Ljubljana/implementation

From 2007.igem.org

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For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). Basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of split ubiquitin assay is described <a href="#">here</a>.<br><br>
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For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). Basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br>
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. Releasing of only a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br>
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. Releasing of only a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br>
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When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane, translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react on infection with minute numbers of HIV viruses.<br><br>
When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane, translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react on infection with minute numbers of HIV viruses.<br><br>
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      <h3><span>Split TEV protease</span></h3>
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      <p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from the purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br>
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For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br>
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       <h3><span>Classes of Antiretroviral Drugs</span></h3>
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       <h3><span>Activation based on the HIV Protease Activity</span></h3>
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       <p class="p1">Antiretroviral drugs are mostly inhibitors of different stages in HIV life cycle. They are targeted at different enzymes or events that are typical for HIV infection – entry of the virus into the cell, reverse transcription, polyprotein cleavage... and are divided into seven main classes (REFERENCA!):<br>
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       <p class="p1">The second approach to detect the viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV already infected cells. This protease is required for a cleavage of viral polyprotein and has a defined substrate specificity, similar as above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides <font color="red">(ref NatMed)</font>. The activation step occurs when HIV protease cleaves inside the linker, which connets membrane anchor and a reporter protein.<br><br>
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We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br>
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When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br>
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       <h3><span>Development</span></h3>
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Revision as of 16:57, 25 October 2007

Company Name

Implementation

Activation based on heterodimer formation and reconstitution of split proteins

We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.

Split ubiquitin system

For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). Basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of split ubiquitin assay is described HERE.

It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. Releasing of only a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.

T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in cellular antiviral defense system were placed under this promoter.

When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane, translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react on infection with minute numbers of HIV viruses.

Split TEV protease

TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from the purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described HERE.

For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.

Activation based on the HIV Protease Activity

The second approach to detect the viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV already infected cells. This protease is required for a cleavage of viral polyprotein and has a defined substrate specificity, similar as above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (ref NatMed). The activation step occurs when HIV protease cleaves inside the linker, which connets membrane anchor and a reporter protein.

We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.

When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.