HIV-1 integrase crosslinked oligomers are active in vitro

Abstract

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.

Figure 1

Concerted integration reaction catalyzed by yeast recombinant HIV-1 IN. (A) Gel analysis of the integration products. Reactions were performed in the presence (+) or in absence (−) of 250 nM of IN. The target DNA is indicated as pBSK receptor plasmid. The donor DNA was 5′-end labeled. Products were submitted to 1% agarose gel electrophoresis. nCI, CI: respectively non-concerted and concerted full-site integration products. SI: Self-integration products. M: Markers (base pairs). The structure of the expected products are also described in this figure. (B) Effect of reaction conditions on the integration reaction catalyzed by recombinant HIV-1 IN. For each condition one parameter was changed independently (MgCl₂ or MnCl₂ concentration, presence of DMSO or PEG). The percentage of the total integrated substrate was reported on the graph. Results are the mean +/− SD of three separate experiments.

Figure 2

(A) Gel filtration chromatography of crosslinked HIV-1 IN. IN (150 pmol) was crosslinked for different lengths of time and products were loaded on a Superose 12 column. Migration of the molecular weight markers is indicated. Monomers (Mo), dimers (Di) and tetramers (Te) of IN are indicated by comparison with markers. The fractions containing monomers (IN^Mo), dimers (IN^Di) and tetramers (IN^Te) of IN were pooled and used to assay the IN activity. (B) Size of the IN oligomers determined by gel filtration chromatography. 25 μl aliquots of the fractions IN^Mo, IN^Di and IN^Te obtained in A were chromatographed on a Superose 12 column under similar conditions. The elution position of each fraction was reported (shown as triangles) in this figure and compared to elution profile of known molecular weight markers (shown as clubs in the figure): catalase (232 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa).

Figure 3

In vitro activities of the HIV-1 IN oligomers. Reactions were performed under standard conditions using no protein (Mock), 250 nM of non-crosslinked IN (IN^NC), monomers (IN^Mo), or crosslinked dimers (IN^Di) and tetramers (IN^Te) purified by gel filtration. (A) In vitro concerted integration assay; (B) number of zeocin-resistant clones obtained after cloning of the total reaction products in MC1061/P3 E.coli strain (mean +/− SD of three experiments; (C) processing and strand transfer assays.

Figure 4

Effect of reaction conditions on concerted integration activity. Standard conditions described in Materials and Methods section were used, but either DMSO or PEG or both were omitted. The total integrated donor DNA into the receptor plasmid was determined by quantifying the bands corresponding to form I (nCI + CI) and form II (CI) using the NIH software.

Figure 5

SDS-PAGE analysis of crosslinked IN–DNA complexes. IN (5 pmol) was preincubated with the 5 ′-end radiolabeled 294 bp DNA substrate (1 pmol) carrying either no viral LTR (A), one viral LTR (B) or two viral LTRs sequences (C) for 0 to 60 min (lanes 0 to 60) in the presence of AHDAP at 37°C (final volume 20 μl). After 1 h of DNase 1 treatment, products were separated by electrophoresis on 12% SDS-PAGE gel. The gel was then dried and autoradiographed. The positions of bands a, b and c were compared with the migration of protein weight markers (BIO-RAD) submitted to electrophoresis under the same conditions and reported in the right part of each gel. The migration front of monomers, dimers and tetramers of IN obtained by crosslink of the enzyme by AHDAP is also reported (stars in the figure).

Figure 6

Model for the in vitro formation of an active IN–DNA complex. HIV-1 IN exists in solution as an equilibrium between monomers and dimers (A). Each LTR extremity might bind to a correctly folded monomer (B), allowing the correct dimerization of IN (C). The 3′-processing of each extremity might be performed by dimers (D). Both dimers might interact together (E), forming a tetramer ready to integrate the two LTRs in the target DNA (F). Black circles indicate the 3′-processed viral ends.