Retroviral DNA integration: HIV and the role of LEDGF/p75

To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.     

Molecular Mechanism of LEDGF/p75 Dimerization

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FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.     

Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five alpha-helices. Based on folding topology, the IBD is structurally related to a diverse family of alpha-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-beta. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.     

Identification of the LEDGF/p75 binding site in HIV-1 integrase

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.     

HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

Background

The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.

Findings

Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs).

Conclusions

These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.     

LEDGF/p75 Proteins with Alternative Chromatin Tethers Are Functional HIV-1 Cofactors

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi''s sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.     

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.