Development of a novel antiviral strategy directed against the nuclear import of HIV.

Insertion of the viral genome into host cell chromatin is a hallmark ofHuman Immunodeficiency Virus type 1 (HIV-1) replication. Catalyzed by the viral integrase (IN) enzyme, this step irreversibly links the fate ofthe provirus with that of the cell. IN however, has proved to be a tough nut for researchers to crack. Nevertheless, a number of milestone discoveries and insights accumulating over the last decade have propelled the field forward. In this thesis are bundled five research manuscripts reflecting distinct lines of research on HIV-1 IN, yet all focusing on itsstructure, function and inhibition.

Since the approval of raltegravir in 2007, IN strand transfer inhibitors (INSTIs) have become an integral part of antiretroviral therapy. Three are in clinical use today, potently inhibiting viral replication with a dramatic drop in viral load.However, the emergence of resistance to these drugs underscores the need to develop next-generation IN catalytic site inhibitors with improved resistance profiles. In chapter 3, we present a 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivative (MB-76) which potently blocks integration of wild type as well as raltegravir-resistant viruses. A crystal structure of MB-76 bound to the Prototype Foamy Virus (PFV) intasome reveals an overall binding mode identical to that of INSTIs but with a number of unique features. Its characterization highlights MB-76 as a promising candidate for further development.

Chapters 4 and 5 describe an entirelydifferent approach to target IN. HIV-1 is capable of infecting non-dividing cells and therefore needs to be imported into the nucleus prior to integration. Transportin-SR2 (TRN-SR2) is a cellular β-karyopherin thought to import the viral preintegration complex (PIC) into the nucleus through a direct interaction with IN. As for most nuclear import cargoes, the driving force behind PIC import is likely a gradient of theGDP- and GTP-bound forms of Ran, a small GTPase. In chapter 4 we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. We demonstrate stable complex formation of TRN-SR2 and RanGTP in solution.

Consistent with the behavior of normal nuclearimport cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. While in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2–RanGTP complex was significantly morecompact. We present a homology model of the TRN-SR2–RanGTP complex, which is in excellent agreement with the experimental small-angle X-ray scattering data and represents a stepping stone towards modulation of TRN-SR2 function for therapeutic purposes. In chapter 5 we describe the development and use of a high-throughput screening pipeline for small molecule inhibitors of the HIV-1 IN–TRN-SR2 interaction that block viral nuclear import and replication. We identify and confirm 5 active compound families from a 25,608-compound library. Modest antiviral activities are detected across all 5 classes. Two representative compounds significantly reducethe percentage of nuclear PICs in a fluorescence-based HIV-1 nuclear import assay. These compounds represent the first small molecule inhibitors of HIV-1 nuclear import and corroborate the role of the IN–TRN-SR2interaction for HIV-1 nuclear import.

As IN requires a dynamic equilibrium between at least dimers and tetramers for its catalytic activities, another candidate drug target is IN oligomerization. In chapter 6, we develop, characterize and validate an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits proved to act as allosteric IN inhibitors, are devoid of cross-resistance with INSTIs and may become true next-generationIN inhibitors. Additionally, the assay offers a platform to study IN dimerization and unravel the full mechanism of action of LEDGINs, allosteric inhibitors recognizing the Lens epithelium-derived growth factor/p75 (LEDGF/p75) site on HIV-1 IN.

Finally, in chapter 7 we identify the amino acids in HIV-1 IN that directly contact target DNA bases and affect local integration site sequence selection during viral replication.These residues also determine the propensity of the virus to integrate into flexible sequences. Remarkably, natural polymorphisms INS119G and INR231G redirect viral integration away from gene dense regions. Precisely these variants are associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.