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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-mediated autophagy and lysosomal gene expression is dependent upon TFEB.(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) 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. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.
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ppat.1005018.g006: HIV-mediated autophagy and lysosomal gene expression is dependent upon TFEB.(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) 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. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.

Mentions: Finally, we analyzed the transcriptional activity of TFEB post-HIV exposure. For this, we exposed macrophages to both infectious HIV and AT-2-inactivated HIV and assessed the expression of the known TFEB targets ATG9B, UV radiation resistance associated gene (UVRAG) (both autophagy genes), and mucolipin 1 (MCOLN1) (a lysosomal gene) after 24 h using qRT-PCR. Both infectious HIV and AT-2-inactivated HIV increased the transcription of ATG9B, UVRAG, and MCOLN1 suggesting activation of TFEB by HIV does not require productive infection (Fig 6A). Moreover, when TLR8 was silenced, expression of these genes post-HIV exposure was similar to the mock-infected controls (Fig 6C). Collectively, these data suggest that exposure of macrophages to HIV induces TLR8-dependent TFEB dephosphorylation and nuclear translocation that induces autophagy, and that this is not dependent upon a productive infection.


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-mediated autophagy and lysosomal gene expression is dependent upon TFEB.(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) 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. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.
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ppat.1005018.g006: HIV-mediated autophagy and lysosomal gene expression is dependent upon TFEB.(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) 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. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.
Mentions: Finally, we analyzed the transcriptional activity of TFEB post-HIV exposure. For this, we exposed macrophages to both infectious HIV and AT-2-inactivated HIV and assessed the expression of the known TFEB targets ATG9B, UV radiation resistance associated gene (UVRAG) (both autophagy genes), and mucolipin 1 (MCOLN1) (a lysosomal gene) after 24 h using qRT-PCR. Both infectious HIV and AT-2-inactivated HIV increased the transcription of ATG9B, UVRAG, and MCOLN1 suggesting activation of TFEB by HIV does not require productive infection (Fig 6A). Moreover, when TLR8 was silenced, expression of these genes post-HIV exposure was similar to the mock-infected controls (Fig 6C). Collectively, these data suggest that exposure of macrophages to HIV induces TLR8-dependent TFEB dephosphorylation and nuclear translocation that induces autophagy, and that this is not dependent upon a productive infection.

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