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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 is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).
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fig6: Nef is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).

Mentions: We next investigated intracellular distribution of Nef in relationship to autophagy regulators. Nef did not colocalize with mTOR (Fig. S3 B), so it is unlikely that it affects Tor directly. Nef showed a partial colocalization with 2xFYVE-GFP (Fig. S3 C), a probe binding to membranes containing phosphatidylinositol 3-phosphate (PI3P), the enzymatic product of type III PI3K hVPS34 that plays a critical role in autophagy when complexed with Beclin 1 (Kihara et al., 2001; Furuya et al., 2005; Pattingre et al., 2005; Zeng et al., 2006). Nef showed colocalization with autophagy factors Atg7 and Atg12 (Fig. S3, D and E), and colocalized (Figs. 6 A and S3 F) with the autophagic protein Beclin 1, which is the central regulator of autophagy at multiple stages (Liang et al., 1999; Pattingre et al., 2005). Immunoprecipitation of Beclin 1 in extracts from cells transfected with Nef-GFP resulted in the presence of Nef-GFP in the precipitated protein complexes (Fig. 6 B, top left). GFP was absent from the control samples when Beclin 1 was immunoprecipitated from cells transfected with GFP alone (Fig. 6 B, top right). A converse experiment using immunoprecipitation of GFP revealed the presence of Beclin 1 in immune complexes in cells transfected with Nef-GFP (Fig. 6 B, bottom left) but not in extracts from cells transfected with GFP alone (Fig. 6 B, bottom right). In a different configuration, using cells transfected with C-terminally myc epitope–tagged Nef, Beclin 1 was found in immunoprecipitates generated with myc antibodies (Fig. 6 C). In all immunoprecipitation experiments, IgG control showed negative results for the specific proteins analyzed (Fig. 6). The blots shown with the IgG control were developed until a very faint band (representing background in any type of immunoprecipitation experiments) was revealed when possible; shorter development times left IgG controls completely blank, whereas the specifically coimmunoprecipitated bands were still detected. Importantly, HIV Nef also coimmunoprecipitated with Beclin 1 in extracts from cells infected with HIV virus (Fig. 6 D), demonstrating that Nef–Beclin 1 complexes form during viral infection. Thus, Beclin 1 and Nef colocalize (Fig. 6 A) and are present in a shared protein complex (Fig. 6, B–D), associating directly or indirectly via an intermediate partner. Furthermore, Nef affected hVPS34 distribution (Fig. 6, E and F), as a consequence of its association with Beclin 1, resulting in an increased presence of hVPS34 on membranes.


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 is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).
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fig6: Nef is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).
Mentions: We next investigated intracellular distribution of Nef in relationship to autophagy regulators. Nef did not colocalize with mTOR (Fig. S3 B), so it is unlikely that it affects Tor directly. Nef showed a partial colocalization with 2xFYVE-GFP (Fig. S3 C), a probe binding to membranes containing phosphatidylinositol 3-phosphate (PI3P), the enzymatic product of type III PI3K hVPS34 that plays a critical role in autophagy when complexed with Beclin 1 (Kihara et al., 2001; Furuya et al., 2005; Pattingre et al., 2005; Zeng et al., 2006). Nef showed colocalization with autophagy factors Atg7 and Atg12 (Fig. S3, D and E), and colocalized (Figs. 6 A and S3 F) with the autophagic protein Beclin 1, which is the central regulator of autophagy at multiple stages (Liang et al., 1999; Pattingre et al., 2005). Immunoprecipitation of Beclin 1 in extracts from cells transfected with Nef-GFP resulted in the presence of Nef-GFP in the precipitated protein complexes (Fig. 6 B, top left). GFP was absent from the control samples when Beclin 1 was immunoprecipitated from cells transfected with GFP alone (Fig. 6 B, top right). A converse experiment using immunoprecipitation of GFP revealed the presence of Beclin 1 in immune complexes in cells transfected with Nef-GFP (Fig. 6 B, bottom left) but not in extracts from cells transfected with GFP alone (Fig. 6 B, bottom right). In a different configuration, using cells transfected with C-terminally myc epitope–tagged Nef, Beclin 1 was found in immunoprecipitates generated with myc antibodies (Fig. 6 C). In all immunoprecipitation experiments, IgG control showed negative results for the specific proteins analyzed (Fig. 6). The blots shown with the IgG control were developed until a very faint band (representing background in any type of immunoprecipitation experiments) was revealed when possible; shorter development times left IgG controls completely blank, whereas the specifically coimmunoprecipitated bands were still detected. Importantly, HIV Nef also coimmunoprecipitated with Beclin 1 in extracts from cells infected with HIV virus (Fig. 6 D), demonstrating that Nef–Beclin 1 complexes form during viral infection. Thus, Beclin 1 and Nef colocalize (Fig. 6 A) and are present in a shared protein complex (Fig. 6, B–D), associating directly or indirectly via an intermediate partner. Furthermore, Nef affected hVPS34 distribution (Fig. 6, E and F), as a consequence of its association with Beclin 1, resulting in an increased presence of hVPS34 on membranes.

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