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HIV-1 Vpr triggers mitochondrial destruction by impairing Mfn2-mediated ER-mitochondria interaction.

Huang CY, Chiang SF, Lin TY, Chiou SH, Chow KC - PLoS ONE (2012)

Bottom Line: The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation.Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria.Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.

ABSTRACT
Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4(+) T lymphoblast cell line SupT1, or human primary CD4(+) T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1.

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Enforced expression of Mfn2 and DRP1 relieves Vpr-induced apoptosis.After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. Results are the means ± S.D. of three independent experiments. For panels C and D, † (p<0.05) and †† (p<0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.
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pone-0033657-g009: Enforced expression of Mfn2 and DRP1 relieves Vpr-induced apoptosis.After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. Results are the means ± S.D. of three independent experiments. For panels C and D, † (p<0.05) and †† (p<0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.

Mentions: As noted above that the reduction of Mfn2 protein and the decreased cytoplasmic distribution of DRP1 were the primary effects of Vpr, we therefore investigated whether Mfn2 and DRP1expression may relieve the cell death induced by Vpr. HEK 293 and SupT1 cells were respectively transfected with Mfn2, DRP1, and Mfn2/DRP1 expression plasmids for 24 hours, and infected with Vpr-expressing lentivirus for 72 hours. To evaluate the apoptotic cell death, we further examined the 89 kDa cleavage fragment of poly(ADP-ribose) polymerase (PARP), an apoptotic marker [29]. Vpr induced the apoptotic cleavage of PARP whereas the enforced expression of Mfn2 and DRP1 greatly reduced PARP cleavage in Vpr-positive HEK 293 cells (Fig. 9A). Similarly, overexpression of Mfn2 and DRP1 decreased the cleavage of PARP in Vpr-expressing SupT1 cells (Fig. 9B). Moreover, the losses of MMP and cell death induced by Vpr were also alleviated in Mfn2- and DRP1-expressing HEK 293 and SupT1 cells (Fig. 9C and 9D).


HIV-1 Vpr triggers mitochondrial destruction by impairing Mfn2-mediated ER-mitochondria interaction.

Huang CY, Chiang SF, Lin TY, Chiou SH, Chow KC - PLoS ONE (2012)

Enforced expression of Mfn2 and DRP1 relieves Vpr-induced apoptosis.After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. Results are the means ± S.D. of three independent experiments. For panels C and D, † (p<0.05) and †† (p<0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.
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Related In: Results  -  Collection

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pone-0033657-g009: Enforced expression of Mfn2 and DRP1 relieves Vpr-induced apoptosis.After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. Results are the means ± S.D. of three independent experiments. For panels C and D, † (p<0.05) and †† (p<0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.
Mentions: As noted above that the reduction of Mfn2 protein and the decreased cytoplasmic distribution of DRP1 were the primary effects of Vpr, we therefore investigated whether Mfn2 and DRP1expression may relieve the cell death induced by Vpr. HEK 293 and SupT1 cells were respectively transfected with Mfn2, DRP1, and Mfn2/DRP1 expression plasmids for 24 hours, and infected with Vpr-expressing lentivirus for 72 hours. To evaluate the apoptotic cell death, we further examined the 89 kDa cleavage fragment of poly(ADP-ribose) polymerase (PARP), an apoptotic marker [29]. Vpr induced the apoptotic cleavage of PARP whereas the enforced expression of Mfn2 and DRP1 greatly reduced PARP cleavage in Vpr-positive HEK 293 cells (Fig. 9A). Similarly, overexpression of Mfn2 and DRP1 decreased the cleavage of PARP in Vpr-expressing SupT1 cells (Fig. 9B). Moreover, the losses of MMP and cell death induced by Vpr were also alleviated in Mfn2- and DRP1-expressing HEK 293 and SupT1 cells (Fig. 9C and 9D).

Bottom Line: The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation.Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria.Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.

ABSTRACT
Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4(+) T lymphoblast cell line SupT1, or human primary CD4(+) T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1.

Show MeSH
Related in: MedlinePlus