Commitment to apoptosis in CD4(+) T lymphocytes productively infected with human immunodeficiency virus type 1 is initiated by lysosomal membrane permeabilization, itself induced by the isolated expression of the viral protein Nef

Laforge M, Petit F, Estaquier J, Senik A
Source: J Virol
Publication Date: (2007)
Issue: 81(20): 11426-40
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
Resting human T cells were nuclefected with Cat-D mRNA targeting siRNA, followed by HIV infection. Also transfection of activated T cells with expression vectors encoding Nef-WT, Nef-G2A mutant of the antisense Nef, co-transfection of pmaxGFP to validate transfection efficiency (26 +/- 5 % from preliminary experiments).
Primary CD4(+) T lymphocytes, supporting in vitro human immunodeficiency virus type 1 (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 Percoll density gradients to isolate a nonapoptotic CD4(+) T-cell subset with a high inner mitochondrial transmembrane potential (DeltaPsim) 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 small interfering RNA 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 is 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, with 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.