The lentiviral integrase binding protein LEDGF/p75 and HIV-1 replication

Abstract

Retroviral replication proceeds through a stable proviral DNA intermediate, and numerous host cell factors have been implicated in its formation. In particular, recent results have highlighted an important role for the integrase-interactor lens epithelium-derived growth factor (LEDGF)/p75 in lentiviral integration. Cells engineered to over-express fragments of LEDGF/p75 containing its integrase-binding domain but lacking determinants essential for chromatin association are refractory to HIV-1 infection. Furthermore, both the levels of HIV-1 integration and the genomic distribution of the resultant proviruses are significantly perturbed in cells devoid of endogenous LEDGF/p75 protein. A strong bias towards integration along transcription units is a characteristic feature of lentiviruses. In the absence of LEDGF/p75, HIV-1 in large part loses that preference, displaying concomitant integration surges in the vicinities of CpG islands and gene promoter regions, elements naturally targeted by other types of retroviruses. Together, these findings highlight that LEDGF/p75 is an important albeit not strictly essential cofactor of lentiviral DNA integration, and solidify a role for chromatin-associated LEDGF/p75 as a receptor for lentiviral preintegration complexes. By now one of the best characterized virus-host interactions, the integrase-LEDGF/p75 interface opens a range of opportunities for lentiviral vector targeting for gene therapy applications as well as for the development of novel classes of antiretroviral drugs.

Figure 1. Retroviral Early Events and the Mechanism of HIV-1 DNA Integration.

(A) The early phase of the retroviral lifecycle is divided into nine steps, spanning from step 1, when the extracellular virus initially engages its cellular receptor, to step 9, with the completion of provirus formation. Steps 5–8, which encompass IN 3′ processing of the nascent reverse transcript in the cytoplasm to the formation of the DNA strand transfer reaction product in the nucleus (highlighted in blue font), could potentially be affected by the IN–LEDGF/p75 interaction. Among these steps, lentiviruses display unique nuclear import and chromatin targeting properties (blue boxes). (B) Details of phosphodiester bond breakage and joining during HIV-1 integration. Small vertical arrows denote the bonds cleaved by IN, using water as the chemical nucleophile for 3′ processing, and the OH groups at the 3′ ends of the processed viral DNA for DNA strand transfer . Results of in vitro experiments indicate that a dimer of HIV-1 IN suffices to process each viral DNA end, whereas a tetramer is required for DNA strand transfer activity –. IN is known to function as a multimer during infection ,; notably, though, its functional PIC-associated multimeric form has not been determined. A tetramer is represented at each step here for simplicity. The open and filled triangles at the ends of the viral DNA represent U3 and U5 sequences, respectively, important for IN function (reviewed in [97]). Host cell factors are likely to repair the single strand gaps present within the DNA recombination intermediate (reviewed in [5]).

Figure 2. Domain Organization of LEDGF/p75 and HIV-1 IN Proteins.

(A) LEDGF/p75 and related proteins. The binding of LEDGF/p75 to DNA in vitro is mediated by the NLS and a nearby dual copy of the AT-hook DNA binding motif , whereas the N-terminal PWWP domain supplies a critical chromatin recognition function ,. Charged regions (CRs) 1–3 work in concert with the PWWP domain and AT-hooks to affect the wild-type chromatin binding phenotype as determined by biochemical fractionation . The function of the HRP2 AT-hooks, identifiable though sequence gazing , has not been established experimentally. A patch of conserved amino acids known as homology region III (HR3) was also identified via aligning multiple HRP2 orthologs . The LEDGF/p75 IBD is critical for stimulation of HIV-1 IN function in vitro ,,,,, and for HIV-1 infection ,. The N-terminal 325 residues within LEDGF/p75 and LEDGF/p52 are identical, whereas the p52 isoform harbors a unique 8–amino acid residue tail . (B) The three IN domains as defined by a number of structural and functional studies are shown (refer to and for details). Amino acid residues within each domain that are conserved across Retroviridae are indicated. The Asp and Glu residues highlighted in red within the CCD coordinate Mg ions for catalysis during 3′ processing and DNA strand transfer.

Figure 3. Crystal Structure of the LEDGF/p75-IN Interaction.

(A) Cartoon representation of the CCD-IBD complex (the complete asymmetric unit) . IN CCD molecules are colored green and blue, whereas the LEDGF/p75 IBDs are magenta and yellow. The side chains of IN active site residues Asp-64, Asp-116, and Glu-152 (Figure 2B) are shown as red sticks. The region within the dashed box is expanded in (B). (B) Details of the CCD-IBD interface. LEDGF/p75 hotspot residues Ile-365 and Asp-366, situated at the base of the loop between IBD helices 1 and 2, project into a pocket at the CCD dimer interface. The bidentate hydrogen bond contact between Asp-366 and the backbone amides of IN residues Glu-170 and His-171 is critical for the protein–protein interaction in vitro , in yeast cells , and during HIV-1 infection . Ile-365 is buried into a hydrophobic pocket predominantly formed by IN residues Ala-128, Trp-132, Leu-102, and Met-178. Hydrogen bonds and salt bridges are shown as dotted lines. The figure was drawn using PyMOL .

Figure 4. Model of LEDGF/p75 Function during HIV-1 Integration.

(A) The basic unit of chromatin, the nucleosome, is depicted as a gray oval of histone proteins in association with chromosomal DNA (orange lines). LEDGF/p75 might engage chromatin via its NLS and AT-hook motifs (A/T h) binding directly to DNA and/or by the PWWP domain and AT-hooks mediating protein interactions with histone proteins or other currently unknown chromatin factors (labeled X) ,,. Upon engaging the IBD, PIC-bound IN is encouraged to integrate the viral cDNA at a nearby position, statistically favoring the indicated palindromic target DNA sequence (indicated by short black lines, and expanded to denote the sequence) , –. The short vertical lines indicate the sites of integration on the two chromosomal DNA strands. (B) The PIC can still engage the consensus target DNA sequence in the absence of LEDGF/p75, though overall levels of HIV-1 integration are reduced ∼10-fold under this condition (represented by the relative size of the arrow in [A] and [B]) .