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Project

Validation of novel Cellular cofactors of retroviral nuclear import and integration.

Replication of the human immunodeficiency virus type-1 (HIV-1) relies on the presence of cellular proteins or so-called cofactors to complete its replication cycle. Interactions between these cofactors and viral proteins are often essential and as such interesting targets for drug development. This work focuses on the validation and characterization of HIV-1 integrase (IN)-host interactions as novel therapeutic targets for drug discovery and the evaluation of small molecules targeting the interaction between HIV integrase and its cellular cofactor LEDGF/p75.

In 2003, our group validated the cellular protein LEDGF/p75 as a cofactor of HIV-1 IN. LEDGF/p75 targets viral integration towards active chromatin and influences IN multimerization and catalytic activity. In 2010 our group reported on the discovery of LEDGINs, potent small molecules targeting the LEDGF/p75 binding pocket on HIV IN. LEDGINs are currently in early clinical development. LEDGINs displace LEDGF/p75 from HIV IN, preventing targeting to active chromatin, and allosterically inhibit HIV integrase activity. LEDGINs do not only block the integration step, but surprisingly also affect the infectivity of newly produced viral particles. To unravel the mechanism behind this late block, we investigated two hypotheses: 1.) The late activity of LEDGINs is mediated by their effect on IN (or Pol) multimerization 2.) LEDGINs prevent incorporation of LEDGF/p75 in viral particles.

In chapter 3 we discuss the first hypothesis. We demonstrate that LEDGINs potently inhibit the late stages of HIV replication, with half maximal effective concentrations (EC50’s) in the nanomolar range. Furthermore, we show that addition of LEDGINs during virus production leads to defective viruses with a nucleoprotein complex outside the capsid cone, due to aberrant multimerization of HIV IN or its precursor protein Pol.

In chapter 4 we investigate the second hypothesis. While LEDGF/p75 depletion in producer cells does not result in altered LEDGIN potency, we demonstrate that LEDGF/p75 is recruited into HIV-1 particles through a direct interaction with the viral IN (or the Pol precursor protein). We show that LEDGF/p75 can be processed by the viral protease in viral particles. The importance of LEDGF/p75 or processed fragments for viral replication is however not clear.

In chapter 5 we validated the cellular protein YB-1, Y-box-binding protein 1, as a new cofactor of HIV replication. YB-1 depletion resulted into hampered HIV-1 replication in different cell lines. Our data revealed hampered virion production due to interference with the viral RNA metabolism. In addition, using quantitative qPCR and a microscopy based nuclear import test, we demonstrated that YB-1 knockdown leads to a block in nuclear import of HIV-1. We confirmed an interaction with HIV integrase using co-immunoprecipitation and Western blotting. Yet, a direct interaction using recombinant proteins could not be confirmed since YB-1 seemed to bind most proteins non-specifically. Together, our results indicate that YB-1 affects multiple stages of HIV replication.

In conclusion, in the context of virus-host interactions we unraveled the mechanism of LEDGINs, showed the presence of LEDGF/p75 in viral particles and characterized YB-1 as a cofactor of HIV-1 replication.

Date:13 Sep 2011 →  12 May 2016
Keywords:Cofactors, Cellular, Retroviral
Disciplines:Microbiology, Systems biology, Laboratory medicine, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering
Project type:PhD project