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Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration.

Campbell GR, Rawat P, Bruckman RS, Spector SA - PLoS Pathog. (2015)

Bottom Line: RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy.Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB.To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

No MeSH data available.


Related in: MedlinePlus

HIV induces TFEB nuclear localization in human macrophages through TLR8.(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
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ppat.1005018.g004: HIV induces TFEB nuclear localization in human macrophages through TLR8.(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.

Mentions: To investigate the role of TFEB in regulating autophagy activation upon exposure to HIV, we initially examined TFEB localization using immunoblotting (Fig 4A). The results show that TFEB localizes predominantly in the cytoplasm of uninfected macrophages. Moreover, almost all TFEB ran at a higher molecular size than in the HIV-exposed samples suggesting that a substantial fraction of TFEB is phosphorylated under basal conditions. Exposure to purified HIV for 24 h led to a significant and dose-dependent dephosphorylation and activation of TFEB as monitored by its more rapid mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and increased nuclear accumulation (Fig 4A). We then assessed the effect of long-term productive HIV infection on TFEB localization. The dephosphorylation and activation of TFEB lasted until at least 72 h post-infection and by 5 d post-infection, although levels were elevated, TFEB was localized predominantly to the cytoplasmic fraction. By 7 d TFEB localization was similar to the mock-infected controls (Fig 4B). To confirm further the nuclear translocation of TFEB, we monitored TFEB sub-cellular localization using confocal immunofluorescence microscopy. Macrophages were cultured in the presence of HIV and the sub-cellular distribution of TFEB was evaluated using 4',6-diamidino-2-phenylindole nuclear staining and an anti-TFEB antibody (Fig 4C). In untreated macrophages, TFEB localized predominantly to the cytoplasm whereas in HIV exposed macrophages TFEB translocated to the nucleus by 72 h. However, by 6 d post-infection, this effect had dissipated and TFEB localization was again similar to the mock-infected controls (Fig 4C).


Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration.

Campbell GR, Rawat P, Bruckman RS, Spector SA - PLoS Pathog. (2015)

HIV induces TFEB nuclear localization in human macrophages through TLR8.(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
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ppat.1005018.g004: HIV induces TFEB nuclear localization in human macrophages through TLR8.(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Mentions: To investigate the role of TFEB in regulating autophagy activation upon exposure to HIV, we initially examined TFEB localization using immunoblotting (Fig 4A). The results show that TFEB localizes predominantly in the cytoplasm of uninfected macrophages. Moreover, almost all TFEB ran at a higher molecular size than in the HIV-exposed samples suggesting that a substantial fraction of TFEB is phosphorylated under basal conditions. Exposure to purified HIV for 24 h led to a significant and dose-dependent dephosphorylation and activation of TFEB as monitored by its more rapid mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and increased nuclear accumulation (Fig 4A). We then assessed the effect of long-term productive HIV infection on TFEB localization. The dephosphorylation and activation of TFEB lasted until at least 72 h post-infection and by 5 d post-infection, although levels were elevated, TFEB was localized predominantly to the cytoplasmic fraction. By 7 d TFEB localization was similar to the mock-infected controls (Fig 4B). To confirm further the nuclear translocation of TFEB, we monitored TFEB sub-cellular localization using confocal immunofluorescence microscopy. Macrophages were cultured in the presence of HIV and the sub-cellular distribution of TFEB was evaluated using 4',6-diamidino-2-phenylindole nuclear staining and an anti-TFEB antibody (Fig 4C). In untreated macrophages, TFEB localized predominantly to the cytoplasm whereas in HIV exposed macrophages TFEB translocated to the nucleus by 72 h. However, by 6 d post-infection, this effect had dissipated and TFEB localization was again similar to the mock-infected controls (Fig 4C).

Bottom Line: RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy.Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB.To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

No MeSH data available.


Related in: MedlinePlus