HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Poe, Jerrod A.; Smithgall, Thomas E.

doi:10.1016/j.jmb.2009.09.047

Cited by 68 publications

(156 citation statements)

References 70 publications

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“…Nef-E24 is located in the N-terminal alpha helix within a region (amino acids 17 to 26) that, when deleted and engineered along with a V10E substitution, renders Nef defective for HLA downregulation (69); it also lies four codons downstream of M20, which, when artificially mutated to alanine, produces a severe HLA downregulation defect (50). Nef-Q107 lies adjacent to arginine residues R105/R106 that are essential for several Nef functions, including dimerization (70)(71)(72); it is therefore possible that the creation of a triple arginine (RRR 105-107 ) via a Q107R substitution may alter this protein-protein interface. Mutations at the adjacent residue 106 (e.g., R106K and R106L) also confer modest to severe HLA class I downregulation defects (73).…”

Section: Discussionmentioning

confidence: 99%

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Shahid

Olvera

Anmole

et al. 2015

J Virol

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HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1 NL4.3 , and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag 147 , was identified in p24Gag (GQMVHQAI Gag140 -147 ). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-Nef E24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1 NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCEProtective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

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Section: Discussionmentioning

confidence: 99%

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Shahid

Olvera

Anmole

et al. 2015

J Virol

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“…To investigate whether interaction of Nef with the AP-1 complex during CD4 downregulation occurs in subcellular sites where γ2 is present, we used bimolecular fluorescence complementation (BiFC). This technique has been broadly used to study Nef dimerization (Poe and Smithgall, 2009;Poe et al, 2014) and the interaction of Nef with host cell proteins (Amorim et al, 2014;Dikeakos et al, 2012;Dirk et al, 2015). To this end, we generated constructs encoding Nef and μ1A fused to the N-or C-terminal halves of the Venus fluorescent protein, respectively (Nef-VNt and μ1A-VCt).…”

Section: Knockdown Of γ2 Alleviates Surface Depletion Of Cd4 By Nefmentioning

confidence: 99%

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

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“…The SH3 domain mutation (E93A) was then introduced into these vectors via site-directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Construction of the complementary BiFC expression vector for HIV-1 Nef (Nef-VN) has been described elsewhere (43).…”

Section: Mammalian Expression Vectors For Bimolecular Fluorescence Comentioning

confidence: 99%

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment

Alvarado

Tarafdar

Yeh

et al. 2014

Journal of Biological Chemistry

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HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Cited by 68 publications

References 70 publications

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment

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