Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.
Abstract for nonspecialists
HIV-1 virus is one of the most difficult targets for therapy because it hijacks the cells of our immune system and particularly because the virus mutates rapidly making it drug resistant. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously.
We propose a different strategy, where we target a specific FUNCTION of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.
The effect of mutations can thus be avoided since those mutations that cause the loss of the function also render the virus harmless. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on the viral maturation. In our system activation of any of them activates the antiviral cell defense or alternatively kills the infected cells, preventing further spread of infection. The same approach could be implemented for defense against other viral infections.
Animation of split-ubiquitin system.
We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. Two essential viral functions have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease.
Binding of virus to human T-cells causes formation of CD4-CCR5 heterodimers, which in our system reconstitutes the split ubiquitin. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of versatile effector genes, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same viral function was successfully utilized in the implementation of the split TEV protease system.
The second implementation of this idea was to utilize the activity of HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 RNA polymerase. T7 RNA polymerase released from the membrane subsequently activates the defense similar to that described with the split protein system. All three systems work in human cells. We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.
PLEDGE: All experimental work on this project was performed from May to October 2007 by the undergraduate students participating in the team under the tutorial of instructors. All the students participated at iGEM for the first time.