Two Sorting Motifs, a Ubiquitination Motif and a Tyrosine Motif, Are Involved in HIV-1 and Simian Immunodeficiency Virus Nef-Mediated Receptor Endocytosis

Cai, Catherine Yi; Zhang, Xiaoping; Sinko, Patrick J.; Burakoff, Steven J.; Jin, Yongmei M.

doi:10.4049/jimmunol.1003506

Cited by 9 publications

(10 citation statements)

References 60 publications

(72 reference statements)

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“…Further, it is reported that K144 in HIV-1 Nef is di-ubiquitinated for downregulation of CD4 and MHC-I (Cai et al, 2011). However, little is known as to whether Nef indeed plays an essential role in regulating the stability of HIV-1 viral and cellular proteins and consequent HIV-1 replication and survival of HIV-infected cells, and if so, how and to what degree Nef orchestrates overall HIV-1 and host cell protein fates during pathogenesis.…”

Section: Introductionmentioning

confidence: 99%

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

et al. 2016

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HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation.

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Section: Introductionmentioning

confidence: 99%

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

et al. 2016

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“…Immunoblotting was performed as described previously. 18 HIV-1 Nef was blotted with anti-HIV-1 Nef rabbit serum (1:10,000) (NIH). AP1 and AP2 were blotted with anti-AP1 (c) and anti-AP2 (a) mAb (1: 500).…”

Section: Immunoblottingmentioning

confidence: 99%

“…Abrogation of CD4 downregulation by mutations in the hydrophobic region is due to impairment of the Nef-AP2 interaction It is well documented that Nef-mediated CD4 downregulation is AP2 dependent. 4,[10][11][12][13][14][15][16][17][18] To confirm that the above mutations that impaired the CD4 downregulation abrogate the Nef-AP2 interaction, we analyzed the Nef- The results indicated that the M 168 S 169 /KK mutation impaired the Nef-AP2 interaction but not the Nef-AP1 interaction. The Nef-AP1 interaction is known not to be required in Nef-mediated CD4 downregulation.…”

Section: The Hydrophobic Region Downstream Of the Dileucine Motif (L mentioning

confidence: 99%

“…9 In CD4 downregulation, the Nef motif W 57 L 58 binds to the cytoplasmic tail of CD4 whereas the dileucine motif (ENNSL 164 L 165 ) in the Nef C-terminal flexible loop (C-loop) interacts with the clathrin adaptor protein complex AP2, connecting CD4 to the clathrin-coated vesicles for endocytosis. 4,[10][11][12][13][14][15][16][17][18] However, it is unclear why the AP2 interaction of the dileucine motif in the Nef C-terminal flexible loop resulted in the constitutive endocytosis of Nef-connected receptors whereas non-Nef-mediated cell surface receptor endocytosis interacting with AP2 through their own dileucine motif usually resulted in a recycled endocytosis without stimulation. [19][20][21][22] For example, cell surface CD4 is expressed at a stable level and strong downregulation of CD4 was observed with PMA-induced phosphorylation of the serine residues proximal to the dileucine motif in the CD4 tail.…”

Section: Introductionmentioning

confidence: 99%

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Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation

Jin

Cai²,

Mezei³

et al. 2013

AIDS Research and Human Retroviruses

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HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

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“…Both pathways are dependent on Nef 's N-terminal myristoylation site to assemble within lipid rafts in the plasma membrane. It appears that the "signalling mode" for MHC modulation is utilized in response to acute infection over the first 48 hours that specifically targets the 'mature' MHC-I proteins that are present at the cell surface [8], while the stoichiometric mode is a distinct mechanism utilized by the virus after day 3 of infection and blocks the transport of newly synthesized MHC-I proteins from the ER to the cell surface [8,[47][48][49]. In this way the HIV-1 virus can effectively block immune recognition of virus infected cells by antigen-specific CD8 + CTLs.…”

Section: Mhc Class I Downregulation By Nef Occurs By Two Different Pamentioning

confidence: 99%

The Role of the Nef Protein in MHC-I Downregulation and Viral Immune Evasion by HIV-1

Elliott¹,

Hoyne²

2015

J Clin Cell Immunol

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The Nef protein is a major determinant of pathogenicity caused by the human immunodeficiency virus (HIV) and is encoded by the nef gene within the genomes of primate lentiviruses HIV-1, HIV-2 and simian immunodeficiency virus (SIV). The HIV Nef protein subverts the intracellular membrane traffic to mediate endocytosis of a number of cell surface receptors to accelerate their degradation. In this review we will examine how the multifunctional Nef can mediate down regulation of the Major histocompatibility Complex (MHC) I complex proteins from the surface of infected cells as a means of immune evasion by HIV. By selectively downregulating MHC-I HLA-A and HLA-B haplotypes, while maintaining the expression of HLA-C, HLA-E and HLA-G the HIV virus is able to avoid recognition by both the NK and cytotoxic CD8 + T cell effector responses. This protects the virus from cell lysis and enables it to hide from the cell-mediated immune system.

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Two Sorting Motifs, a Ubiquitination Motif and a Tyrosine Motif, Are Involved in HIV-1 and Simian Immunodeficiency Virus Nef-Mediated Receptor Endocytosis

Cited by 9 publications

References 60 publications

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation

The Role of the Nef Protein in MHC-I Downregulation and Viral Immune Evasion by HIV-1

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