15th Conference on Retroviruses and Opportunistic Infections Boston, Massachusetts - February 3-6, 2008

Conf Retrovir Opportunistic Infect 2008 Feb 3-6;15: (abstract no. 49)

Nathalie Arhel and P Charneau
Pasteur Inst, Paris, France

BACKGROUND: Emerging real-time imaging techniques, coupled with fine ultrastructural studies of viral infections, provide powerful tools for understanding viral-host cell interaction dynamics and viral mechanisms.

METHODS: To visualize the early steps of HIV-1 infection in a quantitative manner, until integration of the provirus in the cellular DNA, we labeled HIV-1 integrase with a small tetracysteine tag, which, while entirely preserving virus infectivity, allows both intracytoplasmic and intranuclear HIV-1 complexes to be quantitatively tracked in 3 dimensions through time (4 dimensions; 4D) in human cells, and enables the analysis of HIV-1 kinetics by automated 4D quantitative particle tracking.

RESULTS: In the cytoplasm, HIV-1 complexes underwent movements kinetically characteristic of microtubule- followed by actin-dependent transport before reaching the nuclear membrane. Docked complexes then adopted vibratory movements, followed by diffuse movements once inside the nucleus. We also sought to understand the mechanisms by which the central DNA Flap mediates HIV-1 nuclear import. We found that reverse transcription occurs within an intact capsid shell, independently of the rapid routing process towards the nuclear membrane, and that decapsidation is not an immediate post-fusion event, but rather occurs at the nuclear pore upon completion of reverse transcription. We provide the first observation with ultrastructural resolution of intact intracellular HIV-1 capsid shells by scanning electron microscopy.

CONCLUSIONS: In the absence of central DNA Flap formation, decapsidation is impaired and linear DNA remains trapped within an integral capsid shell precluding translocation through the nuclear pore. We therefore show that DNA Flap formation, the very last event of HIV-1 reverse transcription, acts as a viral promoting element for the uncoating of HIV-1 at the nuclear pore.

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2008-02-03
49

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