Structure and function of nucleoprotein complexes involved in retroviral integration

Upon infection, a retrovirus aims to insert its genome into the host DNA. This process, commonly referred to as “integration”, is catalysed by the retroviral integrase enzyme (IN). The property to introduce foreign DNA into a host genome is at the basis of gene therapy applications, and provides a means for pathogenic retroviruses (such as the human immunodeficiency virus HIV) to hijack the cellular transcription machinery. In these contexts, it is important to note that in vivo, retrovirusses typically integrate non-randomly throughout the host chromatin. For example, HIV favours integration in the bodies of actively transcribed genes. These genomic regions were recently found to correlate with substantial levels of negative supercoiling. Whether and how the physical state of the host DNA/chromatin affects retroviral strand transfer catalysis, has not been reported before. Nevertheless, several lines of evidence suggest that tension and torque might play an important role during retroviral integration in vivo.

The goal of this doctoral thesis is to unravel the role of DNA structure, mechanics, and topology in retroviral integration, thereby focussing on the HIV-1 virus. Experiments in vitro with purified recombinant proteins will be carried out in singulo using single-molecule fluorescence and atomic force microscopy, and with magnetic tweezers. These studies will result in a detailed understanding of the nanoscale structure and mechanics of retroviral integration. In a second step, these in vitro techniques will be employed to study the interaction of purified pre-integration complexes with DNA, and serve as an intermediate between the in vitro and in vivo situation. Finally, these results will be validated in vivo, using a combination of integration site sequencing and psoralen crosslinking.

We anticipate that this unique combination of experiments will yield unprecedented insights in the mechanochemistry of retroviral integration, at increasing levels of complexity.