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Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages.

Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, Federico M, Panganiban A, Vergne I, Deretic V - J. Cell Biol. (2009)

Bottom Line: This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation.The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy.The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

ABSTRACT
Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

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Nef inhibits autophagic maturation. (A) Nef blocks maturation of early autophagic organelles into autolysosomes. 293T cells were infected with VSV-G–pseudotyped HIV or HIVΔNef and transfected with mRFP-GFP-LC3 for 48 h, then immunostained for Gag and analyzed by confocal microscopy. Based on differential pH sensitivity of RFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autolysosomes (red+green−; red in merged images). (B and C) Quantification of (red+green+) R+G+ and R+G− puncta per cell, respectively. (D) Analysis of Lamp2 association with RGP-GFP-LC3 profiles in HIV-infected 293T cells (HIV infection of >90% determined by staining with antibody to Gag). L+, percentage of Lamp2-positive profiles; L−, percentage of Lamp2-negative profiles. Data are from 42 cells from three slides. (E) LC3-II levels in Nef-transfected cells in the presence or absence of bafilomycin A1. 293T cells were transfected with GFP alone or Nef-GFP for 48 h. Cells were then incubated with or without bafilomycin A1 (Baf A1 or Baf) for 4 h and immunoblotted for LC3 and GAPDH. (E, bottom) Quantification: LC3/GAPDH ratios, representative of one of two experiments. (F) Inhibition of autophagic flux/maturation protects Nef- virus from degradation. U937 cells were infected with VSV-G–pseudotyped HIVΔNef for 72 h, then washed and treated with rapamycin or rapamycin plus bafilomycin A1 (100 nM) for 5 h. VLP and cell lysates were subjected to immunoblot analysis. (F, right) Quantification of cellular p24 (n = 3). Data indicate means; error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; †, P ≥ 0.05 (ANOVA).
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fig5: Nef inhibits autophagic maturation. (A) Nef blocks maturation of early autophagic organelles into autolysosomes. 293T cells were infected with VSV-G–pseudotyped HIV or HIVΔNef and transfected with mRFP-GFP-LC3 for 48 h, then immunostained for Gag and analyzed by confocal microscopy. Based on differential pH sensitivity of RFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autolysosomes (red+green−; red in merged images). (B and C) Quantification of (red+green+) R+G+ and R+G− puncta per cell, respectively. (D) Analysis of Lamp2 association with RGP-GFP-LC3 profiles in HIV-infected 293T cells (HIV infection of >90% determined by staining with antibody to Gag). L+, percentage of Lamp2-positive profiles; L−, percentage of Lamp2-negative profiles. Data are from 42 cells from three slides. (E) LC3-II levels in Nef-transfected cells in the presence or absence of bafilomycin A1. 293T cells were transfected with GFP alone or Nef-GFP for 48 h. Cells were then incubated with or without bafilomycin A1 (Baf A1 or Baf) for 4 h and immunoblotted for LC3 and GAPDH. (E, bottom) Quantification: LC3/GAPDH ratios, representative of one of two experiments. (F) Inhibition of autophagic flux/maturation protects Nef- virus from degradation. U937 cells were infected with VSV-G–pseudotyped HIVΔNef for 72 h, then washed and treated with rapamycin or rapamycin plus bafilomycin A1 (100 nM) for 5 h. VLP and cell lysates were subjected to immunoblot analysis. (F, right) Quantification of cellular p24 (n = 3). Data indicate means; error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; †, P ≥ 0.05 (ANOVA).

Mentions: The role of Nef in inhibiting degradative stages of autophagy was further examined in human cells using the RFP-GFP-LC3 probe, a specialized tool for investigation of the autophagic flux, i.e., the maturation of autophagic organelles into degradative autolysosomal compartments (Kimura et al., 2007). Based on the sensitivity of GFP fluorescence to acidic pH and insensitivity of RFP fluorescence to low pH, it is possible to differentiate early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autophagic organelles (red+green−; red in merged images; Kimura et al., 2007). In cells infected with Nef+ HIV, there was a pronounced accumulation of red+green+ (yellow) puncta, compared with uninfected cells or cells infected with ΔNef HIV (Fig. 5, A–C). This is in keeping with the conclusion that Nef blocks maturation of early autophagic organelles into acidified, degradative autolysosomes. Of the Nef-dependent red+green+ puncta, 85% were negative for the lysosomal protein Lamp2 (Fig. 5 D). All red+green− puncta (representing 31% of the total mRFP-GFP-LC3 puncta) were Lamp2 positive (Fig. 5 D). Expression of Nef-GFP resulted in an increase of LC3-II (Fig. 5 E). This was not or only slightly enhanced in the presence of bafilomycin A1 (Fig. 5 E, graph), an inhibitor of autophagosomal/autolysosomal acidification used to differentiate between effects on autophagy induction versus maturation (Mizushima and Yoshimori, 2007), which suggests that the bulk of Nef effects on autophagy were based on blocking autophagic flux.


Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages.

Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, Federico M, Panganiban A, Vergne I, Deretic V - J. Cell Biol. (2009)

Nef inhibits autophagic maturation. (A) Nef blocks maturation of early autophagic organelles into autolysosomes. 293T cells were infected with VSV-G–pseudotyped HIV or HIVΔNef and transfected with mRFP-GFP-LC3 for 48 h, then immunostained for Gag and analyzed by confocal microscopy. Based on differential pH sensitivity of RFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autolysosomes (red+green−; red in merged images). (B and C) Quantification of (red+green+) R+G+ and R+G− puncta per cell, respectively. (D) Analysis of Lamp2 association with RGP-GFP-LC3 profiles in HIV-infected 293T cells (HIV infection of >90% determined by staining with antibody to Gag). L+, percentage of Lamp2-positive profiles; L−, percentage of Lamp2-negative profiles. Data are from 42 cells from three slides. (E) LC3-II levels in Nef-transfected cells in the presence or absence of bafilomycin A1. 293T cells were transfected with GFP alone or Nef-GFP for 48 h. Cells were then incubated with or without bafilomycin A1 (Baf A1 or Baf) for 4 h and immunoblotted for LC3 and GAPDH. (E, bottom) Quantification: LC3/GAPDH ratios, representative of one of two experiments. (F) Inhibition of autophagic flux/maturation protects Nef- virus from degradation. U937 cells were infected with VSV-G–pseudotyped HIVΔNef for 72 h, then washed and treated with rapamycin or rapamycin plus bafilomycin A1 (100 nM) for 5 h. VLP and cell lysates were subjected to immunoblot analysis. (F, right) Quantification of cellular p24 (n = 3). Data indicate means; error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; †, P ≥ 0.05 (ANOVA).
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fig5: Nef inhibits autophagic maturation. (A) Nef blocks maturation of early autophagic organelles into autolysosomes. 293T cells were infected with VSV-G–pseudotyped HIV or HIVΔNef and transfected with mRFP-GFP-LC3 for 48 h, then immunostained for Gag and analyzed by confocal microscopy. Based on differential pH sensitivity of RFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autolysosomes (red+green−; red in merged images). (B and C) Quantification of (red+green+) R+G+ and R+G− puncta per cell, respectively. (D) Analysis of Lamp2 association with RGP-GFP-LC3 profiles in HIV-infected 293T cells (HIV infection of >90% determined by staining with antibody to Gag). L+, percentage of Lamp2-positive profiles; L−, percentage of Lamp2-negative profiles. Data are from 42 cells from three slides. (E) LC3-II levels in Nef-transfected cells in the presence or absence of bafilomycin A1. 293T cells were transfected with GFP alone or Nef-GFP for 48 h. Cells were then incubated with or without bafilomycin A1 (Baf A1 or Baf) for 4 h and immunoblotted for LC3 and GAPDH. (E, bottom) Quantification: LC3/GAPDH ratios, representative of one of two experiments. (F) Inhibition of autophagic flux/maturation protects Nef- virus from degradation. U937 cells were infected with VSV-G–pseudotyped HIVΔNef for 72 h, then washed and treated with rapamycin or rapamycin plus bafilomycin A1 (100 nM) for 5 h. VLP and cell lysates were subjected to immunoblot analysis. (F, right) Quantification of cellular p24 (n = 3). Data indicate means; error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; †, P ≥ 0.05 (ANOVA).
Mentions: The role of Nef in inhibiting degradative stages of autophagy was further examined in human cells using the RFP-GFP-LC3 probe, a specialized tool for investigation of the autophagic flux, i.e., the maturation of autophagic organelles into degradative autolysosomal compartments (Kimura et al., 2007). Based on the sensitivity of GFP fluorescence to acidic pH and insensitivity of RFP fluorescence to low pH, it is possible to differentiate early, nonacidified autophagosomes (red+green+; yellow in merged images) from acidified, degradative autophagic organelles (red+green−; red in merged images; Kimura et al., 2007). In cells infected with Nef+ HIV, there was a pronounced accumulation of red+green+ (yellow) puncta, compared with uninfected cells or cells infected with ΔNef HIV (Fig. 5, A–C). This is in keeping with the conclusion that Nef blocks maturation of early autophagic organelles into acidified, degradative autolysosomes. Of the Nef-dependent red+green+ puncta, 85% were negative for the lysosomal protein Lamp2 (Fig. 5 D). All red+green− puncta (representing 31% of the total mRFP-GFP-LC3 puncta) were Lamp2 positive (Fig. 5 D). Expression of Nef-GFP resulted in an increase of LC3-II (Fig. 5 E). This was not or only slightly enhanced in the presence of bafilomycin A1 (Fig. 5 E, graph), an inhibitor of autophagosomal/autolysosomal acidification used to differentiate between effects on autophagy induction versus maturation (Mizushima and Yoshimori, 2007), which suggests that the bulk of Nef effects on autophagy were based on blocking autophagic flux.

Bottom Line: This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation.The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy.The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

ABSTRACT
Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

Show MeSH
Related in: MedlinePlus