Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

Abstract

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.

Figure 1

Stereo views of the nine lowest-energy structures of U5-term generated from classical scalar coupling and NOE restraints. These were optimized by relaxation matrix refinement using: (a) uncertainties of ±20%; (b) very weak, weak, medium and strong distance restraint categories.

Figure 2

Stereo views of the U5-term structures corresponding to: (a) 36 RDC-structures, i.e. four families of nine best structures each family being generated from a different value of alignment tensor (see Table 2); (b) nine lowest-energy structures generated from classical NOE and scalar coupling restraints combined to RDC restraints (Da = −22 Hz; R = 0.1).

Figure 3

Structural parameters of U5-term. Profiles of: (a) inter base (intrastrand) rolls in the upper strand; (b) global base–base openings; (c) inter base twists in the upper strand; (d) global base–base propellers; (e) global base–base buckles. Values were extracted from outputs of CURVES (39) for: in left, a set of nine structures generated from NOE restraints classified into very weak, weak, medium and strong distance categories; in middle, a set of nine structures generated from NOE restraints with uncertainties of ±20%; and in right, the 36 RDC structures (see legend in Figure 2).

Figure 4

(a) Profiles of the local number of B-DNA structures (Zp value inferior to 0.5 Å) among the 36 RDC structures [calculated with 3DNA (42)]. A schematic definition of Zp is given in insert (41); (b) Profile of the global base pair axis inclinations of the 36 RDC structures (see legends in Figure 3 and Table 2), values being calculated with CURVES (39); (c) Profiles of local inter base (intrastrand) tilts in the upper strand of the 36 RDC structures (see legend in Figure 3), values being calculated with CURVES (39); (d) Profile of the average interstrand phosphorus–phosphorus (P_m–P_n+3) distances (illustrating the minor groove width) from the 36 RDC structures (see legend in Figure 3), calculated with 3DNA (42); (e) Profiles of intrastrand phosphate–phosphate P_i–P_i+1 distances along the upper strand (left) and the lower strand (right).

Figure 5

A stereo view of the average structure of the five outermost base pairs obtained from the 36 RDC structures of U5-term (see legend in Figure 2) featuring the particular interstrand stacking of purines A15 and G21 in red thick lines and the phosphorus of the scissile bond in CPK.