The mechanism of retroviral integration from X-ray structures of its key intermediates

Abstract

To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA. This process is operated by the intasome, a nucleoprotein complex composed of an integrase tetramer (IN) assembled on the viral DNA ends. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.

Figure 1. Crystal structure of the PFV STC

a, Sequence of the model tDNA. The arrows point to the sites of strand transfer with a 4-bp stagger (boxed). Nucleotide numbers are indicated for both strands. The green diamond indicates the 2-fold symmetry axis. b-c, Surface (b) and cartoon (c) representations of the STC crystal structure, viewed along or perpendicular to the crystallographic two-fold axis, respectively. IN active site carboxylates are shown in red. Protein domains (NTD, N-terminal domain; CTD, C-terminal domain; CCD, catalytic core domain; NED, NTD extension domain) as well as viral (v) and target (t) DNAs are indicated.

Figure 2. Details of DNA conformations, recognition, and active site mechanics during strand transfer

a, Cartoon representation of the viral and target DNAs in the TCC_Apo (left panel) and STC (right panel) structures. b, Mechanics of DNA strand transfer. Superposition of common elements within TCC_ddA, STC, and Mn²⁺-loaded intasome (PDB ID 3L2S). Carbon atoms of viral DNA and IN active site residues are painted orange (3L2S) and yellow (STC), respectively. c, Close in view of the integration site in the STC structure. d, IN interactions with the tDNA backbone. Dashed lines indicate hydrogen bonds.

Figure 3. Sequence analysis of strand transfer reaction products

a-d, Sequence logos representing nucleotide frequencies at PFV integration sites in vitro using wild type (a), R329S (b), R329E (c), or A188S (d) INs. Target DNA sequences as joined to the viral DNA ends were used in the alignments. Arrowheads indicate sites of strand transfer, and dotted lines symmetry axes of the integration site consensi; the strict two-fold symmetry is imposed by the use of intasomes containing pairs of U5 viral DNA ends.