Chromatin tethering and retroviral integration: recent discoveries and parallels with DNA viruses

Abstract

Permanent integration of the viral genome into a host chromosome is an essential step in the life cycles of lentiviruses and other retroviruses. By archiving the viral genetic information in the genome of the host target cell and its progeny, integrated proviruses prevent curative therapy of HIV-1 and make the development of antiretroviral drug resistance irreversible. Although the integration reaction is known to be catalyzed by the viral integrase (IN), the manner in which retroviruses engage and attach to the chromatin target is only now becoming clear. Lens epithelium-derived growth factor (LEDGF/p75) is a ubiquitously expressed nuclear protein that binds to lentiviral IN protein dimers at its carboxyl terminus and to host chromatin at its amino terminus. LEDGF/p75 thus tethers ectopically expressed IN to chromatin. It also protects IN from proteosomal degradation and can stimulate IN catalysis in vitro. HIV-1 infection is inhibited at the integration step in LEDGF/p75-deficient cells, and the characteristic lentiviral preference for integration into active genes is also reduced. A model in which LEDGF/p75 acts to tether the viral preintegration complex to chromatin has emerged. Intriguingly, similar chromatin tethering mechanisms have been described for other retroelements and for large DNA viruses. Here we review the evidence supporting the LEDGF/p75 tethering model and consider parallels with these other viruses.

Figure 1:

A) Domain organization of LEDGF/p75. The N terminal domain ensemble (NDE) and the integrase binding domain (IBD) are indicated. The PWWP domain and AT hooks are the primary determinants of chromatin binding [35, 36]. However restoration of triton resistant chromatin attachment requires additional charged regions (CR2 and/or CR3) [35]. A transferable NLS was mapped to amino acids 146–156 [37, 38]. The IBD extends from amino acids 347 to 429 [37, 44]. This domain is present in one other HDGF member, HRP2, which binds IN in vitro [44], but does not appear to compensate for LEDGF/p75 deficiency in human cells [71]. B) LEDGF/p75 tethers lentiviral IN to chromatin LH4 cells are 293T cells where LEDGF/p75 is knocked down by plasmid mediated shRNA, and stably express low levels of a myc tagged HIV-1 IN. IN is detectable in the cytoplasm of these cells (top panel). When LEDGF/p75 is re-expressed IN is stabilized [62], is exclusively nuclear, and is tethered to mitotic chromatin.

Figure 2:

A) Testing the tether hypothesis To recapitulate LEDGF/p75 cofactor function, an alternatively tethered IBD should minimally restore IN chromatin attachment. The N terminal 31 amino acids of LANA were fused to GFP-IBD, which contains only amino acids 347–429 of LEDGF/p75 [89]. LH4 cells (described in Figure 1) were transiently transfected with either GFP-IBD or LANA31-GFP-IBD, and the effect on IN localization was examined by immunofluorescent staining and confocal microscopy. In the top panel, GFP-IBD interacts with IN, forming discrete structures, but most of IN remains cytoplasmic. In contrast in the lower panel LANA31GFP-IBD binds IN, tethering it to chromatin even in mitotic cells (circled). Images from [89]. B) LEDGF/p75 IBD molecules with alternative chromatin tethers are functional lentiviral cofactors. In the graph on the left, knockdown of LEDGF/p75 (TL3 cells) or expression of untethered GFP-IBD in SupT1 cells inhibits HIV-1 infection, and combining both is multiplicative (TL4 cells). The deficit in HIV-1 infection here is 3 logs. In contrast, as is seen on the graph on the right the tethered LANA31GFP-IBD restores HIV-1 infectivity by 2 logs, comparably to native LEDGF/p75. Both graphs modified from [89]. See [89] for additional data on the LEDGF/p75 knockdown rescuing properties of other LANA31 chimeras.

Figure 2:

A) Testing the tether hypothesis To recapitulate LEDGF/p75 cofactor function, an alternatively tethered IBD should minimally restore IN chromatin attachment. The N terminal 31 amino acids of LANA were fused to GFP-IBD, which contains only amino acids 347–429 of LEDGF/p75 [89]. LH4 cells (described in Figure 1) were transiently transfected with either GFP-IBD or LANA31-GFP-IBD, and the effect on IN localization was examined by immunofluorescent staining and confocal microscopy. In the top panel, GFP-IBD interacts with IN, forming discrete structures, but most of IN remains cytoplasmic. In contrast in the lower panel LANA31GFP-IBD binds IN, tethering it to chromatin even in mitotic cells (circled). Images from [89]. B) LEDGF/p75 IBD molecules with alternative chromatin tethers are functional lentiviral cofactors. In the graph on the left, knockdown of LEDGF/p75 (TL3 cells) or expression of untethered GFP-IBD in SupT1 cells inhibits HIV-1 infection, and combining both is multiplicative (TL4 cells). The deficit in HIV-1 infection here is 3 logs. In contrast, as is seen on the graph on the right the tethered LANA31GFP-IBD restores HIV-1 infectivity by 2 logs, comparably to native LEDGF/p75. Both graphs modified from [89]. See [89] for additional data on the LEDGF/p75 knockdown rescuing properties of other LANA31 chimeras.

Figure 3:. The tether model applies across different classes of viruses.

A) Ty5 retrotransposon utilizes Sir4p to target integration to telomeres and silent mating loci. A specific 6aa targeting domain at the C-terminus of IN binds Sir4p. Sir4p binds Rap1p, a telomeric repeat binding protein, thereby directing the entire complex to telomeres. B) Latency associated nuclear antigen (LANA) binds to terminal repeats in the DNA of KSHV episome and a groove formed by Histones 2A and 2B, tethering the DNA to chromatin and allowing persistence in latently infected cells. C) Tethering model for lentiviruses. The cDNA, IN, LEDGF/p75 complex is illustrated. LEDGF/p75 binds an IN dimer. Attachment persists through mitosis. The chromatin ligand of the PWWP domain not known.