Ljubljana

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

The main problem with treatment of HIV infection is rapid mutation rate which makes the virus resistant against different drugs. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously. This however means that this therapy is very extensive and has to be used for life. Our approach was to construct a defense system based on the principles of synthetic biology, which uses the viral function to activate defense response in the attacked cell. Mutations that cause the loss of its function also render the virus also harmless at the same time. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on viral maturation. 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 as well.

Scientific abstract

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 and processing of viral proteins by its own protease. Binding of virus to human cells causes formation of CD4-CCR5 heterodimers, which reconstitutes the split TEV protease. This protease cleaves the membrane-anchored T7 RNA polymerase off 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 function was successfully implemented with split ubiquitin system. Our second approach was to exploit the viral protease, which cleaves the specific linker introduced between the T7 RNA polymerase and a cellular membrane anchor. Released polymerase subsequently activates the defense similar to that described with the split protein system. All three systems have been shown to work in human cells and have potential also for defense against other infections.

Executive summary

HIV-1 virus is one of the most difficult targets for therapy because it hijacks our immune system and particularly because the virus mutates rapidly. This allows the selection of mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Currently the most effective therapy consists of an inhibitor cocktail that decreases the probability of HIV to overcome all target sequences at the same time.

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.

For implementation of this idea we have selected two viral functions: viral attachment to the cellular co-receptors CD4 and CCR5 and viral proprotein processing by its own protease. Binding of virus to T-cells leads to the formation of CD4-CCR5 heterodimer. In our device, formation of this heterodimer triggers reconstitution of a split protein (split ubiquitin or TEVP) which activates the specific proteolytic activity. This releases the T7 RNA polymerase from the membrane anchor and leads to transcription of the effector gene, which prevents the spread of viral infection.
The second implementation of this idea was to utilize the 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 polymerase. Activation of viral protease similarly as above releases the T7 polymerase and starts the defense program.

Important points of both approaches are to use the amplification of the signal, achieved by the use of T7 polymerase and the system's versatility, as we can select any number of different genes to be activated by the detection of viral function. The crucial aspect of our approach is that this system is not sensitive to viral mutations and is not activated only in case where the mutation leads to the loss of the viral function, which however also renders virus harmless at the same time.

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.

National Institute of Chemistry

University of Ljubljana

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