Ljubljana/summary

Achievements

• Design and implementation of three functional systems of activation of anti-HIV defense based on:

  - the detection of viral interactions with the cells and

  - cleavage of specific substrate by viral protease.

• Successful detection of CD4-CCR5 heterodimer formation through:

  - split tobacco etch virus (TEV) protease,

  - split ubiquitin system.

• Construction of BioBricks for heterodimerization, which represent a powerful platform for engineering cell processes based on protein-protein interactions.
• Introduction of the versatile amplification platform based on the use of T7 RNA polymerase in mammalian cells.
• Activation device based on the proteolytic cleavage and release of membrane anchored T7 RNA polymerase from the membrane into the cell nucleus.
• Anchoring of T7 RNA polymerase to the plasma membrane either by:

  - fusion to CD4 or

  - addition of a myristoylation signal.

• Implementation of two different types of effector molecules for:

  - induction of cell death (caspase-3)or

  - activation of antiviral defense (interferon beta).

Prospects for future work and real world applications

• The idea of relying on viral functions could be applied also to other HIV-specific processes (such as genome integration, reverse transcription…).
• Our system based on BioBricks has been useful to prove the principle, but for the real application cellular delivery would be much better applied using vectors based on viral delivery.
• In the case of using viral vectors, therapy could be delivered specifically to the relevant cells that can be infected by HIV-1 (e.g. similar as T-cell-directed retroviral delivery systems described by David Baltimore at SB2.0).
• Selection of different effectors will (and can) be made based on further experiments – apoptosis can only be used in 100% leak-proof systems, while antiviral and immune response effectors are not as sensitive.
• Similar systems can be prepared for therapy of many other pathogens, particularly viruses (e.g. yellow fever, SARS, foot and mouth disease…) which contain specific proteases and rely on defined cellular receptors.