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VISUALIZATION OF HIV-1 DNA IN INFECTED CELLS USING A NEW FLUORESCENT VIRUS-BASED REPORTER SYSTEM Cristina Di Primio (1), Valentina Quercioli (1), Marina Lusic (2, Awatef Allouch (1), Zeger Debyser (3), Frauke Christ (3), Mauro Giacca (2), Anna Cereseto (4) (1) Scuola Normale Superiore, Molecular Biology Laboratory, Pisa, Italy; (2) Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; (3) Laboratory of Molecular Virology and Gene Therapy, Leuven, Belgium; (4) Laboratory of Molecular Virology, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy Fluorescent microscopy represents a new powerful tool in HIV-1 research. In fact imaging-based approaches were crucial in unraveling transient viral events occurring at different stages of the viral replication such as fusion, cytoplasmic trafficking and budding. Nevertheless, several aspects of the nuclear biology of HIV-1 are still unclear and new tools of investigation are required to understand the viral interactions with the nuclear compartment. Recently a new imaging system was developed allowing the analysis of the trafficking of the pre-integration complexes within the nuclear compartment. However, there is still no experimental approach to image the viral DNA. Here we developed a new virus-based fluorescent reporter system that allows for the first time the visualization of HIV-1 DNA genomes. We engineered the HIV-1 genome by including a heterologous endonuclease target site which can be visualized by immunofluorescence (IF) following its cleavage. 3D Immuno-DNA FISH, antiviral drugs tests and trasportin-SR2 knock down experiments confirmed that the IF signal correspond to HIV-1 DNA and experiments with integration competent and not-competent viruses demonstrated that this system allows specifically the detection of HIV-1 DNA integrated into the host genome. We demonstrated that this system can be used as a clear-cut readout to study the timing of integration and to test known and new antiviral compounds. The integration of this reporter system with high throughput screening platforms could offer unprecedented perspectives for HIV-1 drug discovery.