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126 PML Nuclear Bodies Determine the Repressive Environment and Restrict Viral Gene Expression in Primary Human Lymphocytes

Marini, Bruna1; Giacca, Mauro1; Lusic, Marina1,2

JAIDS Journal of Acquired Immune Deficiency Syndromes: April 2011 - Volume 56 - Issue - p 51
doi: 10.1097/01.qai.0000397314.01966.b8

1ICGEB, Padriciano 99, Trieste, Italy; and 2AIDS Immunophatogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Instititute, Milan, Italy

Various members of tripartite motif (TRIM) protein family display antiviral properties, targeting retroviruses in particular. The activity of TRIM19, better known as promyelocytic leukemia protein (PML) against several viruses has been well documented, yet its role in HIV-1 infection remains elusive.

P-TEFb kinase complex, composed of CDK9 and CyclinT1, is one of the most important cellular partners playing a key role in HIV-1 transcription. We have previously demonstrated that both members of P-TEFb localize inside the PML Nuclear Bodies (PML NBs) and that the acetylated and enzymatically inactive form of CDK9 interacts with PML. Since acetylated CDK9 was found to bind the integrated and transcriptionally silent viral genome, we hypothesized that the latter might reside in the proximity of PML NBs.

To test this hypothesis, we developed 3D Immuno DNA FISH on latently infected Jurkat cells; we observed close proximity between silent HIV-1 provirus and PML NBs, whereas transcriptional activation induced by TNF-α or TPA led to a significant displacement of PML NBs. This result was confirmed by ChIP, showing PML occupancy at the HIV LTR in resting conditions and its dynamic release after induction. To establish the functional role for PML in HIV-1 repression, we disrupted PML NBs by arsenic trioxide treatment or by stably knocking-down PML protein with a lentiviral vector; both treatments induced a very significant increase in the levels of viral transcription. Finally, we confirmed the association of HIV with PML and the role of these bodies in a primary model of HIV latency, using CD4+ cells purified from human blood and successively infected in vitro.

Thus, provirus resides in the close proximity to PML NBs, nuclear neighborhood that represses viral gene expression and contributes to viral latency.

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