Ljubljana/implementation
From 2007.igem.org
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.
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.
Split ubiquitin system
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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