During the early stages of retroviral replication, the virus travels from the cellular plasma membrane across the nuclear pore to finally integrate its viral cDNA into the host cell genome. These early events first require the reverse transcription of the viral RNA into a linear double strand cDNA copy by the viral reverse transcriptase (RT). Once synthesized, this cDNA becomes part of a large nucleoprotein complex, called the pre-integration complex (PIC) (reviewed in ). PICs from Moloney murine leukemia virus (MLV) [2–4] or Human immunodeficiency virus (HIV) [5–7] can be partially purified after cell infection and can efficiently integrate their associated reverse transcribed viral cDNA into heterologous DNA targets in vitro. The integration reaction is mediated by the retroviral integrase (IN) [9–11]. Within the PIC, IN binds to viral cDNA ends [12–14] and catalyzes the DNA cutting and joining reactions. First, the 3′ processing reaction consists in the hydrolysis of a dinucleotide at each end of the viral cDNA [4, 15, 16]. Then, exposed recessed 3′ hydroxyl groups of the viral cDNA are joined to the 5′ ends of the cut host target DNA [4–6, 15]. At this stage, cellular enzymes are probably in charge of removing the 5′ unpaired viral DNA ends and subsequently catalyze the gap filling and ligation reactions of host-viral DNA junctions . Human immunodeficiency virus type-1 (HIV-1) integration produces a 5 bp duplication of the DNA host sequence at each end of the integrated provirus .
Retroviral PICs are large nucleoprotein complexes that contain several viral and cellular proteins in addition to IN and viral cDNA. Biochemical studies indicate that HIV-1 PICs contain the viral nucleocapsid (NC), matrix (MA), Vpr and RT proteins [19–25]. In contrast with MLV [2, 26], HIV-1 PICs were shown to be devoid of CA [19–25]. In addition, cellular proteins including barrier to auto-integration factor (BAF), high mobility group protein HMGA, Ku and LEDGF/p75 have been found to associate with partially purified HIV-1 PICs [20, 27–29].
HIV and other lentiviruses have the ability to infect non-dividing cells, such as terminally differentiated macrophages. Therefore, this large viral nucleoprotein complex (>50 nm) must pass through the nuclear pore with the active participation of cellular factors involved in nucleo-cytoplasmic shuttling. Although several viral proteins within the HIV-1 PIC contain karyophilic signals (MA, Vpr and IN), their exact role during PIC translocation into the nucleus is still controversial . The central polypurine track (cPPT), a cis-acting sequence that forms a short triple stranded DNA structure (the central DNA Flap) during reverse transcription, is also implicated in the nuclear import of HIV PICs [31, 32]. Importantly, reports show that the HIV-1 capsid (CA) is the dominant viral determinant for HIV-1 infection of non-dividing cells, and the kinetic of dissociation of CA from the viral core appears to be a critical step in controlling nuclear import [33, 34].
Among the HIV-dependency factors involved in HIV-1 replication, TNPO3 was recently shown to be involved in a nuclear import and/or preintegration step [35–40]. TNPO3 is a karyopherin from the importin-ß family that mediates transport of serine/arginine rich (SR) proteins into the nucleus in a phosphorylation-dependent manner . Using yeast two-hybrid screenings, we identified TNPO3 as a binding partner of IN [36, 42]. Although a direct interaction between HIV-1 IN and TNPO3 has been clearly established [36, 43, 44], recent reports indicated that HIV CA is one of the viral determinants important for TNPO3 requirement [8, 38–40, 44, 45].
Once inside the nucleus, IN catalyzes viral cDNA integration into the genome of the host cell [46, 47]. HIV-1 integration occurs preferentially in transcription units (TUs) of transcriptionally active genes whereas CpG islands and promoter regions are disfavored. Importantly, the targeting of viral integration to specific regions of the host chromosome is under the control of LEDGF/p75 [48–50]. LEDGF/p75 is a key factor of HIV-1 integration that was identified as an IN interacting factor [51–53]. LEDGF/p75 is a cellular chromatin-associated protein presumably involved in transcriptional regulation of cellular genes [54–56]. LEDGF/p75 is tightly associated to chromatin, and the molecular basis of this interaction involves its conserved PWWP and AT-hook domains in the N-terminal region of the protein [57, 58]. LEDGF/p75 plays an important role in lentiviral cDNA integration, as demonstrated by mutagenesis [52, 59–61], over-expression of LEDGF/p75 IBD (Integrase Binding Domain) [62, 63] as well as RNAi and knock-out studies [49, 50, 62–65]. Structural studies revealed the roles of both the catalytic core domain dimeric interface and the N-terminal domain of IN for high affinity binding to IBD [60, 66, 67]. Albeit not strictly essential for replication, LEDGF/p75 tethers PIC-associated IN to chromatin to presumably stimulate its enzymatic activity at the site of integration [57, 58].
In this study, we explored at early times post infection the dynamics of interaction between IN and its cellular and viral partners. However, the detection of IN in infected cells remains technically challenging. We took advantage of a previously characterized virus carrying an active tagged-IN with the HA epitope at the C-terminus  to purify and characterize IN complexes in the context of infected lymphocytes. Our results shed light on the stability and distribution of IN during early steps of HIV-1 infection. We show that IN is rapidly degraded in a proteasome-dependent manner upon virus entry into the cell. Immunoprecipitation experiments allowed us to detect interactions between IN and its cofactors LEDGF/p75 and TNPO3 at 6 h post-infection (p.i.). Using size exclusion chromatography, we uncover that IN exists in at least two distinct complexes in infected cells: a high molecular weight complex that co-fractionates with viral cDNA and integration activity, and a low molecular weight complex devoid of viral cDNA that is found exclusively in the nucleus and depends on LEDGF/p75 expression.