transfected population was positively skewed and spread over three log decades of signal intensities (101 –104). Since the distribution of the transfected population was extremely heterogeneous and widely dispersed, it was necessary to treat the data as a frequency distribution. To compute the sample mean of individual data values, it is necessary to sum all the values and divide by ‘n’, the sample size. To do this, the entire distribution was divided into 4 decades or class intervals (markers M1 –M4), as the fluorescence signal intensity was expressed over a range of four logarithms of signal amplification (100 –104). The midpoint (median) of each class i, was denoted by Mi. The frequency of class i, was denoted by fi, and the sum of items in class i, was approximated by fiMi. Summing these values over all classes, we obtained fi Mi, which

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approximates the sum of all the data values. Once this sum was obtained an approximation of the sample mean is computed by dividing the sum by ‘n’. Since the percentage of cells in each interval was used instead of the frequency as fi, it was not necessary to divide by ‘n’, and the summation of pi Mi was used as the approximation of the median of the population, where pi is the proportion or percentage of cells in class i, (pi =fi /  fi ). The mean of the population has algebraic properties, but for our purpose, the median was also treated similarly and considered to have algebraic properties. The absolute value of the median of the histogram of the mock transfected cells was subtracted from the population of cells transfected with LTR-EGFP-1 either in the absence or in the presence of Tat. The ratio of the median fluorescent intensity (of the histograms) of cells transfected with LTR-EGFP-1 in the presence of Tat and in the absence of Tat were used to compute the degree of Tat transactivation. In this report, whenever the term ‘mean’ is used, it denotes the median of the population (quadrant) multiplied by the percentage of cells in that population (quadrant). To determine the level of significance between two data sets, the students two tailed t-test of unequal variance was performed. A P value B0.05 was considered statistically significant.

2.5. CAT analysis CAT assay was carried out essentially as described previously (Gorman et al., 1982; Rosenthal, 1987) with minor modifications. Briefly, 48 h after transfection, cells were washed two times with 1×PBS. 1 ml of TEN buffer (40 mM Tris – HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl) was added and the cells were incubated for 5 min, before harvesting from the plate. Cells were centrifuged at 14 000× g for 1 min and the pellet was resuspended in 200 ml of 0.25 M Tris – HCl and subjected to three rapid freeze/thaw cycles (Rosenthal, 1987). The lysate was heated at 60°C for 10 min to inactivate endogenous deacetylase activity. The heating step does not inactivate CAT, but may inactivate other enzymes like bgalactosidase, that have been co-transfected into

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the cells. Consequently a fixed aliquot of the lysate was removed to measure the b-galactosidase activity, and the rest was subjected to heat inactivation. The extract was centrifuged at 14 000× g for 2 min and the supernatant was transferred to a fresh tube. Equal amount of the extract (50 ml) from each lysate was mixed with 150 mCi of 14C-chloramphenicol (57.0 mCi/mmol), 5 ml of n-butyryl co-enzyme A (5 mg/ml), and 0.25 M Tris–HCl (pH 8.0) to make a final volume of 125 ml. The entire mixture was incubated for 1 h at 37°C. The reactions were terminated by adding 500 ml of ethyl acetate. Acetylated 14C (CAT activity) was measured by thin layer chromatography (TLC) assay (Gorman et al., 1982; Rosenthal, 1987). For normalization, plasmid containing bgalactosidase was used as an internal control (Rosenthal 1987). As a negative control, HeLa cells were transfected with pCAT-Basic vector, which contains no promoter upstream to the CAT gene.

3. Results

3.1. DNA dose response, transfection efficiency and fluorescence intensity To demonstrate the usefulness of GFP as a marker for transfection efficiency, different amounts of pCMV-EGFP-1 were transfected into HeLa cells and 48 h later the cells were analyzed by FACS. Fig. 1a depicts a density plot of HeLa cells transfected with pCMV-EGFP-1. Electronic ‘gating’ allowed the exclusion of FL3 high cells which represent dead cells that were stained with 7-A.A.D. To estimate the percentage of transfected cells, the fluorescence emitted from the live population (region R1 in Fig. 1a) of transfected cells was compared with that of mock transfected cells. Fig. 1b depicts the histogram of the fluorescence intensity of the gated (live) population. Marker bounds (M1 –M4) were set to divide the fluorescence intensity of the population into 4 quadrants. The FL1 photomultiplier tubes voltage was adjusted such that autofluorescence signals from cells lie within marker M1 (fluorescent signal equivalent to 101). The percentage of cells within

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quadrant M2 –M4 were added to estimate the percentage of transfected cells. Fig. 2a shows that the linear range of transfection efficiency was up

to 300 ng, where the number of transfectants increased in proportion to the amount of DNA used, until approximately 300 ng and plateaued thereafter. Similar to the transfection efficiency, the fluorescence intensity (median) emitted from the transfected cells which is proportional to the amount of GFP produced by the transfected cells, progressively increased in proportion to DNA concentration (Fig. 2b). However, unlike the transfection efficiency, the median fluorescence intensity did not plateau when the DNA concentration was over 300 ng. Similar pattern of DNA dose response was observed when the assay was repeated with the HIV-1 LTR constructs, 601EGFP-1 and 1E17-EGFP-1 (Fig. 2c, d).

3.2. GFP ser6es as its own internal control

Fig. 1. (a) Dot Plot Analysis of HeLa Cells Transfected with pCMV-EGFP-1. 6 ×105 HeLa cells were transfected with 500 ng pCMV-EGFP-1 plasmid using lipofectamine. Forty eight hours after transfection cells were trypsinized and incubated with 7 A.A.D (1: 50 dilution) for 1 h. Cells were washed with 1×PBS and prepared for FACS analysis. Gating of live cells (region R1) was possible with the help of a dot plot using the parameter FL3 (plotted in the Y-axis), since red fluorescence of the high 7 A.A.D stained dead cells can be detected in the FL3 channel and Side Scatter (plotted in the X-axis). Each dot represents a single cell. (b). Histogram of the cells within region R1. The data was grouped into 4 intervals or classes (M1 –M4). The number of cells are shown in the vertical axis, whereas the relative amount of emitted fluorescence from each cell is shown in the horizontal axis. The percentage of cells within marker bounds M2 –M4 were added to obtain the percentage of transfected cells, and the median fluorescent intensity (MFI) of the 4 class intervals are shown, the population median fluorescent intensity was calculated as described in Section 2.

Use of CAT reporters to monitor transcription required additional plasmids, such as a b-galactosidase expression vector (Rosenthal, 1987; Alam and Cook, 1990) as a control for normalizing transfection efficiency. However, using the GFP reporter alleviates the need for additional control plasmids necessary to normalize for transfection efficiencies. Using FACS, it was possible to distinguish transfected cells from untransfected cells by the fluorescence intensity of the cells. Analysis using the flow cytometric software capable of displaying the data in histograms, allow visualization and setting of markers to determine the frequency or percentage of cells that are transfected (Fig. 2a). Simultaneously the relative median fluorescence intensity of the transfected population (Fig. 2b) can be determined. Since the relationship between the fluorescent signal and the number of emitting chromophores per cell is linear, a higher fluorescence signal equates to higher expression of the reporter. Comparison of the median fluorescence intensities of different transfected populations allow the evaluation of transcription efficiencies of different promoter constructs, however this needs to be done only under similar transfection efficiencies and as well as within the linear range of transfection. The transcriptional activity of two different LTRs from two primary isolates of HIV-1, 1E17 and 1E6 (p1E17-EGFP and p1E6-EGFP) were

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Fig. 2. (a) DNA dose response curve of the cytomegalovirus early promoter pCMV-EGFP-1. 6 ×105 cells were transfected with various amounts of plasmid pCMV-EGFP-1. Forty eight hours later, cells were trypsinized and prepared for FACS analysis. The amount of DNA used (in nanograms) is shown in the X-axis and the percentage of cells transfected is shown in the Y-axis. Experiments were done in triplicate. The arrow bars show the standard deviations. (b) Relationship between median fluorescence intensity and DNA dose response of pCMV-EGFP-1. 6 × 105 cells were transfected with various amounts of the plasmid pCMV-EGFP-1. Forty eight hours later cells were trypsinized and prepared for FACS analysis. The median fluorescent intensity of the transfected population is shown in the Y-axis, and the amount of DNA used is shown in the X-axis. (c) DNA dose response curve of the LTRs 601 and 1E17-EGFP-1. (d) Relationship between median fluorescence intensity and DNA dose response of LTR 601-EGFP-1 and 1E17-EGFP-1.

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transcription (Tat independent) of 1E17 was significantly higher than 1E6 when 500 ng of DNA was used (PB 0.05), however at the 250 ng range the difference in basal transcription between 1E17 and 1E6 was not significant (P= 0.0625) (Fig. 3a). Tat activated transcription from LTR 1E17 was significantly higher than 1E6 (PB0.05), when as little as 50 ng of LTR construct was co-transfected with 50 ng of Tat-expression DNA (Fig. 3b). These results demonstrated that the GFP reporter can be used to measure simultaneously transfection efficiency and transcriptional activity. When CAT was used as a reporter it was necessary to use the b-galactosidase expression vector to normalize the transfection efficiency for comparing CAT activity directed by different LTRs (data not shown).

3.3. Effect of HIV-1 Tat on LTR-directed EGFP expression Whether HIV-1 LTR directed GFP expression can be activated by Tat was studied by co-transfection experiments, with pSV2tat72 (Sakai et al. 1988) and different LTR-EGFP-1 constructs (p1E6-, 1E17-, 4A8- and 4A23-EGFP-1). LTRs of p1E17-, 4A8- and 4A23- showed different degrees of Tat transactivation, whereas LTR 1E6 was unresponsive to Tat. When 50 ng of Tat expression vector was co-transfected with 50 ng of pLTR-EGFP-1 constructs, the fold increase in median fluorescence intensity over basal level expression was significantly higher for LTRs 4A8, 4A23 and 1E17 (P B0.05) than that of LTR 1E6

Fig. 3.

compared by fluorescence intensity of the transfected population. At the linear range of transfection, the ratio of the fluorescence intensity and transfection efficiency was calculated to normalize for variation in transfection efficiency. Basal level

Fig. 3. (a) Comparison of Tat independent transcription from LTR of two HIV-1 primary isolates, 1E6 and 1E17 using the GFP reporter. Different amounts of plasmid p1E17-EGFP-1 and p1E6-EGFP-1 was used to transfect HeLa cells. Forty eight hours later cells were analyzed by FACS. The ratio of the median fluorescent intensity to the transfection efficiency is shown in the Y-axis, and the amount of DNA used in the X-axis. (b) Comparison of Tat dependent transcription from LTR of two HIV-1 primary isolates, 1E6 and 1E17 using the GFP reporter. Different amounts of plasmid p1E17-EGFP-1 and p1E6-EGFP-1 was used to transfect HeLa cells. Forty eight hours later cells were analyzed by FACS. Experiments were done in triplicate, the data shown are the mean values of three experiments.

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Fig. 4. (a) Tat dependent transcription from LTRs of four primary isolates of HIV-1 1E6, 1E17, 4A8 and 4A23. 6 ×105 HeLa cells were transfected with 50 ng of plasmid constructs p4A8-EGFP-1, p4A23-EGFP-1, p1E17-EGFP-1 and p1E6-EGFP-1 in the presence of 50 ng of pSV2tat72. Forty eight hours later cells were analyzed by FACS for the expression of GFP. The fold increase in the mean fluorescence intensity after addition of Tat is shown here in the Y-axis. Data shown are the mean values of three experiments. (b) DNA dependent dose response of Tat transactivation. 6 × 105 HeLa cells were transfected with 50 ng of LTR 1E17-EGFP-1, in the presence of increasing amount of Tat (1 – 500 ng). The fold increase in the median fluorescence intensity after addition of Tat is shown here in the Y-axis, and the different amounts of Tat co-transfected in the presence of 50 ng of 1E17-EGFP-1 is shown in the X-axis. The data shown are the mean values of three experiments.

(Fig. 4). p1E6-EGFP-1 could not be transactivated even using 500 ng of Tat DNA (data not shown). These observations were consistent with previous results where CAT was used as the reporter gene (Peterlin et al. 1986). The median fluorescent intensity of GFP expression increased in the presence of increasing amounts of Tat DNA (Fig. 4), suggesting that GFP response to Tat transactivation was also DNA dependent. These properties of the reporter validates further the assay.

3.4. Sensiti6ity of CAT 6ersus GFP To compare reporter sensitivity of GFP and CAT, various amount of p1E17-EGFP-1 or p1E17-CAT were transfected in HeLa cells in the presence and absence of Tat. GFP expression can be detected using as little as 1 ng of the p1E17EGFP-1 in the absence of Tat (Table 1). At least 50 ng of the CAT construct was required to

observe basal CAT activity. Tat-activated expression was detected in the presence of 10 ng of 1E17-EGFP-1 and 1E17-CAT, and the degree of transactivation from both reporters using 1 and 5 ng of Tat DNA were similar (Table 2) and significant (PB 0.05). However when 50 ng of p1E17Table 1 Comparison of sensitivity of Tat-independent transcription using GFP and CAT reporters DNA amount (ng)

GFP expressiona

CAT expressiona

1 10 50 100

1.32 6.61 8.69 10.3

NDb ND 3.13 12.56

a GFP and CAT expression units are arbitrary. Experiments were done in triplicate and the mean values are shown in the table. b ND, not detected.

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Table 2 Comparison of sensitivity of detection by Tat transactivation using GFP and CAT reporters 1E17-EGFP-1/CAT (ng)

1 10 50 a b

GFP expressiona

CAT expressiona

Tat (1 ng)

Tat (5 ng)

Tat (1 ng)

Tat (5 ng)

1.64 1.86 2.5

2.79 6.28 9.74

NDb 4.12 76.66

ND 5.86 190.03

GFP and CAT expression units are arbitrary. Experiments were done in triplicate and the mean value are shown in the table. ND, not detected.

CAT was co-transfected with either 1 or 5 ng of Tat, the degree of transactivation was several folds higher than that of 1E17-EGFP-1 (Table 2).

4. Discussion It was established in this study that GFP is a useful reporter to quantitatively measure HIV-1 LTR promoter activity in a transient expression assay. The major advantage of using GFP over other reporters is the ability to detect gene expression in live cells by fluorescence without the need for cellular lysis and fixation, and to measure the transfection efficiency at the same time without co-transfection of a reference expression plasmids. Flow cytometric methods enabled us to analyze a large number of individual cells, and to evaluate the functional characteristics of the transfected viable cells. In a transient transfection assay, one expects that the expression of the transfected gene would be heterogeneous. FACS enabled us to visualize the heterogeneity in the level of GFP expression within the transfected population. The transfection efficiency was observed to be proportional linearly to the DNA transfected, but it eventually plateaued. However, median fluorescence intensity of the transfected population increased with the DNA amount without plateau, suggesting that further increase in the DNA concentration beyond the linear range of transfection efficiency, led to increases in the DNA copy number of the transfected cell population. Finding the linear dose response range of transfection is necessary for accuracy and sensitivity of the assay.

When comparing basal level transcription from the HIV-1 LTR using the GFP reporter versus the CAT reporter we observed that under very low concentration of DNA, sensitivity of detection by GFP was better than CAT. It is postulated that ‘gating’ of viable cells using FACS makes it possible to detect fluorescent signals from very few transfectants, which is not possible in a CAT assay. Targeting the transfected population, instead of using the entire cell homogenate for measuring the transcriptional activity, makes this assay more accurate, and also increases the sensitivity of detection of a positive signal. When Tat transactivated transcription was compared, it was noted that at lower levels of DNA, the degree of Tat transactivation was similar using either CAT or GFP. However, with increasing DNA amount the degree of CAT activity increased several folds over basal level in comparison to GFP. This suggests that the linear range of detection by GFP is more sensitive than CAT under low DNA concentration. However at higher concentration of DNA, CAT gives a stronger signal. This is because CAT assay utilizes the enzymatic properties of the reporter. As a result of the amplification of signal, the enzyme based reporter maybe more sensitive under certain conditions, but less under others. Since there is a linear relationship between fluorescent signal and the number of emitting chromophores, GFP fluorescence is a direct assay for protein expression, while CAT artificially enhances the signal, in that respect GFP expression system maybe more precise for comparing activities between two promoters.

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Acknowledgements We wish to thank the generous support of Department of Microbiology and Immunology and University of Maryland School of Medicine. We also wish to thank Dr David Volsky for providing the HIV-1 LTR clones, 601 and 602 and Dr Buel Dantese Rogers for comments on the manuscript. Reagents obtained from NIH AIDS Research and Reference Reagent Program are acknowledged.

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