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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.(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (C) 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. Cells were then 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 = 3.
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ppat.1005018.g001: HIV induces autophagy in human macrophages.(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (C) 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. Cells were then 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 = 3.

Mentions: Whereas exposure of primary human macrophages to HIV envelope proteins has no autophagy inducing effect, exposure to MOLT-4 cells chronically infected with HIV induces autophagy by day 3 post-co-culture [14]. However, the autophagic status of macrophages after exposure to purified HIV virions is unknown. Therefore, the effect of HIV on autophagy induction in human macrophages was determined using RNase/DNase I treated virus purified through an iodixanol velocity gradient (purified HIV), which effectively separates extracellular proteins and microvesicles from the virus (S1 Fig) [15]. During autophagy, cytosolic microtubule-associated protein 1 light chain 3 beta (LC3B)-I is converted to LC3B-II by a ubiquitin-like system that involves autophagy related (ATG) 7, ATG3 and the ATG12–ATG5 complex. The ATG12–ATG5 complex ligates LC3B-II to the nascent autophagosome membrane through phosphatidylethanolamine with the LC3B-II associated with the inner membrane degraded after fusion of the autophagosome with lysosomes. Therefore, the conversion of LC3B-I to LC3B-II and its turnover is an indicator of autophagy induction and flux [16]. Exposure of macrophages to purified HIV led to a significant dose-dependent increase in LC3B-II after 24 h (Fig 1A) in the absence of significant cytotoxic effects (P > 0.05; S2A Fig).


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.(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (C) 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. Cells were then 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 = 3.
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ppat.1005018.g001: HIV induces autophagy in human macrophages.(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB 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. (C) 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. Cells were then 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 = 3.
Mentions: Whereas exposure of primary human macrophages to HIV envelope proteins has no autophagy inducing effect, exposure to MOLT-4 cells chronically infected with HIV induces autophagy by day 3 post-co-culture [14]. However, the autophagic status of macrophages after exposure to purified HIV virions is unknown. Therefore, the effect of HIV on autophagy induction in human macrophages was determined using RNase/DNase I treated virus purified through an iodixanol velocity gradient (purified HIV), which effectively separates extracellular proteins and microvesicles from the virus (S1 Fig) [15]. During autophagy, cytosolic microtubule-associated protein 1 light chain 3 beta (LC3B)-I is converted to LC3B-II by a ubiquitin-like system that involves autophagy related (ATG) 7, ATG3 and the ATG12–ATG5 complex. The ATG12–ATG5 complex ligates LC3B-II to the nascent autophagosome membrane through phosphatidylethanolamine with the LC3B-II associated with the inner membrane degraded after fusion of the autophagosome with lysosomes. Therefore, the conversion of LC3B-I to LC3B-II and its turnover is an indicator of autophagy induction and flux [16]. Exposure of macrophages to purified HIV led to a significant dose-dependent increase in LC3B-II after 24 h (Fig 1A) in the absence of significant cytotoxic effects (P > 0.05; S2A Fig).

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