Targeting HIV-1 DNA integration by swapping tethers.

R etroviruses replicate by integrating a DNA copy of their genome into cellular DNA (1). The integrated DNA is replicated along with cellular DNA during each celldivision cycle and is the template for transcription of RNAs required for production of progeny virus. DNA integration is mediated by the virally encoded integrase enzyme and occurs at essentially any location in the host DNA, but each group of retroviruses exhibits distinct regional preferences (2). For example, HIV1 and closely related retroviruses called lentiviruses preferentially integrate in active transcription units. The mechanistic basis of the target-site preference of HIV1 DNA integration is not well understood, but studies suggest that a cellular protein called lens epithelium-derived growth factor (LEDGF) plays a key role (3). LEDGF binds to both HIV-1 integrase and chromatin, and in a popular model, it tethers the viral integration machinery to chromatin (4). In this issue of PNAS, Ferris et al. (5) provide strong support for this model and show that the targeting preference of HIV-1 can be changed simply by swapping the chromatin-binding domain (CBD) of LEDGF. Retroviral DNA is made by reverse transcription in the cytoplasm after infection and forms part of a large nucleoprotein complex termed the preintegration complex (PIC). Integrase, which catalyzes the key DNA-cutting and -joining steps of integration, is tightly associated with the viral DNA within the PIC. In addition to integrase, many other viral and cellular proteins have been reported to be components of the PIC, although in most cases their functional role, if any, is unclear. Integrase is an obvious candidate for determining target specificity because the target site must juxtapose the active site of the enzyme during the catalytic steps. Indeed, it has been long established that retroviral integrases do not catalyze random integration into DNA but rather use some sites in DNA preferentially as targets (see ref. 6 and references therein). However, the specificity directed by integrase and target DNA alone is local, accounting for nonrandom distribution of integration within a few hundred base pair stretches of DNA; it cannot account for the regional preferences of integration into chromatin discussed here. A paradigm for thinking about how integration can be targeted comes from studies of other mobile genetic elements that exhibit a very high degree of specificity for their sites of integration. Wellunderstood examples include the yeast Ty3 retrotransposon, which integrates almost exclusively into transcription initiation sites of genes transcribed by RNA polymerase III, and Ty5, which integrates into heterochromatin. This specificity is directed by tethering the integrase proteins to chromatin via different transcription factors (7, 8). HIV-1 integrase binds LEDGF (9), making this protein a prime candidate for a role in target-site selection. A domain in the C-terminal region of LEDGF, the integrase-binding domain (IBD), binds integrase, and the interface has been determined by x-ray crystallography (10, 11). The N-terminal part of LEDGF contains a proline-tryptophan-tryptophan-proline (PWWP) domain and adenine-thymine (AT) hooks that bind chromatin (12). Importantly, a viable mouse embryo fibroblast LEDGF knockout (MEF-KO) cell line exists that supports HIV-1 replication, albeit at a reduced level, enabling the potential role of this protein in directing integration specificity to be explored (13). Deletion of the PWWP chromatin-binding region of LEDGF reduces HIV-1 replication to the level observed in the absence of this cofactor, but replacing the PWWP domain with other chromatin-binding peptides restores efficient replication (14). In the simplest model, LEDGF is an adaptor that enhances integration by tethering integrase within the PIC to chromatin. The study by Ferris et al. (5) tests this hypothesis by replacing the PWWP and AT hooks of LEDGF with other CBDs with known specificity (Fig. 1): the plant homeodomain finger from inhibitor of growth protein 2 (ING2), which binds histone 3 lysine 4 trimethylations (H3K4me3), and the chromodomain of heterochromatin protein 1-α (HP1α), which binds histone 3 that is methylated on lysine 9 (H3K9me2,3). The H3K4me3 modification is common in the 5′ regions of active genes (15), whereas H3K9me2,3 is widely distributed throughout chromatin (16). Not only do these LEDGF fusion proteins restore HIV-1 replication when expressed in MEF-KO cells; they also direct integration to the target sites of their respective CBDs. How does LEDGF exert such a strong influence on the targeting of HIV-1 DNA integration? The stimulation of integration observed upon expression of LEDGF in MEF-KO cells and the redirection of integration sites directed by fusions with heterologous CBDs suggest that LEDGF Fig. 1. Tethering of PICs to chromatin by LEDGF fusion proteins. The viral DNA within the PIC is shown in green with the ends associated with a tetramer of integrase (IN). The IBD of LEDGF (yellow) binds to integrase within the PIC. The CBD of LEDGF tethers the PIC to its binding sites in chromatin; the binding partner in chromatin is unknown. PICs can be tethered to different regions of chromatin by substituting other CBDs that bind different partners. The tether spatially restrains the PIC, and integration occurs in the vicinity of attachment.