1 / 24

BST2/Tetherin: a restriction factor of HIV-1.

BioCIHP Meeting, Cochin Institute 23rd, April 2012. Nicolas Roy 3eme année de Doctorat. BST2/Tetherin: a restriction factor of HIV-1. Implication of the ubiquitination pathway in its downregulation by the viral protein Vpu. Laboratory director : Dr. Clarisse Berlioz-Torrent

dalila
Download Presentation

BST2/Tetherin: a restriction factor of HIV-1.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. BioCIHP Meeting, Cochin Institute 23rd, April 2012 Nicolas Roy 3eme année de Doctorat BST2/Tetherin: a restriction factor of HIV-1. Implication of the ubiquitination pathway in its downregulation by the viral protein Vpu. Laboratory director: Dr. Clarisse Berlioz-Torrent Thesis supervisor: Dr. Katy Janvier Laboratoire « Interactions Hôte-Virus » Institut Cochin INSERM U1016 – CNRS UMR 8104 Département «3I»

  2. The Human Immunodeficiency virus (HIV) is the causal agent responsible for the Acquired Immuno-deficiency syndrome or AIDS Globally, 34.0 million people are living with HIV. 1.7 million people worldwide died from AIDS‑related causes, down 24% from the peak in 2005 Public Health and HIV (Report on the world AIDS epidemic agency, ONUSIDA, 2012)

  3. HIV-1 replication cycle Early steps Late steps Release HIV-1 HIV-1 Assembly Vpu CD4 Gag ARN Co-receptor Gag-Pol Entry Reverse-transcription Translation Transcription Enveloppe glycoprotein Integration  Molecular and cellular mechanisms involved in the release of HIV-1 particles by Vpu

  4. Vpu: an accessory protein of HIV-1 Genome of HIV-1 Rev R U5 GAG Vif Tat R U3 POL Vpr Vpu ENV Nef Vpu • Vpu is an accessory protein • Vpu is a small transmembrane protein (16kDa) Vpu is encoded by HIV-1 and some strains of SIV

  5. Vpu: an accessory protein of HIV-1 • Vpu is involved in CD4 degradation and in the release of HIV-1 particles. • Vpu promotes an efficient release of viral particles by counteracting the cellular protein BST2, recently described as a restriction factorof HIV release (Vandamme et coll, 2008; Neil et coll, 2008) From Perez-Caballero et coll, Cell, 2009  The protein BST2 tethers mature viral particlesat the plasma membrane(Perrez-Caballero et coll, Cell, 2009) BST2 From Bienasz et coll, Cell Hostand Microbe, 2009 Vpu restores HIV release through down-regulation of BST2 expression • Decrease of BST2 cell surface expression • Targeting of BST2 for degradation Vandamme et coll, Cell Host and Microbe, 2008 Miyagi et coll, PNAS, 2009

  6. The modulation of BST2 expression induced by Vpu restores an efficient release of viral particles Release -/+ Release +++ Infected cells (Vpu-) Infected cells(Vpu+) The molecular mechanisms involved in this process remain to be deciphered.

  7. HRS (ESCRT-0) is required for an efficient release of HIV-1 particles Infected cells (Vpu+) Decreasing of BST2 level on the cell Endocytosis through AP-2 Recycling decreased Endosome + Vpu ESCRT Ub HRS Late endosomes MVB Recruitment by the ESCRT machinery Lysosome Increased rate of BST2 degradation From Stenmark et coll, Nature, 2009 Janvier et coll., PloS Pathogen, 2011 Implication of the ubiquitination pathway in BST2 downregulation by Vpu

  8. NL4.3 Udel NL4.3 Udel NL4.3 WT NL4.3 WT Flag-BST2 + + + + + + + + HA-Ub - + - + - + - + HA-Ub Flag-BST2 Lysates Flag-IP BST2 is ubiquitinated and Vpu increases its ubiquitination Flag-BST2 - - + + - - + + HA-Ub - + - + - + - + HA-Ub Flag-BST2 Lysates Flag-IP

  9. E3 E2 E2 Ub Ub Ub Ub Ub Ub Ub Ub K K K K E3 ligases : siRNA screening ATP E3 E1 + E1 Substrat siRNA Screening HECT E3-ligases: Nedd4 Nedd4-L WWP1 ITCH RING E3-ligases: RNF189 RNF138 CBL β-TrCp ½ UBAP1 Role of the E3 ligase on BST2-regulation in a non-infectious context Role of the E3 ligase on BST2-regulation in an infectious context

  10. siRNA E3 ligase J1 HeLa J5 Cell lysis Western blot analysis Effect of E3-ligases depletion on BST2 expression level Western-blot analyses of E3 depletions Western-blot analyses of BST2 expression - siRNF138 - siRNF189 - siNedd4-L - siUBAP1 - siβ-TrCp - siNedd4 - siWwp1 - siITCH - siCBL - siCT - siNedd4-L - siRNF189 - siRNF138 - siβ-TrCp - siUBAP1 - siNedd4 - siWwp1 - siITCH - siCBL - siCT Nedd4 - Nedd4-L - BST2 CBL - ITCH - Tubulin Wwp1 - UBAP1 - RT-PCR analyses of depletions Odyssee quantification - si β-TrCp1 - siCT β-TrCp1 - - si β-TrCp2 - si RNF189 BST2 expression level (% of siCD) - siCT - siCT β-TrCp2 - RNF189 - siCD - siCBL - siITCH - siNedd4 - siWwp1 - siβ-TrCp - siUBAP1 - siRNF189 - siRNF138 - siNedd4-L -

  11. siRNA E3 ligase J1 HeLa J5 Immunofluorescence microscopy Effect of E3-ligases depletion on BST2 cellular localization si RNF189 si β-TrCp si CT si Nedd4 BST2 BST2 BST2 BST2 HRS HRS HRS HRS BST2 HRS BST2 HRS BST2 HRS BST2 HRS  RNF189 depletion leads to the accumulation of BST2 in HRS-positive compartments

  12. Effects of E3-ligases overexpression on BST2 expression level BST2 BST2 BST2 BST2 HeLa J1 pCI-HA Nedd4 RNF189 β-TrCp pHA-E3 Ligases J2 J3 RNF189 β-TrCp BST2 BST2 Nedd4 BST2 BST2 Immunofluorescence microscopy  RNF189 overexpression seems to decrease intracellular pool of BST2

  13. Effect of E3-ligases overexpression on BST2 cell-surface expression level  RNF189 overexpression decreases BST2 cell-surface expression

  14. J1 pHA-E3 ligase J2 24h Cell lysis Flag pull-down Western blot analysis Interaction of BST2 with E3 ligases Co-immunoprecipitation analyses Flag-BST2: - + - + Flag-BST2: - + - + HA-Nedd4 - HA-RNF189 - HeLa Flag-BST2 { { Flag-BST2 Flag-BST2 Lysates IP Lysates IP Flag-BST2: - + - + Flag-BST2: - + - + HA-WWP1 - HA-HRS - { Flag-BST2 { Flag-BST2 Lysates IP Lysates IP Flag-BST2: - + - + HA-βTrCp -  BST2 interacts with Nedd4 and RNF189 { Flag-BST2 Lysates IP

  15. siRNA E3 ligase - + + + + + + + + HeLa Flag-BST2 J1 HA-Ub - + + + + + + + + J4 J5 - siNedd4-L - siRNF189 - siβ-TrCp - siNedd4 - siWwp1 - siITCH - siCBL - siCT - siCT Cell lysis Flag pull-down Western blot analysis Effect of E3-ligases depletion on BST2 ubiquitination Ubiquitination assay Western-blot analyses of E3 depletions - siNedd4-L - siNedd4-L - siRNF189 - siRNF189 - siβ-TrCp - siβ-TrCp - siNedd4 - siWwp1 - siNedd4 - siWwp1 - siITCH - siITCH - siCBL - siCBL - siCT - siCT - siCT - siCT HA-Ub Nedd4 - HA-Ub Nedd4-L - CBL - ITCH - Flag-BST2 Wwp1 - Lysates Flag-IP  Nedd4 and RNF189 are involved in BST2 ubiquitination

  16. siRNA E3 ligase siCD siNedd4 siRNF189 siβTrCp J1 CHX (h) 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4 HeLa BST2 CHX Chase J5 Tubuline Cell lysis Western blot analysis Effects of E3-ligases depletion on BST2 turnover Cycloheximide chase: Western-blot analyses  Nedd4 & RNF189 regulate BST2 turn-over

  17. Conclusions (1) - In a non-infectiouscontext Nedd4 & RNF189 are involved in BST2 ubiquitination Nedd4 & RNF189 are involved in BST2 turnover

  18. E3 E2 E2 Ub Ub Ub Ub Ub Ub Ub Ub K K K K E3 ligases : siRNA screening ATP E3 E1 + E1 Substrat siRNA Screening HECT E3-ligases: Nedd4 Nedd4-L WWP1 ITCH RING E3-ligases: RNF189 RNF138 CBL β-TrCp UBAP1 Role of the E3 ligase on BST2-regulation in a non-infectious context Role of the E3 ligase on BST2-regulation in an infectious context

  19. siRNA E3-ligase J1 HeLa HIV (NL4.3 WT) J3 J5 Immunofluorescence microscopy Effects of E3-ligases depletion on Vpu-induced BST2 down-regulation si RNF189 si β-TrCp si CT si Nedd4 BST2 BST2 BST2 BST2 Env Env Env Env Env Env Env Env BST2 BST2 BST2 BST2 • Nedd4 &RNF189 are not involved in Vpu-inducedtargeting of BST2 for degradation • But, β-TrCp is required for this activity

  20. Effects of Vpu and TrCp interactions on BST2 downregulation - si RNF189 - si RNF189 • Infection by NL4.3 Vpu2.6 strain leads to an unability for Vpu to target BST2 for degradation • The loss of interaction between Vpu and TrCp leads to stabilisation of an undeterminated form of BST2

  21. J1 pHA-E3 pFlag-BST2 eGFP-Vpu J2 24h Cell lysis Flag pull-down Western blot analysis Effects of Vpu overexpression on BST2 and E3-ligases interactions

  22. Conclusions (2)  In a non-infectiouscontext: • Nedd4 & RNF189 are involved in the regulation of BST2 ubiquitination and turnover HOWEVER,  In an infectiouscontext: • Nedd4 & RNF189 do not seem to berequiredin Vpu-induceddownregulation of BST2 • Interestingly the E3 ligase β-TrCp is necessary for BST2 down-regulation by the viral protein Vpu • TrCp is specifically hijacked by Vpu to interact with BST2 Vpudoes not hijack cellular proteinnaturallyinvolved in BST2 regulation but rather use anotherproteinwith a similarfunction

  23. Clarisse Berlioz-Torrent’s group Stéphane Emiliani’s group Katy Janvier Clarisse Berlioz-Torrent Sophie Lambert Stéphane Frémont Grégory Pacini Ursula Madjo Sophie Abélanet Vanessa Gourhand Platforms Microscopy Sequencing Flow Cytometry

  24. HIV-1 (NL4.3 VSV-G) Viral production siRNA E3-ligases HeLa  CAp24 quantification  WB  Infectivity 48h 24h Effects of E3-ligases depletion on viral production • Neither Nedd4, RNF189 nor β-TrCp are required for HIV-1 release

More Related