The interaction of HIV-1 and HIV-2 with cellular protein trafficking pathways

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

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A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

10.1101/2021.02.22.432252 ◽

2021 ◽

Author(s):

Charlotte A. Stoneham ◽

Simon Langer ◽

Paul D. De Jesus ◽

Jacob M. Wozniak ◽

John Lapek ◽

...

Keyword(s):

Ascorbate Peroxidase ◽

Membrane Trafficking ◽

Protein Trafficking ◽

Immune Surveillance ◽

Cellular Membrane ◽

Cellular Protein ◽

Wild Type ◽

Membrane Systems ◽

Innate Defense ◽

Hiv 1

AbstractThe HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.Author SummaryVpu triggers the degradation or mis-localization of proteins important to the host’s immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the protein’s location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpu’s presence at the limiting membranes of multi-vesicular bodies.

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Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

Journal of Virology ◽

10.1128/jvi.75.23.11336-11343.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11336-11343 ◽

Cited By ~ 70

Author(s):

Romi Ghose ◽

Li-Ying Liou ◽

Christine H. Herrmann ◽

Andrew P. Rice

Keyword(s):

T Lymphocytes ◽

Rna Polymerase Ii ◽

Interleukin 2 ◽

Cellular Protein ◽

Peripheral Blood Lymphocytes ◽

Tat Protein ◽

Necrosis Factor Alpha ◽

Cyclin T1 ◽

Protein Levels ◽

Hiv 1

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

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A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription

Nucleic Acids Research ◽

10.1093/nar/gkz937 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11057-11068 ◽

Cited By ~ 8

Author(s):

Emanuela Ruggiero ◽

Sara Lago ◽

Primož Šket ◽

Matteo Nadai ◽

Ilaria Frasson ◽

...

Keyword(s):

Long Terminal Repeat ◽

Antiviral Drug ◽

Cellular Protein ◽

Terminal Repeat ◽

Proviral Genome ◽

Stem Loop ◽

Hnrnp K ◽

Induced Folding ◽

Ltr Promoter ◽

Hiv 1

Abstract I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

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The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010534 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5081-5094

Author(s):

Evan Chaudhuri ◽

Sabyasachi Dash ◽

Muthukumar Balasubramaniam ◽

Adrian Padron ◽

Joseph Holland ◽

...

Keyword(s):

Molecular Mechanisms ◽

Mammalian Species ◽

Cellular Protein ◽

Luciferase Reporter ◽

Down Regulation ◽

Host Interaction ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Viral Dna Integration ◽

Hiv 1

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3′UTR of CPSF6 contains a miR-125b–binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3′UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3′UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3′UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3′UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

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SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles

Journal of Virology ◽

10.1128/jvi.00594-20 ◽

2020 ◽

Vol 94 (20) ◽

Cited By ~ 1

Author(s):

Austin Featherstone ◽

Christopher Aiken

Keyword(s):

Transmembrane Domain ◽

Antiviral Effect ◽

Cellular Protein ◽

Cell Protein ◽

Specific Effects ◽

Glycoprotein Gp120 ◽

Env Protein ◽

Hiv 1 ◽

Soluble Cd4 ◽

Viral Accessory Protein

ABSTRACT SERINC5 is a 10-transmembrane-domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3 loop, a soluble CD4 (sCD4)-induced epitope, and an N-linked glycan. In contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes. IMPORTANCE SERINC5 is a host cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here, we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins and that this action is correlated with SERINC5’s ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

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HIV-1 Nef-GCC185 interaction regulates assembly of cellular protein complexes at TGN targeting MHC-I downregulation

Life Sciences ◽

10.1016/j.lfs.2019.04.008 ◽

2019 ◽

Vol 229 ◽

pp. 13-20

Author(s):

Sushila Kumari ◽

Manjeet Kumar ◽

Richa Verma ◽

Jimut Kanti Ghosh ◽

Raj Kamal Tripathi

Keyword(s):

Protein Complexes ◽

Cellular Protein ◽

Mhc I ◽

Hiv 1

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Association of HIV-1 Tat with the cellular protein, Puralpha , is mediated by RNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.20.11572 ◽

1999 ◽

Vol 96 (20) ◽

pp. 11572-11577 ◽

Cited By ~ 40

Author(s):

G. L. Gallia ◽

N. Darbinian ◽

A. Tretiakova ◽

S. A. Ansari ◽

J. Rappaport ◽

...

Keyword(s):

Cellular Protein ◽

Hiv 1

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Basic Residues in the Nucleocapsid Domain of Gag Are Required for Interaction of HIV-1 Gag with ABCE1 (HP68), a Cellular Protein Important for HIV-1 Capsid Assembly

Journal of Biological Chemistry ◽

10.1074/jbc.m507255200 ◽

2006 ◽

Vol 281 (7) ◽

pp. 3773-3784 ◽

Cited By ~ 64

Author(s):

Jaisri R. Lingappa ◽

Julia E. Dooher ◽

Michael A. Newman ◽

Patti K. Kiser ◽

Kevin C. Klein

Keyword(s):

Cellular Protein ◽

Capsid Assembly ◽

Hiv 1

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Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

Journal of Virology ◽

10.1128/jvi.03584-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3988-4001 ◽

Cited By ~ 25

Author(s):

Christopher Ritchie ◽

Isabel Cylinder ◽

Emily J. Platt ◽

Eric Barklis

Keyword(s):

Lipid Metabolism ◽

Rna Processing ◽

Protein Trafficking ◽

Cellular Proteins ◽

Wild Type ◽

Gag Protein ◽

Gag Proteins ◽

Biotin Ligase ◽

Close Proximity ◽

Hiv 1

ABSTRACTWe have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with theEscherichia colipromiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins intrans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference.IMPORTANCEWe have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1polgene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.

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