Single-Cell Imaging of HIV-1 Proviruses

HIV proviruses at the periphery of the nucleus Technical developments in imaging-based techniques have greatly improved our understanding of HIV–host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus–host interplay during all steps of HIV-1 replication cycle. Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment, which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA.

A new paper describes a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. A 3D topological analysis demonstrated that integrated viral DNA localizes at the periphery of the nuclei revealing important insights in the nuclear biology of HIV-1.

 

Single-Cell Imaging of HIV-1 Provirus (SCIP). PNAS USA 19 March 2013, doi: 10.1073/pnas.1216254110
Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.

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