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The LEDGF-integrase interaction as a new target for ART

The LEDGF-integrase interaction as a new target for ART. Linos Vandekerckhove , Jan De Rijck, Rik Gijsbers, Myriam Witvrouw, Frauke Christ and Zeger Debyser Molecular Medicine, K.U.Leuven, Belgium. HIV-1 replication cycle and role of IN. entry attachment fusion. reverse transcription.

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The LEDGF-integrase interaction as a new target for ART

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  1. The LEDGF-integrase interaction as a new target for ART Linos Vandekerckhove, Jan De Rijck, Rik Gijsbers,Myriam Witvrouw,Frauke Christ and Zeger Debyser Molecular Medicine, K.U.Leuven, Belgium

  2. HIV-1 replication cycle and role of IN entry attachment fusion reverse transcription nuclear import integration transcription translation budding maturation

  3. LEDGF/p75, a novel co-factor of HIV integration • Identified as binding partner of HIV-1 IN (Cherepanov et al., 2003; Maertens et al., 2003) • LEDGF/p75 is lentiviral specific and increases the affinity of IN for DNA (Busschots et al., 2005)

  4. The cellular function LEDGF/p75 • Splice variant p52 • Hepatoma derived growth factors (Conserved PWWP domain) • LEDGF/p75 increases the resistance against environmental stress (Upregulates stress reponse genes)

  5. Importance of LEDGF/p75 in HIV replication • The IN Q168A interaction mutant is defective for replication (Emiliani et al., 2005). • Knock-down using RNAi inhibits HIV replication significantly (Vandekerckhove et al., 2006). • Overexpression of C-terminal fragments

  6. The dominant binding partner of HIV-1 integrase (IN) in human cells Potential targeting factor of lentiviral DNA to the chromosomes Potential role of LEDGF during HIV replication Human DNA histones LEDGF LEDGF LEDGF LEDGF IN ViralDNA

  7. C-terminal fragments of LEDGF/p75 p52 LEDGF/p75 p75 1 91 148 156 325 429 530 aa 347 IBD PWWP AT NLS integrase binding domain DNA binding domain D366A (Cherepanov et al., 2004) 325 530 aa D 325 IBD eGFP Puro IRES IBD 429 aa 347 IBD Puro eGFP IRES

  8. In vitro pull-down MBP-D325 D366A MBP-D325 D366A LEDGF/p75 LEDGF/p75 MBP- D325 MBP- D325 MBP-IBD MBP-IBD - LEDGF/p75 70 kDa - - MBP-D325 55 kDa - - MBP-IBD 45 kDa - - His6-IN

  9. ∆325 inhibits interaction of IN with LEDGF/p75

  10. HeLaP4 cell lines stably overexpressing C-terminal LEDGF fragments HeLaP4 eGFP-D325 D366A HeLaP4 eGFP-IBD HeLaP4 eGFP-D325 HeLaP4 eGFP HeLaP4

  11. Cytopathogenic effect of HIV-1on HeLaP4 cells HeLaP4 HeLaP4eGFP HeLaP4eGFP-Δ325D366A HeLaP4eGFP-Δ325 HeLaP4eGFP-IBD

  12. HIV replication is strongly inhibited by over expression of C-terminal LEDGF fragments (Vandekerckhove, De Rijck et al., submitted for publication)

  13. Tet-Off regulatable IBD expressionin HeLaP4 cells

  14. MT4- breakthrough experiment strain (MOI:1) HIV-1 NL4.3 HIV-1 IIIb

  15. Q-PCR to pinpointthe replication block Reverse transcription Non-Integrated Nuclear Viral DNA Integrated proviral DNA

  16. MT4-breakthrough experimentDKA resistant strain (MOI:1) HIV-1 L-708,906

  17. Conclusions • LEDGF/p75 has a key-role in HIV replication • IBD competes with LEDGF/p75 for IN binding • potent inhibition of HIV replication at the integration step • proof-of-concept for LEDGF-based anti-HIV therapy

  18. Acknowledgements Molecular Medicine – K.U.Leuven, Belgium Jan De Rijck, Rik Gijsbers, Frauke Christ, Katrien Busschots, Koen Bartholomeeusen, Barbara Podessu, Martine Michiels, Myriam Witvrouw, Zeger Debyser Laboratory for Biomolecular Dynamics – K.U.Leuven, Belgium Jo Vercammen, Jelle Hendrickx, Yves Engelborghs Institut Cochin - Paris, France Stephane Emiliani, Nicole Kubat,Richard Benarous

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