Ljubljana/results

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<title>Company Name</title>
<title>Company Name</title>
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       <h3><span>Current Disease treatment</span></h3>
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       <h3><span>Results</span></h3>
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       <p class="p1"><span><b>HIV is a typical retrovirus that upon entry into host cell reverse transcribes its RNA to DNA which integrates into the host genome. Viral genes are expressed as polyprotein that is cleaved into functional proteins by HIV protease. Viral coat is composed of lipids and two predominant proteins, gp41 and gp120, which are essential for binding onto the host cell receptor CD4. Several drugs were developed that target crucial stages of HIV entry into the host cell, its reverse transcription, integration and processing of the polyprotein. Current standard therapy consists of taking a combination of at least three different antiretroviral drugs.</b></span></p>
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       <p class="p1"><b>Before testing the full functional devices we had to do some research on individual BioBricks to prove their expression and activity of their protein products in mammalian cell cultures. Preliminary testing included control of surface expression of transmembrane receptors with flow cytometry and testing and optimization of the T7 promoter system in mammalian cells.<br>
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We continued with testing of individual subsystems; fluorescent reporter proteins were used to prove the functionality of subsystems with confocal microscopy and luminescence for luciferase reporters. Western blotting was used to proteolytic cleavage of test split and TEV protease subsystems. Final stage was testing of effector protein activity – caspase 3. Its expression was determined by ELISA test and its activity, resulting in cell apoptosis was detected by flow cytometry.<br>
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We have shown that anti-HIV defense based on HIV entry or its activity in infected cell can lead to transcription and activation of effector proteins that either trigger apoptosis or enhance antiviral immune response.<br>
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Detailed information on results of our experiments can be found in the following chapters.<span></b>
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      <h3><span>Virus Life Cycle</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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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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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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       <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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      <h3><span>Classes of Antiretroviral Drugs</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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      <h3><span>Development</span></h3>
 
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     <div id="footer">______________________________________<br />
     <div id="footer">______________________________________<br />
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<a href="https://2007.igem.org/Ljubljana/model">Model</a>     
|<a href="https://2007.igem.org/Ljubljana">Home</a>|
|<a href="https://2007.igem.org/Ljubljana">Home</a>|
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<a href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>
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<a href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>
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         <h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3>
         <h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3>
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          <li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li>
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         <h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3>
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         <h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3>
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          <li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li>
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        <li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> 
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<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li>
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<div id="ldiscussion">
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         <h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3>
         <h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3>
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<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Terms & References</a>&nbsp; </li>
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Terms & References</a>&nbsp; </li>
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li>
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li>
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Latest revision as of 18:48, 23 November 2007

Company Name

Results

Before testing the full functional devices we had to do some research on individual BioBricks to prove their expression and activity of their protein products in mammalian cell cultures. Preliminary testing included control of surface expression of transmembrane receptors with flow cytometry and testing and optimization of the T7 promoter system in mammalian cells.
We continued with testing of individual subsystems; fluorescent reporter proteins were used to prove the functionality of subsystems with confocal microscopy and luminescence for luciferase reporters. Western blotting was used to proteolytic cleavage of test split and TEV protease subsystems. Final stage was testing of effector protein activity – caspase 3. Its expression was determined by ELISA test and its activity, resulting in cell apoptosis was detected by flow cytometry.
We have shown that anti-HIV defense based on HIV entry or its activity in infected cell can lead to transcription and activation of effector proteins that either trigger apoptosis or enhance antiviral immune response.
Detailed information on results of our experiments can be found in the following chapters.























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