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Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells.

Natesampillai S, Nie Z, Cummins NW, Jochmans D, Bren GD, Angel JB, Badley AD - PLoS Pathog. (2010)

Bottom Line: The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells.These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication.Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

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Related in: MedlinePlus

Discordance Associated Mutations (DAMs) within HIV-1PR alters the loss of mitochondrial transmembrane potential.YFP HIV protease or the indicated protease point mutants were expressed in HeLa cells, and the YFP positive cells were analyzed for loss of mitochondrial transmembrane potential (A, B). *Indicates p value ≤0.05. Data reflective of at least three independent replicates. Bars with solid color represent controls, bars with angled hatching represent DAMs, and bars with horizontal hatching represent non-DAMs.
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ppat-1001213-g003: Discordance Associated Mutations (DAMs) within HIV-1PR alters the loss of mitochondrial transmembrane potential.YFP HIV protease or the indicated protease point mutants were expressed in HeLa cells, and the YFP positive cells were analyzed for loss of mitochondrial transmembrane potential (A, B). *Indicates p value ≤0.05. Data reflective of at least three independent replicates. Bars with solid color represent controls, bars with angled hatching represent DAMs, and bars with horizontal hatching represent non-DAMs.

Mentions: We have previously shown that expression of HIV-1 PR causes cell death which is associated with loss of mitochondrial transmembrane potential, caspase 3 activation and nuclear fragmentation [16]. After transfection with YFP and YFP protease, He-La cells were assessed for loss of mitochondrial transmembrane potential in the YFP-PR positive cells by TMRE staining, which is a hydrophobic dye, taken up by mitochondria with intact transmembrane potential. We specifically evaluated TMRE loss in the YFP positive cells, indicating expression of protease. Expression of YFP alone or YFP with catalytically inactive YFP D25G PR resulted in minimal loss of transmembrane potential. Conversely, YFP-WT PR or treatment with CCCP, which uncouples mitochondrial transmembrane potential, resulted in significant loss of TMRE retention (Figure 3A). Strikingly, expression of YFP-L63P PR, YFP-D30N PR, YFP-K20R-PR, or YFP-L90M PR caused high degrees of transmembrane potential loss, similar to WT Protease. In contrast, YFP-PR fusions containing the DAMs I54V, V82A caused a lesser degree of transmembrane potential loss than WT-PR, that was similar to that of catalytically impaired T26S (Figure 3B).


Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells.

Natesampillai S, Nie Z, Cummins NW, Jochmans D, Bren GD, Angel JB, Badley AD - PLoS Pathog. (2010)

Discordance Associated Mutations (DAMs) within HIV-1PR alters the loss of mitochondrial transmembrane potential.YFP HIV protease or the indicated protease point mutants were expressed in HeLa cells, and the YFP positive cells were analyzed for loss of mitochondrial transmembrane potential (A, B). *Indicates p value ≤0.05. Data reflective of at least three independent replicates. Bars with solid color represent controls, bars with angled hatching represent DAMs, and bars with horizontal hatching represent non-DAMs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991267&req=5

ppat-1001213-g003: Discordance Associated Mutations (DAMs) within HIV-1PR alters the loss of mitochondrial transmembrane potential.YFP HIV protease or the indicated protease point mutants were expressed in HeLa cells, and the YFP positive cells were analyzed for loss of mitochondrial transmembrane potential (A, B). *Indicates p value ≤0.05. Data reflective of at least three independent replicates. Bars with solid color represent controls, bars with angled hatching represent DAMs, and bars with horizontal hatching represent non-DAMs.
Mentions: We have previously shown that expression of HIV-1 PR causes cell death which is associated with loss of mitochondrial transmembrane potential, caspase 3 activation and nuclear fragmentation [16]. After transfection with YFP and YFP protease, He-La cells were assessed for loss of mitochondrial transmembrane potential in the YFP-PR positive cells by TMRE staining, which is a hydrophobic dye, taken up by mitochondria with intact transmembrane potential. We specifically evaluated TMRE loss in the YFP positive cells, indicating expression of protease. Expression of YFP alone or YFP with catalytically inactive YFP D25G PR resulted in minimal loss of transmembrane potential. Conversely, YFP-WT PR or treatment with CCCP, which uncouples mitochondrial transmembrane potential, resulted in significant loss of TMRE retention (Figure 3A). Strikingly, expression of YFP-L63P PR, YFP-D30N PR, YFP-K20R-PR, or YFP-L90M PR caused high degrees of transmembrane potential loss, similar to WT Protease. In contrast, YFP-PR fusions containing the DAMs I54V, V82A caused a lesser degree of transmembrane potential loss than WT-PR, that was similar to that of catalytically impaired T26S (Figure 3B).

Bottom Line: The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells.These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication.Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

Show MeSH
Related in: MedlinePlus