We studied human immunodeficiency virus, type 1 (HIV-1) integrase (IN) complexes derived from nuclei of human cells stably expressing the viral protein from a synthetic gene. We show that in the nuclear extracts IN exists as part of a large distinct complex with an apparent Stokes radius of 61 Å, which dissociates upon dilution yielding a core molecule of 41 Å. We isolated the IN complexes from cells expressing FLAG-tagged IN and demonstrated that the 41 Å core is a tetramer of IN, whereas 61 Å molecules are composed of IN tetramers associated with a cellular protein with an apparent molecular mass of 76 kDa. This novel integrase interacting protein was found to be identical to lens epithelium-derived growth factor (LEDGF/p75), a protein implicated in regulation of gene expression and cellular stress response. HIV-1 IN and LEDGF co-localized in the nuclei of human cells stably expressing IN. Furthermore, recombinant LEDGF robustly enhanced strand transfer activity of HIV-1 IN in vitro. Our findings indicate that the minimal IN molecule in human cells is a homotetramer, suggesting that at least an octamer of IN is required to accomplish coordinated integration of both retroviral long terminal repeats and that LEDGF is a cellular factor involved in this process.

We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.

The INK4a/ARF tumor suppressor locus, a key executor of cellular senescence, is regulated by members of the Polycomb group (PcG) of transcriptional repressors. Here we show that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3 and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activates INK4a, but not ARF, and causes p16^INK4a-dependent arrest. In mouse embryo fibroblasts, Jmjd3 activates both Ink4a and Arf and elicits a p53-dependent arrest, echoing the effects of RAS in this system. Our findings directly implicate JMJD3 in the regulation of INK4a/ARF during oncogene-induced senescence and suggest that JMJD3 has the capacity to act as a tumor suppressor.

Retroviruses such as HIV need to insert their genome into the host genome in order to replicate. This process is mediated by a tetramer of the virus-encoded integrase protein. Earlier this year, Peter Cherepanov and colleagues reported the structure of a related integrase from prototype foamy virus bound to the cleaved viral DNA ends, a complex known as the intasome. Now they have solved the structure of the intasome interacting with the target host DNA, before and after it is cleaved. This work reveals new details of the integration process that could be useful in designing improved inhibitors of HIV. Insertion of retrovirus genome into host genome to replicate is mediated by a tetramer of the virus-encoded integrase protein. The structure of a related integrase from prototype foamy virus bound to the cleaved viral DNA ends, a complex called the intasome, was previously revealed. These authors solve the structure of the intasome interacting with the target host DNA both before and after it is cleaved, revealing new details of the integration process that may help in designing improved inhibitors of HIV. To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA1,2. This process is operated by the intasome, a nucleoprotein complex composed of an integrase tetramer (IN) assembled on the viral DNA ends3,4. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported4,5,6, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.

Significance: Transportin 3 (Tnpo3) was shown to orchestrate nuclear import of splicing factors over a decade ago, but how it recognizes these cargoes remained unknown. Furthermore, the recently discovered role for Tnpo3 as a cofactor of HIV-1 replication requires mechanistic clarification. We show that Tnpo3 associates with a wide range of proteins involved in mRNA metabolism, the majority of which contain serine/arginine-rich domains. Using X-ray crystallography we determined the three-dimensional structures of Tnpo3 in its key functional states, explaining how this nuclear import factor binds and releases its cargoes. We also show that Tnpo3 mutants that are not able to interact with cleavage and polyadenylation specificity factor 6 do not facilitate HIV-1 infectivity, suggesting a potential route of pharmacological intervention in the treatment of AIDS.