Figure 1.
A, Schematic of the IN fusion proteins. NED, N-terminal extension domain NTD, N-terminal domain; CCD, catalytic core domain; CTD, C-terminal domain. B, Comparison of the solubilities of wild-type HIV-1 IN and Sso7d-IN. Proteins were incubated at the indicated NaCl concentrations in 20 mM HEPES pH 7.5, 10% glycerol, 5 mM DTT and 1 mM EDTA, centrifuged and the supernatants and pellets were analyzed by SDS PAGE. C, Schematic of the in vitro integration reaction with a double stranded oligonucelotide mimicking viral LTR-U5 and a circular target DNA. D, Strand transfer reaction carried with either wild-type HIV-1 IN or Sso7d-IN with an 11 amino acid linker and a fluorescently labeled viral DNA substrate (U5-25) in 20 mM HEPES pH 7.5, 10 mM DTT, 5 mM MgCl2, 4 µM ZnCl2, 100 mM NaCl, 300 ng pGEM-9zf and 0.5 µM viral DNA substrate. The position of concerted and half-site integration products is indicated. The same gel was visualized by either ethidium bromide staining (left panel) or a Typhoon 8600 fluorescence scanner (right panel).
Figure 2.
Optimization of reaction conditions with Sso7d-IN and oligonucleotide DNA substrates.
Concerted integration bands are indicated with arrows. A, Effect of donor DNA length. The reactions were carried with 1 µM Sso7d-IN (Gly-11) and 0.5 µM viral DNA substrate containing a “GC rich” motif in 20 mM HEPES pH 7.5, 10 mM DTT, 5 mM MgCl2, 4 µM ZnCl2, 100 mM NaCl, and 300 ng pGEM-9zf. B, Reactions were carried with 1 µM Sso7d-IN differing in the length of the glycine linker. C, Concerted integration under optimized conditions. The ratio of Sso7d-IN (Gly-11) to donor DNA (U5-25) was kept constant at 2∶1. Sso7d-IN concentrations are 0.4 µM (lane 1), 1.0 µM (lane 2), 2.0 µM (lane 3), 4.0 µM (lane 4) and 8.0 µM (lane 5). 25% glycerol was included in the reaction buffer. The DNA smear (S) below the linear concerted integration product results from multiple integrations into the same target DNA (depicted in D).
Figure 3.
Sequence analysis of concerted integration products.
A, Cone presentation of target duplication size distribution or deletions in concerted integrants. 36 clones contained a 5 bp duplication. 10 clones contained a short terminal deletion, likely resulting from contaminating DNA from the smear below the concerted integration product. B, Weblogo representing nucleotide base frequencies at the junction of concerted integration products (n = 72). The arrow indicates the middle position of the 5 bp target site duplication. Alignment of integration site revealed a weak consensus target sequence (GTA/TAC). The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each nucleotide at that position. The figure was created by WebLogo.
Figure 4.
EMSA of intasomes assembled with Sso7d-IN (Gly-11) and a 25 bp DNA substrate (FAM labeled U5-25).
To prevent non-specific DNA binding, 10 µg/ml of heparin was added to the reaction mixture after intasome assembly as well as into 3% agarose gels. A, Intasomes assemble with Sso7d-IN (lane 3), but not with wild-type HIV-1 IN (lane 1) or the Sso7d domain alone (lane 2). B, Sso7d-IN specifically assembles intasomes on LTR-U5 sequence (lane 1), but not on “CA/GT mut” (lane 2) or “3 bp mismatch” (lane 3) DNAs. In the “CA/GT mut” DNA, the conserved “CA” dinucleotide is replaced by “GT” (highlighted in the sequence). “3 bp mismatch” was prepared by replacing of “ACT” with “TGA” at the 5′ end of the non-joining strand.
Figure 5.
Size-exclusion chromatography of intasomes assembled with Sso7d-IN (Gly-11) and 25 bp DNA (U5-25 DNA).
A, Elution profile of intasomes on Superdex 200 2.3/30. The peak labeled free protein as expected elutes in the presence of zinc at a position consistent with a tetramer of Sso7d-IN. The intasome peak elutes at greater than 400 kD relative to protein standards, which is greater than expected for monomeric intasomes. B, Fractions 1 to 15 (F1–F15), corresponding to 13.75 min to 32.5 min elution time (highlighted with blue bar) were analyzed by SDS PAGE (panel B) and 3% agarose gel electrophoresis (panel C), and visualized by silver staining and ethidium bromide staining, respectively. D, Fractions F5 to F20 (highlighted in red), corresponding to 18.75 min to 38.75 min elution time, were tested for strand transfer activity in the presence of Mg2+ and supercoiled plasmid DNA. Note that the bulk of strand transfer activity co-elutes with the protein-DNA complex at around 23–28 min elution time (F8–F11). Concerted integration products are indicated by the arrow. The smear resulting from multiple concerted integration events is indicated by the square brackets.
Figure 6.
Sso7d-IN is functional in virions.
The assay is based on the ability of IN expressed as a Vpr fusion protein to transcomplement N/N virus lacking a functional integrase. A, HIV-1 infectivity normalized to the level obtained with Vpr-IN complementation. The Vpr fusions used for complementation and the infections that were conducted in the presence of RAL are indicated. Sso7d(mut) contains the mutations W24A and R43E which abrogate DNA binding. Graphed are averages with standard deviation for n = 3 (infections with RAL or Vpr-IN-D64A) or n = 6 independent experiments. B, Western blot of IN deletion mutant virus produced with indicated Vpr fusions probed for IN (left panel) and p24 (right panel). All Vpr-IN constructs yielded similar levels of packaged IN protein. The anti-IN antibody 8E5 recognizes the C-terminus (262–271) of IN [18] while the anti-p24 was from Abcam.