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Surface expression of transmembrane receptors CD4, CCR5 and CXCR4

Surface expression of transmembrane receptors was measured by flow cytometry. We have confirmed expression of the following versions of transmembrane receptors at the surface of cell membrane: CD4(HA)-CUb-GFP, CCR5(c-myc)-NUb and CXCR4(c-myc)-NUb (Fig. 1). Additional constructs with transmembrane receptors were also tested and found to be located at the cell membrane. Expression of transmembrane receptors at the cell surface was crucial for our split systems since it shows appropriate protein folding, thus suggesting that receptors are also functional. Both split-ubiquitin and split-TEV system are activated by heterodimerization of transmembrane receptors after their interaction with viral particles. The following experiments were done only with CD4 and CCR5 constructs (used by macrophage-tropic viruses). CXCR4 constructs represent receptors for another group of HIV (lymphotropic or dual-tropic viruses) that utilize CXCR4 receptors for entry into cells. Thus we can expand the range of usefulness of our idea to other viral strains.

Fig. 1. Transmembrane receptor constructs CD4, CCR5 and CXCR4 are expressed at the cell surface. Transmembrane receptors were tagged with peptide tags, which allow detection with labeled antibodies. All versions of CD4 were tagged with HA-tag, CXCR4 and CCR5 were tagged with c-myc-tag at the extracellular side of the receptors. Cells were transfected with different receptor constructs and incubated. Surface expressed receptors were detected by using anti-HA (mouse) and anti-c-myc (rabbit) antibodies, followed by fluorescently labelled secondary antibodies (anti-mouse-PE and anti-rabbit-DyeMer). Receptors were detected by flow cytometry. Fluorophores were excited with 488 argon laser and emitted light was detected at 575 and 610 nm, respectively. 10000 events were collected and results are presented as mean fluorescence values at defined emission wavelenght. In comparison with non-transfected cells we can see that our transmembrane receptors are expressed at the cell surface.

T7 promoter (pT7) in mammalian cells

T7 promoter is one of the most important aspects of our system since all our reporter and effector proteins are put under its control. Split-ubiquitin, split TEV and HIV protease systems all release T7 RNA polymerase after viral activated heterodimerization of receptors or HIV protease cutting at specific sequence. T7 RNA polymerase with NLS (nuclear localisation sequence) is translocated into the nucleus. T7 RNA polymerase transcribes genes that are controlled by the T7 promoter (reporter, effector and self-amplifying genes).
We wanted to show that:

  • T7 RNA polymerase system is specific enough to be active only when the system is activated by the presence of HIV (by viral heterodimerization of receptors or by HIV protease),
  • T7 RNA polymerase is not active when bound to the cell transmembrane receptors,
  • T7 RNA polymerase is activated when it is cut off from the transmembrane receptor.

We have tested T7 promoter by adding different reporter genes under its control. Constructs pT7-fLuc and pT7-mCer were prepared to test our system with lmeasurement of the uciferase luminiscence and by confocal microscopy.
Luciferase measurements were used to determine an optimal amount of an effector gene under the T7 promoter and the activity of T7 RNA polymerase. The reason for optimization was observation of a small amount of luciferase expression under the T7 promoter even in absence of T7 RNA polymerase when cells were transfected with high amount of the cinstruct under the T7 promoter. With the system consisting of CMV-T7pol and pT7-fLuc in different ratios we have demonstrated that at optimal ratios of pT7-effector gene and T7 RNA polymerase we can guarantee specific expression of effector genes (Fig. 2). In the final stage of our project the luciferase is replaced with an effector gne, such as caspase-3. If T7 RNA polymerase is specifically activated by HIV receptor dimerization or by protease activity, caspase-3 should be expressed only in cells under attack and cause their apoptosis.

Fig. 2. Expression of luciferase under T7 promoter is proportional to the amount of added T7 RNA polymerase. HEK293 cells were transfected with 50 ng of pT7-fLuc construct and increasing amounts of stimuli independent CMV-T7 RNA polymerase. After incubation cells were lysed and the amount of expressed luciferase was measured. 50 ng of pT7-fLuc was low enough to prevent spontaneous transcription from T7 promoter. By adding T7 RNA polymerase we triggered specific transcription of luciferase. Higher amount of added T7 RNA polymerase resulted in stronger activation of the system was observed. T-test: p<0.005, ****.

To confirm the results with with luciferase reporters additional experiment was performed with construct a pT7-mCer, which is a fluorescent protein. Confocal microscopy showed that in the absence of T7 RNA polymerase mCer is not formed. After addition of T7 RNA polymerase we can detect expression of mCer in the cytosol (data not shown).

The next step was to show that T7 RNA polymerase is inactive when bound to the membrane proitein CD4 by a peptide linker containing the HIV protease recognition site (Fig. 3). Cells were co-transfected with different amounts of the following constructs: CD4-HIVpro-T7pol(NLS), pT7-rLuc and HIV protease. In the absence of HIV protease T7 RNA polymerase can not transcribe renilla luciferase gene and luciferase activity is not detectable. After the addition of HIV protease T7 RNA polymerase is released from the membrane anchor. It is activated and stays in the cytoplasm or travels to the nucleus because of its nuclear localization sequence. Genes under the T7 promoter control are transcribed and luciferase activity is observed. Again the reporter gene rLuc is replaced by an effector caspase-3 gene under T7 promoter, which would cause apoptosis only in HIV-infected cells.

Fig. 3. Transcriptional activity of T7 RNA polymerase is activated by cleavage of the linker to the membrane anchor by HIV protease. HEK293 cells were transfected with reporter plasmid pT7-rLuc, transmembrane receptor with T7 RNA polymerase fused to it by the HIV protease recognition site and different amounts of HIV protease plasmid. In cells without HIV protease only weak luciferase activity was observed. Addition of HIV protease cleaved the linker and activated T7 RNA polymerase, which in return transcribed the luciferase under the T7 promoter. T-test: p<0,1, *.

In our model we have proposed a construct pT7-T7pol, which would increase the number of active T7 RNA polymerases in a positive autoloop since entry of HIV into cells or HIV protease can only activate a small amount of T7 RNA polymerase. If we want to have fast and strong effector protein action we must guarantee large amount of active T7 polymerase as soon as possible. Construct pT7-T7pol has been made and deposited into BioBrick database.