Nucleosome assembly on pre-integrated DNA of HIV-1-based vectors: Histone composition and post-translational modifications

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This fellowship proposes to analyze the timing of nucleosome formation and the histone composition of unintegrated retroviral DNA of human immunodeficiency virus type 1(HIV-1)-based and murine leukemia virus (MLV)-based retroviral vectors. Retroviral vectors have become effective tools for gene delivery in basic research as well as in clinical gene therapy. The early steps of the vector-based gene delivery resembles the early life cycle of the respective wild-type retrovirus. Therefore, retroviral vectors can be utilized not only as gene delivery vehicles but also to study retrovirus biology. It has been recognized that extrachromosomal, unintegrated retroviral DNA is very poorly transcribed in virtually all cells, whereas the integrating version is robustly expressed in permissive cells. Silencing or decreased transcriptional gene expression is caused by the host cell as a protection against invading pathogens and is achieved by histone modifications that induce an inactive chromatin state. These transcriptional silencing mechanisms also limit efficient gene expression of integration-deficient retroviral vectors. These vectors can be used as an alternative to integrating retroviral vectors in post-mitotic cells, e.g. retina cells or neurons, or for transient gene delivery purposes, e.g. for the delivery of components of the CRISPR/Cas9 technology. The proposed postdoctoral project will systematically analyze the involved host cell machinery involved in nucleosome assembly onto unintegrated retroviral DNA, the timing of nucleosome formation after nuclear entry and the histone composition and their post-translational modifications. This knowledge will lead to a deeper understanding of retrovirus-host interactions after nuclear entry before integration, and will help to discover the underlying mechanisms for silencing. Analyses with non-viral transfected DNA are also planned to investigate any similarities or differences in histone loading and marking, as compared to viral DNA. This will address the question as to whether there is a potential common extrachromosomal defense mechanism. In conclusion, the findings of this fellowship proposal will reveal the principles of nucleosome formation onto pre-integrated retroviral DNA and will have also implications for integration-deficient retroviral vectors and non-viral transfected DNA. Furthermore, it may define new, broadly utilized anti-viral defense mechanisms.