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Abstract

MLV productively only infects proliferating cells such as T cells and B cells but cannot enter the nucleus and integrate its genome in non-dividing cells, in contrast to HIV-1. Reverse transcription of the viral single-stranded RNA (ssRNA) genome into double- stranded DNA (dsDNA) starts inside the cytoplasm after delivery of the capsid core. The viral nucleic acids are protected from host cell factors by the capsid this process. The capsid is then transported to the nuclear envelope, where the subviral particles wait for cellular division to access the nucleus. Nuclear entry and retention upon mitosis is mediated by p12, a cleavage product of the main structural protein Gag, which anchors the reverse transcription and pre-integration complex (RTC/PIC) to chromatin and retains the viral genome in the nucleus once the nuclear envelope reforms. For integration to occur, the MLV capsid needs to release the viral copy DNA (cDNA) in a process called uncoating. Despite being extensively studied, the exact location, mechanism, and spatio-temporal nature of uncoating as well as its relationship to viral cDNA synthesis and integration have not been completely elucidated. Due to the absence of experimental systems able to visualize MLV cDNA in living and dividing cells, the understanding of early replication events is hindered. To overcome this issue, this thesis, adapted a system (ANCHOR) to visualize single molecules of accessible MLV dsDNA genomes in living and dividing cells and to investigate the dynamics of viral dsDNA synthesis using fluorescence microscopy. Nuclear viral dsDNA was unambiguously detected in daughter cells after cellular division using both fixed and live cell conditions. Notably, subviral complexes comprised of largely complete sets of CA and p12 proteins entered the nucleus, whereas the viral cDNA was devoid of detectable CA and p12 proteins. In contrast to HIV-1, a fluorescently tagged integrase fusion protein (IN.FP) additionally incorporated into the particle was not retained at positions of the CA/p12 complex but accompanied MLV cDNA, likely to integration sites. These data suggest, that MLV capsids remain largely intact until nuclear envelope breakdown and subsequent chromatin attachment. Afterwards, the viral genome moves away from the CA protein shell. The uncoated viral cDNA associated with IN.FP is then likely translocated to integration sites of the host genome. Importantly, this thesis achieved, for the first time, tracking of living cell lineages together with visualization of MLV genomes within the nuclei of multiple cell generations. Using this approach allowed the observation of long lived single nuclear signals that are propagated from a single parent to both daughter cells; these cDNA signals likely represent integrated proviruses.In conclusion, the adaptation and validation of the ANCHOR system for the visualization of MLV dsDNA provided a versatile tool for single particle investigation of productive early replication events and shed light on the relation of capsid uncoating and reverse transcription. Finally, this tool may enable future studies to gain additional insights into retroviral replication.