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J. Virol. doi:10.1128/JVI.00597-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Commitment to apoptosis in productively HIV-1-infected CD4⁺ T lymphocytes is initiated by lysosomal membrane permeabilization, an event which is itself induced by the isolated expression of the viral protein Nef

From INSERM U542, Hôpital Paul Brousse, Villejuif, France; URA CNRS 1930, Institut Pasteur, France, and Unité INSERM 841,Creteil, France

^* To whom correspondence should be addressed. Email: anna.senik{at}creteil.inserm.fr.

	Abstract

Primary CD4⁺ T lymphocytes, supporting in vitro HIV-1 replication, are destined to die by apoptosis. We explored the initial molecular events that act upstream from mitochondrial dysfunction, in CD4⁺ T lymphocytes exposed to the HIV-1_LAI strain. We tracked by immunofluorescence the cells expressing the p24 viral antigen and used density Percoll gradients to isolate a nonapoptotic CD4⁺ T cell subset with a high inner mitochondrial transmembrane potential (m), but no outer mitochondrial membrane (OMM) rupture. In most p24⁺ (but not bystander p24^-) cells of this subset, the lysosomes were undergoing limited membrane permeabilization, allowing the lysosomal efflux of cathepsins (Cat) to the cytosol. This was also induced by HIV-1 isolates from infected patients. Using pepstatin A to inhibit Cat-D enzymatic activity, and Cat-D-siRNA to silence the Cat-D gene, we demonstrate that once released into the cytosol, Cat-D induces the conformational change of Bax and its insertion into the OMM. Inhibition of Cat-D activity/expression also conferred a transient survival advantage upon productively HIV-1-infected cells, indicating that Cat-D was an early death factor. The transfection of activated CD4⁺ T lymphocytes with a Nef expression vector rapidly induced the permeabilization of lysosomes and the release of Cat-D, these two events preceding OMM rupture. These results reveal a previously undocumented mechanism in which Nef acts as an internal cytopathic factor and strongly suggest that this viral protein may behave similarly in the context of productive HIV-1 infection in CD4⁺ T lymphocytes.