Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase

Abstract

Background: HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex.

Methodology/principal findings: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer.

Conclusions/significance: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

Figure 1. Sequences of retroviral LTR-LTR junctions and oligonucleotide substrates.

A) The retroviral sequences found at the LTR-LTR junctions of 2-LTR circles are almost perfect palindromes –. The bases underlined correspond to imperfect palindromic sequences. The 5′-CA dinucleotide (U5-LTR) or its complementary sequence GT (U3-LTR), essential for the 3′-processing reaction, is shown in bold. The vertical dashed line indicates the axis of symmetry. B) Summary of the various DNA substrates used for palindrome cleavage and 3′-processing reactions (only the top strands are shown). The mutations in the Mut1 and Mut2 sequences are underlined.

Figure 2. Endonucleolytic activity of HIV-1 IN on the palindromic U5-U3 junction.

A) IN was incubated with 12.5 nM of 38-mer PalA/PalB duplex mimicking the HIV-1 palindromic U5-U3 junction in the presence of 7.5 mM divalent cation, for 2 h at 37°C. The 38-mer duplex was radiolabelled either on the 5′-extremity of the PalB ODN (top strand) (lanes 1-7) or on the 5′-extremity of the PalA ODN (bottom strand) (lanes 8–10). The metallic cofactor was Mg²⁺ (lanes 2–4, 9–10) or Mn²⁺ (lanes 5–7). Lanes 3, 6 and 9: 1.5 µM IN; Lanes 2, 5 and 10: 3 µM IN. Lanes 4 and 7: 3 µM IN + 1 mM EDTA. Lanes 1 and 8: PalA/PalB DNA substrate digested with ScaI. B) Relative cleavage efficiencies for the various DNA positions in the HIV-1 palindromic junction and 3′-processing substrate. The relative cleavage efficiency corresponds to a ratio between cleavage occurring at one position and total IN cleavage activity. This ratio is directly related to the specificity of the cleavage.

Figure 3. HIV-1 IN is highly specific for its cognate palindromic sequence and fails to cleave PVF and mutated HIV-1 palindromes.

A) Cleavage activity of HIV-1 IN on PFV and HIV-1 palindromes. IN was incubated with DNA substrates in a Mg²⁺-containing buffer for 2 h at 37°C. Lanes 1 and 2: FP53B/FP53A substrate (PFV). Lanes 3 and 4: PalB/PalA substrate (HIV). Lanes 5–8: Mut1 substrate (CA->GT). Lanes 9–12: Mut2 substrate (GTAC->CATG). Lanes 2, 4, 6 and 10: 3 µM IN. Lanes 7 and 11: 1.5 µM IN. Lanes 1, 3, 5 and 9: negative control with 3 µM IN + 1 mM EDTA. Lanes 8 and 12: ScaI activity on mutated DNA duplexes. B) Histogram of cleavage efficiencies for the different DNA positions in the HIV-1 LTR-LTR junction. Black bars, wt. White bars, Mut1.

Figure 4. Differential responses of palindrome cleavage and 3′-processing activities to increasing IN concentrations.

PalB/PalA or HIV38A/HIV38B duplexes (12.5 nM) were incubated with increasing concentrations of IN for 2 h at 37°C. Palindrome cleavage and 3′-processing activities were quantified as indicated in Materials and Methods and plotted versus IN concentration: Palindrome cleavage (straight line); 3′-processing (dashed line). A) Ionic strength increased with IN concentration. B) The experiment was performed as in A, except that ionic strength was kept constant ([NaCl]  =  200 mM final concentration). The time courses of product formation for palindrome cleavage and 3′-processing were compared under conditions of optimal IN concentration (3 µM), with 200 mM NaCl (inset). F_DNA represents the fractional saturation function of DNA sites, as measured by fluorescence anisotropy (see Materials and Methods), using either PalB/PalA or HIV38A/HIV38B duplexes. F_DNA is indicated for two IN concentrations (1.5 and 3 µM).

Figure 5. HIV-1 IN oligomer recruitment to the LTR-LTR junction.

The 38-bp duplex PalB/PalA (1 pmol) was incubated with IN (5 pmol) for 0 to 60 min (lanes 2–7) in the presence of AHDAP (300 µM) in a final volume of 10 µL (yielding DNA and IN concentrations of 0.1 and 0.5 µM, respectively). Crosslinked products were then subjected to SDS-PAGE analysis and gel autoradiography. MW: Molecular weight markers (kDa). Lane 1: no IN. The weak reduction of the signal observed lanes 6 and 7 as comparison to the lane 5 is due to the time-dependent formation of higher-order oligomeric states of IN which are dependent on the AHDAP .

Figure 6. Palindrome cleavage by purified IN oligomers.

The pJCT plasmid (12.5 nM) containing the LTR-LTR junction was incubated alone (lanes 1 and 4) or with 3 µM of either pure crosslinked dimers (Di) (lanes 2–3) or pure crosslinked tetramers (Te) of IN (lanes 5–6) for 4 hours at 37°C. It was then subjected to electrophoresis in an agarose gel (1%, 50V, 30 min) in the presence of BET (50 µg). Plasmid cleavage by IN was followed by partial digestion with EcoRI (lanes 3 and 6). MW: Molecular weight markers (kb).