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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 induction and nuclear translocation of TFEB is inhibited by HIV Nef.(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
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ppat.1005018.g008: HIV-mediated autophagy induction and nuclear translocation of TFEB is inhibited by HIV Nef.(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.

Mentions: HIV, in addition to using basal autophagy for its own replication [1], utilizes the Nef protein to protect itself against autophagic degradation [6]. Nef acts as an anti-autophagic maturation factor through interaction with BECN1 [6], and is required for efficient viral replication and HIV pathogenicity. Therefore, we investigated whether Nef was responsible for the down regulation of autophagy observed during permissive HIV infection. Both complete HIV (HIVNL(AD8)) and Nef deleted HIV (HIVNL(AD8)ΔNef), significantly increased LC3B lipidation and SQSTM1 degradation at 24 h and 72 h post-HIV-exposure (Fig 8A). As was the case for HIVBa-L, the levels of LC3B-II and SQSTM1 in HIVNL(AD8) treated cells were similar to that found in the mock infected controls by 5 d post-exposure and this trend continued through 10 d post-exposure. In contrast, macrophages infected with the Nef deleted HIVNL(AD8)ΔNef maintained significantly greater LC3B lipidation and SQSTM1 degradation at all time points (Fig 8A). Importantly, both HIVNL(AD8) and HIVNL(AD8)ΔNef demonstrated extracellular p24 release over the 10 d infection protocol indicating replication competent virus (S4A Fig). These data suggest that once HIV establishes a productive infection, it inhibits autophagy through a Nef-dependent mechanism. Based on our observations that TLR8 agonists inhibit HIV replication through the induction of autophagy, and that silencing TLR8 inhibits HIV-mediated autophagy, we sought to determine whether the inhibition of HIV-induced autophagy would rescue viral replication of Nef deficient HIV at later time points. Silencing of TLR8 resulted in a marked increase in the release of HIV p24 antigen at later time points by both HIVNL(AD8) and HIVNL(AD8)ΔNef (P < 0.05; S4B 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-mediated autophagy induction and nuclear translocation of TFEB is inhibited by HIV Nef.(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
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ppat.1005018.g008: HIV-mediated autophagy induction and nuclear translocation of TFEB is inhibited by HIV Nef.(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Mentions: HIV, in addition to using basal autophagy for its own replication [1], utilizes the Nef protein to protect itself against autophagic degradation [6]. Nef acts as an anti-autophagic maturation factor through interaction with BECN1 [6], and is required for efficient viral replication and HIV pathogenicity. Therefore, we investigated whether Nef was responsible for the down regulation of autophagy observed during permissive HIV infection. Both complete HIV (HIVNL(AD8)) and Nef deleted HIV (HIVNL(AD8)ΔNef), significantly increased LC3B lipidation and SQSTM1 degradation at 24 h and 72 h post-HIV-exposure (Fig 8A). As was the case for HIVBa-L, the levels of LC3B-II and SQSTM1 in HIVNL(AD8) treated cells were similar to that found in the mock infected controls by 5 d post-exposure and this trend continued through 10 d post-exposure. In contrast, macrophages infected with the Nef deleted HIVNL(AD8)ΔNef maintained significantly greater LC3B lipidation and SQSTM1 degradation at all time points (Fig 8A). Importantly, both HIVNL(AD8) and HIVNL(AD8)ΔNef demonstrated extracellular p24 release over the 10 d infection protocol indicating replication competent virus (S4A Fig). These data suggest that once HIV establishes a productive infection, it inhibits autophagy through a Nef-dependent mechanism. Based on our observations that TLR8 agonists inhibit HIV replication through the induction of autophagy, and that silencing TLR8 inhibits HIV-mediated autophagy, we sought to determine whether the inhibition of HIV-induced autophagy would rescue viral replication of Nef deficient HIV at later time points. Silencing of TLR8 resulted in a marked increase in the release of HIV p24 antigen at later time points by both HIVNL(AD8) and HIVNL(AD8)ΔNef (P < 0.05; S4B 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