Ljubljana/implementation

From 2007.igem.org

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       <h3><span>Implementation</span></h3>
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       <p class="p1"><span><b>POVZETEK. Je potreben?</b></span></p>
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       <h3><span>Activation based on the heterodimer formation and reconstitution of split proteins</span></h3>
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       <h3><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h3>
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       <p class="p1"><span>HIV (human immunodeficiency virus) is a retrovirus, Its genome is composed of two single stranded RNA molecules. It has a gag/pol/env organization; gag genes (group specific antigen) code for structural proteins, env for proteins that build viral envelope, while pol genes are responsible for viral reproduction (they contain genes for reverse transcriptase, integrase and HIV protease).<br><br>
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       <p class="p1"><span>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.<br><br>
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HIV envelope consists of lipids and viral glycoproteins gp120 and gp41, which are crucial for binding of HIV to the host cell membrane and for entering into the cell. Inserted into the lipid bilayer are also other glycoproteins that guarantee firmness and protective function of the viral envelope. Gp120 binds to receptors (CD4) on the host cell surface, but additional co-receptors like chemokine receptors (CCR5, CXCR4) are also required for successful entry of HIV. Mutations in co-receptor genes can cause immunity – if HIV cannot enter host cells, HIV infection is prevented, and AIDS cannot develop.<br><br>
 
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The characteristic retroviral enzyme is reverse transcriptase, which transcripts viral RNA into DNA. Only DNA can integrate into host cell genome – this is the crucial step in expressing viral proteins that are needed for assembly of new viral particles. Viral gag and gag/pol genes are expressed as polyprotein; until this polyprotein is cut into functional units, it exerts no biological function. Polyprotein clipping is done by HIV protease. The resulting polyprotein fragments represent functional enzymes and structural proteins.<br><br>
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      <h3><span>Split ubiquitin system</span></h3>
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Transcription of viral RNA into DNA and processing of the viral polyprotein are two most important steps in HIV replication cycle. These are thus obvious targets for HIV therapeutics. Inhibitors of reverse transcriptase and HIV protease are currently used to treat acute HIV infection.</a></span></p><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 in a frame.<br><br>
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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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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.<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>
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      <h3><span>Current Disease Treatment</span></h3>
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      <p class="p1">One of the first AIDS therapeutics were nucleotide or nucleoside analogues (NRTI – nucleoside-analogue reverse transcriptase inhibitors) – pseudosubstrates, that are during reverse transcription integrated into viral DNA instead of nucleosides and thus block the transcription. These drugs were superseded by non-nucleoside inhibitors (NNRTI) that could inhibit reverse transcriptase by binding into the alosteric site of the enzyme. The third type of drugs is a family of HIV protease inhibitors. In most cases specific inhibitors are very similar to protease substrates - the only difference is that because they cannot be cut, they block the active site by remaining bound into it.<br><br>
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Weakness of all these therapeutics is that they are very sensitive to HIV mutations – HIV can easily mutate and thus become drug resistant. A combination of drugs is used to minimize HIV's potential to develop resistance to each individual drug in the mixture. Some of the drugs induce mutations that have negative effect on the virulence and such drugs can be used in spite of developed resistance.<br><br>
 
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We still do not have a cure for AIDS that would be insensitive to HIV mutations. Our project presents new ways of potential AIDS therapeutics. Our approaches can be considered independent of HIV mutations. We have set up a few of biological ambushes; if HIV manages to avoid them, we presume that it would not be able to infect the cell anyway.</p>
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Revision as of 16:42, 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 in a frame.

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.

Classes of Antiretroviral Drugs

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!):

Development

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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.

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