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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 autophagy in human macrophages through TLR8.(A) 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. (B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm. (D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
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ppat.1005018.g002: HIV induces autophagy in human macrophages through TLR8.(A) 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. (B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm. (D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.

Mentions: In order to infect productively a target cell, HIV envelope protein gp120 binds to CD4, triggering conformational changes in gp120 that ultimately leads to the fusion of the viral and target cell membranes allowing entry of the viral capsid. Within uninfected CD4+ T cells, the fusogenic activity of gp41 induces autophagy [18] leading to the induction of apoptosis [19]. In contrast, uninfected or infected macrophages do not undergo Env-mediated autophagy or apoptosis [14]. In addition to this route of entry, HIV can also enter macrophages through CD4-independent macropinocytosis [20] or a macropinocytosis-like mechanism (the pathway of HIV endocytic entry in macrophages [PHEEM]) [21]. Following entry through a CD4-independent pathway, the uridine rich HIV long terminal repeat (LTR) single-stranded RNA, which contains multiple pathogen-associated molecular patterns (PAMPs), can be recognized by the pattern recognition receptor (PRR) TLR8 expressed in macrophage endosomes [22, 23]. Previously, we demonstrated that ssRNA40, a GU-rich ssRNA derived from the HIV LTR, induces autophagy in human macrophages through a TLR8-dependent mechanism involving vitamin D, and the expression of both the vitamin D (1,25D3) receptor, and cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1), which 1α-hydroxylates the inactive form of vitamin D3, 25-hydroxycholecalciferol (25D3), into the biologically active metabolite 1,25D3, and (VDR) [13]. Therefore, we investigated whether productive infection was required for the induction of autophagy using 2,2′-dithiodipyridine (AT-2)-treated HIV. AT-2 inactivates the infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs (S3 Fig). Exposure of macrophages to AT-2-inactivated HIV for 24 h led to a significant increase in LC3B-II (P = 0.0045) and significant degradation of SQSTM1 (P = 0.006). Interestingly, there was no significant difference in either LC3B lipidation or SQSTM1 between AT-2-inactivated HIV and exposure to infectious HIV indicating that productive infection is not required for the HIV-mediated induction of autophagy (P > 0.05; Fig 1C). To determine the role of TLR8, RNA interference (RNAi) of TLR8 was employed. TLR8 silencing (Fig 2A) significantly inhibited HIV-mediated LC3B lipidation and degradation of SQSTM1 (Fig 2B) in the absence of significant cytotoxic effects (P > 0.05; S2D Fig) suggesting that TLR8 is the mediator of HIV-induced autophagy in macrophages. Although we observed an increase in LC3B lipidation and an increase in SQSTM1 degradation, ingestion of pathogens through TLR1/2, TLR2/6, and TLR4 can trigger the recruitment of LC3B-II to single-membrane phagosomes in a process termed LC3-associated phagocytosis (LAP) [24]. As opposed to canonical autophagy, LAP is the receptor-mediated internalization of extracellular cargo that occurs without the formation of a double membrane. The receptor triggers the ligation of LC3 to the phagosome. These LC3-positive SQSTM1-negative phagosomes then fuse with lysosomes and rapidly mature into a phagolysosomes. The pre-initiation complex that is required for autophagy is dispensable for LAP, as LC3B-II deposition at the phagosome proceeds normally in the absence of RB1-inducible coiled-coil 1, ATG13 and unc-51 like autophagy activating kinase 1 (ULK1) proteins [25]. The ability of HIV and TLR8 ligands to initiate LAP is unknown. Therefore, we analyzed whether HIV exposed macrophages contain more SQSTM1 and LC3B dual positive autophagosomes or LC3B-positive SQSTM1-negative phagosomes harboring HIV particles using confocal immunofluorescence microscopy (Fig 2C). We observed SQSTM1 and LC3B dual positive puncta that were also positive for HIV (cream pixels), but failed to observe the presence of LC3B-positive SQSTM1-negative puncta harboring HIV (orange pixels). We then investigated whether HIV or ssRNA40 induces TLR8-mediated LAP using RNAi for ATG13. Silencing of ATG13 (Fig 2D) abrogated LC3B lipidation and SQSTM1 degradation following ssRNA40 (P < 0.0005) and HIV (P < 0.005) exposure for 24 h suggesting that the autophagy pre-initiation complex is required for LC3B lipidation in response to TLR8 triggering (Fig 2E).


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 autophagy in human macrophages through TLR8.(A) 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. (B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm. (D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
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ppat.1005018.g002: HIV induces autophagy in human macrophages through TLR8.(A) 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. (B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm. (D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
Mentions: In order to infect productively a target cell, HIV envelope protein gp120 binds to CD4, triggering conformational changes in gp120 that ultimately leads to the fusion of the viral and target cell membranes allowing entry of the viral capsid. Within uninfected CD4+ T cells, the fusogenic activity of gp41 induces autophagy [18] leading to the induction of apoptosis [19]. In contrast, uninfected or infected macrophages do not undergo Env-mediated autophagy or apoptosis [14]. In addition to this route of entry, HIV can also enter macrophages through CD4-independent macropinocytosis [20] or a macropinocytosis-like mechanism (the pathway of HIV endocytic entry in macrophages [PHEEM]) [21]. Following entry through a CD4-independent pathway, the uridine rich HIV long terminal repeat (LTR) single-stranded RNA, which contains multiple pathogen-associated molecular patterns (PAMPs), can be recognized by the pattern recognition receptor (PRR) TLR8 expressed in macrophage endosomes [22, 23]. Previously, we demonstrated that ssRNA40, a GU-rich ssRNA derived from the HIV LTR, induces autophagy in human macrophages through a TLR8-dependent mechanism involving vitamin D, and the expression of both the vitamin D (1,25D3) receptor, and cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1), which 1α-hydroxylates the inactive form of vitamin D3, 25-hydroxycholecalciferol (25D3), into the biologically active metabolite 1,25D3, and (VDR) [13]. Therefore, we investigated whether productive infection was required for the induction of autophagy using 2,2′-dithiodipyridine (AT-2)-treated HIV. AT-2 inactivates the infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs (S3 Fig). Exposure of macrophages to AT-2-inactivated HIV for 24 h led to a significant increase in LC3B-II (P = 0.0045) and significant degradation of SQSTM1 (P = 0.006). Interestingly, there was no significant difference in either LC3B lipidation or SQSTM1 between AT-2-inactivated HIV and exposure to infectious HIV indicating that productive infection is not required for the HIV-mediated induction of autophagy (P > 0.05; Fig 1C). To determine the role of TLR8, RNA interference (RNAi) of TLR8 was employed. TLR8 silencing (Fig 2A) significantly inhibited HIV-mediated LC3B lipidation and degradation of SQSTM1 (Fig 2B) in the absence of significant cytotoxic effects (P > 0.05; S2D Fig) suggesting that TLR8 is the mediator of HIV-induced autophagy in macrophages. Although we observed an increase in LC3B lipidation and an increase in SQSTM1 degradation, ingestion of pathogens through TLR1/2, TLR2/6, and TLR4 can trigger the recruitment of LC3B-II to single-membrane phagosomes in a process termed LC3-associated phagocytosis (LAP) [24]. As opposed to canonical autophagy, LAP is the receptor-mediated internalization of extracellular cargo that occurs without the formation of a double membrane. The receptor triggers the ligation of LC3 to the phagosome. These LC3-positive SQSTM1-negative phagosomes then fuse with lysosomes and rapidly mature into a phagolysosomes. The pre-initiation complex that is required for autophagy is dispensable for LAP, as LC3B-II deposition at the phagosome proceeds normally in the absence of RB1-inducible coiled-coil 1, ATG13 and unc-51 like autophagy activating kinase 1 (ULK1) proteins [25]. The ability of HIV and TLR8 ligands to initiate LAP is unknown. Therefore, we analyzed whether HIV exposed macrophages contain more SQSTM1 and LC3B dual positive autophagosomes or LC3B-positive SQSTM1-negative phagosomes harboring HIV particles using confocal immunofluorescence microscopy (Fig 2C). We observed SQSTM1 and LC3B dual positive puncta that were also positive for HIV (cream pixels), but failed to observe the presence of LC3B-positive SQSTM1-negative puncta harboring HIV (orange pixels). We then investigated whether HIV or ssRNA40 induces TLR8-mediated LAP using RNAi for ATG13. Silencing of ATG13 (Fig 2D) abrogated LC3B lipidation and SQSTM1 degradation following ssRNA40 (P < 0.0005) and HIV (P < 0.005) exposure for 24 h suggesting that the autophagy pre-initiation complex is required for LC3B lipidation in response to TLR8 triggering (Fig 2E).

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